asd
Scientific Report
2003
Institute for Genetics
University of Cologne
Published by: Institute for Genetics, University of Cologne
Table of Contents
INTRODUCTION ........................................................................................................ 1
SCIENTIFIC REPORTS
Jens C. Brüning
Mouse Genetics and Metabolism.................................................................. 4
Wim Damen
Evolution and Development in Arthropoda .................................................... 7
Ute Deichmann
History of Biological and Chemical Sciences, 20th Century ......................... 10
Walter Doerfler
Molecular Virology and Medical Genetics ................................................... 14
Jürgen Dohmen
Molecular Biology of the Ubiquitin/Proteasome System,
and SUMO Protein Modification in Yeast ................................................... 18
Jonathan Howard
Novel Large GTPases as Cell-Autonomous Resistance Factors.................. 22
Börries Kemper
Proteins in Genetic Recombination and DNA-Repair .................................. 28
Thomas Klein
Pattern Formation and Neurogenesis During Adult
Development of Drosophila melanogaster .................................................. 32
Michael Knittler
Cell Biology of Antigen Presentation .......................................................... 36
Sigrun Korsching
Development and Function of the Olfactory Nervous System....................... 40
Thomas Langer
Proteolysis in Mitochondria........................................................................ 44
Maria Leptin
Mechanisms of Morphogenesis ................................................................. 50
Benno Müller-Hill
From Lac Repressor to the History of Genetics ......................................... 54
Karin Schnetz
Pleiotropic Regulation in E. coli ................................................................. 56
Frank Sprenger
Cell Cycle Control in Drosophila ................................................................ 60
Diethard Tautz
Molecular Evolution ................................................................................... 64
Ari Waisman
Molecular Immunology ............................................................................... 68
Thomas Wiehe
Bioinformatics and Population Genetics ..................................................... 72
Organization, Facilities and Graduate Training
at the Institute for Genetics
Cologne Spring Meeting .........................................................................................74
Center for Mouse Genetics .....................................................................................76
Microarry Facility .....................................................................................................77
Training and Support for Graduate Students ........................................................ 78
Sonderforschungsbereiche ...................................................................................79
How to contact the professors and group leaders? ............................................ 81
Introduction
The Institute for Genetics began life in 1956, when
Max Delbrück and Joseph Straub, the Professor
of Botany, initiated the planning during a phage
course organised by Delbrück in Cologne. The
governing idea was of an institute that would bring
genetics back to Germany, not the discredited racial
genetics of the Nazi era, but now the new triumphant
phage genetics. In the same year, Carsten Bresch
became Associate Professor for microbiology and
quasi leader of a preparatory group. Thomas
Trautner, Rudolf Hausmann and Peter Starlinger
joined him as assistants. In 1958 Walter Harm
became Associate Professor of radiation biology,
again as a member of the future Genetics Institute.
These groups worked in the Botany Department
and in a small villa next to it that has since been
demolished. In 1961 the new Institute building at
Weyertal 121 was ready and Delbrück, on leave
from Caltech for two years, became its first Director
with Hans Zachau and Ulf Henning as group
leaders. In 1963, after Delbrück’s return to Caltech,
most of the senior figures departed for other
positions and it was left to Peter Starlinger to rebuild
the Institute. In 1965 Starlinger became Professor
of Genetics and Radiobiology, in 1967 Walter
Vielmetter Professor of Genetics and Microbiology,
in 1968 Benno Müller-Hill Professor of Genetics,
in 1970 Klaus Rajewsky Professor of Genetics and
Immunology and finally in 1972 Walter Doerfler
Professor of Genetics and Virology.
In 1990 the Institute for Genetics was extended
to accommodate a sixth chair in Genetics by the
construction of a small modern research building
on the site of the pre-war isolation hospital at
Zülpicher Strasse 47, next to the Institutes for Earth
Sciences. Ostensibly dedicated to Biotechnology,
and still known under this name by parts of the
university administration, this building was the
nucleus, first for the construction in 1996 of the
new Biochemistry Institute, and since 2001 for the
new integrated Institute for Genetics which is now
complete. In the next few years the remaining Institutes of Biology (Botany, Zoology and
Developmental Biology) will move from the
Weyertal site to join Biochemistry and Genetics in
a further new building on the Zülpicher Strasse site
and form a Biocentre with central teaching facilities
and library. When complete, the new plan means
that all biology and biochemistry teaching and
research in the Mathematics and Natural Sciences
Faculty are for the first time physically close to each
other, adjacent to the Earth Sciences, and to the
Institutes for Chemistry and Physics. The single
disadvantage of the new location of the Genetics
Institute is the increased distance from some of our
closest colleagues in the University, namely the
research-oriented Institutes of the University
Medical School, and from the German Central
Medical Library.
From 1972 until 1994, no further recruitments
were made to full Professor in the Institute for
Genetics. With the retirement of all 5 of the original
chairs since that time, the Institute has undergone a
radical change in style and direction. Eleven
professorships are now associated with the Institute for Genetics (six C4, five C3). Of the present
incumbents, all except one were recruited within
the last 10 years, with research interests including
population genetics and evolution, developmental
genetics, immunology, neurobiology, cell biology
and bioinformatics. The recruitment of another C4
Professor in mouse genetics is currently ongoing.
The first time all the new Professors will work
together will be when the Weyertal Institute moves
into the new building, presumably in the next few
months.
The Professors of the Institute form a board (Vorstand), which decides upon all Institute matters. The
Vorstand is chaired by the acting director who is
elected for a two-year term. Since October 2002,
Thomas Langer has been serving in this function.
In general the Vorstand takes its decisions by
consensus rather than by vote.
In addition to the ‘professorial’ groups the Institute currently houses six independent nonprofessorial groups.
1
Introduction
In recent years links have been growing between
the Institute and research oriented groups in the
Medical Faculty. The inter-faculty Centre for
Molecular Medicine Cologne (CMMC), based on
the Medical Faculty, has projects held by Institute
staff, and an independent neurobiology research
group of the CMMC has occupied space in the
Zülpicher Strasse building for several years. The
new SFB653 “Posttranslational Control of Protein
Function” in the Institute for Genetics will contribute
to and profit from the service laboratory of the
CMMC in protein microanalytical studies. Several
other links between the Institute and the Medical
Faculty have developed recently. Most developed
is the joint decision to recruit a new medical C4
Professor in Human Genetics with the research
effort located in the new Institute for Genetics
building.
When the Institute was founded it represented a
new enterprise for Germany, both in its research
programme and in the informal character of its
institutions. Forty years later the Institute for
Genetics is perhaps now closer to the mainstream
with its exclusive use of the English language, the
internationalism of its student body, the
independence of all its Professors and their equal
voice in Institute management. The Institute values
its existence as an integrated structure with its own
atmosphere, its own initiatives and its own voice.
This applies to every aspect of the Institute’s activity,
its recruitment, its staff, its research and its teaching.
The Institute participates in the teaching of
undergraduates for the foundation course (Vordiplom) and in the advanced course.
The Institute for Genetics is proud of its
reputation, of its history, its distinctive place in
German biology and of a sense of identity that has
survived the radical transformation of the Institute
of the last decade and seems likely to persist.
2
Scientific Reports
3
Jens C. Brüning
Professor
Jens Claus Brüning was born in Cologne on
June 4th, 1966. He studied medicine from 1985
to 1992 at the University of Cologne, where
he completed his doctoral thesis under the
supervision of Prof. Dr. M. Pfreundschuh at
the Klinik I für Innere Medizin der Universität
zu Köln (Head: Prof. V. Diehl). He received
his medical doctor in 1993. From 1992 to 1994
he worked as a resident (AIP) at the Klinik II
und Poliklinik für Innere Medizin der Universität zu Köln (Head: Prof. Dr. W. Krone).
Supported by a scholarship of the Deutsche
Forschungsgemeinschaft DFG and a Mary K. Iacocca grant he
continued his studies as postdoctoral fellow from 1994 to 1997 in the lab
of Prof. C. R. Kahn at the Joslin Diabetes Center/Harvard Medical
School, Boston/USA. When he returned to Cologne in July 1997 he
started to establish his own research group working on transgenic mouse
models of insulin resistance at the Klinik II und Poliklinik für Innere
Medizin. Having passed his exams for internal medicine (February 2001)
and endocrinology (December 2001) he became senior physician at the
Klinik II und Poliklinik für Innere Medizin (Head: Prof. W. Krone).
Group members
André Kleinridders, Graduate
Student,
Stephanie Baudler, Postdoc
Jens Alber, Technician,
Julia Baumgartl, Technician
Brigitte Hampel, Technician
Sigrid Irlenbusch, Technician
Ursula Lichtenberg, Department
Manager
In October 2002 he obtained the “venia legendi” for internal medicine
and was offered a tenure position as professor at the Institute for
Genetics, Weyertal, University of Cologne, which he accepted in April
2003. Jens C. Brüning is head of the department of mouse genetics and
metabolism.
Mouse Genetics and Metabolism
The group works on different projects aiming to characterize signaling pathways
responsible for the regulation of energy and glucose homeostasis. The experimental
approach focuses on the generation and characterization of mouse models with targeted
disruption of genes in the leptin, insulin and cytokine signaling pathways. Particularly,
the experiments are based on the establishment of conditional mutants with cell typespecific and/or timely controlled disruption of signaling molecules. From a physiological
point of view, the work focuses on two main areas: first, the characterization of signaling
cascades initiated by insulin, leptin and cytokines in the hypothalamic regulation of energy
homeostasis and second, the interaction of insulin resistance and inflammatory processes
in the development of atherosclerosis
4
Jens C. Brüning
Energy homeostasis of the mammalian organism is
tightly controlled through a balance of energy intake
and energy expenditure. Over the last ten years a
variety of signals has been identified regulating both
food intake and energy expenditure in mammals.
These include the adipocyte-derived hormone
leptin, which signals through leptin receptors
expressed in hypothalamic neurons to inhibit food
intake and to stimulate energy expenditure.
Likewise, the pancreas-derived hormone insulin has
been shown to acutely inhibit food intake through
the activation of neuronal insulin receptors.
Moreover, a variety of interleukin-type signals such
as interleukin-6 and ciliary neurotrophic factor
(CNTF) have been demonstrated to regulate either
food intake or metabolic rate.
However, it is unclear which particular neuronal
population mediates the effects of these signals.
Moreover, the intracellular signaling cascade
responsible for the regulation of energy intake and
expenditure has not fully been elucidated and lastly
the interaction of these different types of signals
remains largely unclear.
Therefore, we have generated mice carrying loxP-flanked alleles for the insulin receptor and the
closely related insulin-like growth factor 1 (IGF1)-receptor. By crossing these animals with mice
expressing the Cre recombinase under
neuropeptide-specific promoters in defined
hypothalamic neuronal sub-populations such as
neuropeptide Y (NPY)-, proopiomelanocortin
(POMC)- and leptin receptor-expressing neurons,
we aim to identify the exact site of insulin action in
the central regulation of energy homeostasis.
Likewise, we have generated mice with targeted
disruption of the gp130 common cytokine signaling
chain in these neurons, to identify the site of action
of IL-6 and CNTF-mediated effects. In addition,
we try to identify novel target genes in these
pathways by high density microarray hybridization
with cDNAs derived from hypothalamic RNA
extracted from these mice.
While leptin’s anorectic effects are mainly
mediated through the activation of the JAK/STAT
pathway, both insulin and leptin appear to regulate
energy homeostasis through activation of the
phosphatidylinositol 3-(PI 3)-kinase pathway in
neurons. In order to physiologically validate these
signaling mechanisms and to define them as potential targets for the development of an anti-obesity
treatment we have generated mice with
hypothalamic inactivation of the PTEN gene, a negative regulator for PI 3-phosphate signaling. The
aim is to assess the effect of these mutations on the
regulation of energy homeostasis, food intake and
the development of obesity. Also in this paradigm
we currently try to identify novel target genes by
high density microarray hybridization.
Insulin resistance in inflammation and
atherosclerosis
Beside in the classical insulin target tissues such as
skeletal muscle, liver and adipose tissue, insulin
receptors are widely expressed throughout the
mammalian organism. Insulin and IGF-1 receptors
are also expressed in lymphocytes and their
expression is regulated through lymphocyte
development. Moreover, macrophages express
both types of receptors and it has been shown that
insulin stimulates the uptake of glycosylated end
products in these cells. Since clinically type 2
diabetes and insulin resistance are closely linked to
the development of atherosclerosis and there is
growing evidence for a role of inflammatory
processes in the development of atherosclerosis,
another focus of our work is to study the role of
insulin signaling in lymphocytes and macrophages.
Along this line, we have reconstituted an insulin
and/or IGF-1 receptor-deficient lymphoid system
in RAG2-deficient mice to study the role of insulin
and IGF-1 receptor signaling in these chimeras.
Moreover, we have generated mice with specific
disruption of these receptors in macrophages and
are currently studying the development of
atherosclerosis in these mice. The aim of these
projects is to characterize the role of macrophage
insulin signaling in the regulation of cholesterol
uptake and metabolism as well as foam cell
formation. We have also isolated ex vivo
immortalized macrophage cell lines from these mice,
which allow inducible inactivation of the insulin
receptor gene in vitro. Again, in these cell systems,
cholesterol uptake and metabolism will be studied
and insulin target genes will be identified. Also in
5
Jens C. Brüning
this research area we will characterize the
intracellular signaling cascade initiated by insulin and
IGF-1 in macrophages determining the regulation
of cholesterol uptake and metabolism in cells
derived from mice deficient for insulin receptor
substrate proteins (IRS-1 - 4). The overall goal is
to define a molecular link for insulin resistance and
inflammatory processes.
Selected Publications
Baudler, S., Krone, W., Brüning, J.C. (2003) Genetic
manipulation of the insulin signaling cascade
in mice - potential insight into the
pathomechanism of type 2 diabetes. Best. Pract.
Res. Clin. Endocrinol. Metab. 17, 431-443.
Schwenk, F., Zevnik, B., Brüning, J., Rohl, M.,
Willuweit, A., Rode, A., Hennek, T.,
Kauselmann, G., Jaenisch, R., Kuhn, R. (2003)
Hybrid embryonic stem cell-derived tetraploid
mice show apparently normal morphological,
physiological, and neurological characteristics.
Mol. Cell. Biol. 23, 3982-3989.
Brüning, J.C., Gillette, J.A., Zhao, Y., Bjorbaeck, C.,
Kotzka, J., Knebel, B., Avci, H., Hanstein, B.,
Lingohr, P., Moller, D.E., Krone, W., Kahn, C.R.,
Müller-Wieland, D. (2000) Ribosomal subunit
kinase-2 is required for growth factor-stimulated
transcription of the c-Fos gene. Proc. Natl.
Acad. Sci. USA 97, 2462-2467.
Brüning, J.C., Gautam, D., Burks, D.J., Gillette, J.,
Schubert, M., Orban, P.C., Klein, R., Krone, W.,
Müller-Wieland, D., Kahn, C.R. (2000). Role of
brain insulin receptor in control of body weight
and reproduction. Science 289, 2122-2125.
Kulkarni, R.N., Brüning, J.C., Winnay, J.N., Postic,
C., Magnuson, M.A., Kahn, C.R. (1999) Tissuespecific knockout of the insulin receptor in
pancreatic beta cells creates an insulin secretory
defect similar to that in type 2 diabetes. Cell 96,
329-339.
Brüning, J.C., Michael, M.D., Winnay, J.N., Hayashi,
T., Horsch, D., Accili, D., Goodyear, L.J., Kahn,
C.R. (1998) A muscle-specific insulin receptor
knockout exhibits features of the metabolic
syndrome of NIDDM without altering glucose
tolerance. Mol. Cell. 2, 559-569.
6
Wim Damen
Group Leader
Wim Damen received his PhD at the University
of Utrecht (the Netherlands) in 1996, where
he worked on the regulation of gene expression
in spiralian embryos. He then joined as a postdoc the lab of Diethard Tautz at the University
of Munich. Here he established the spider
Cupiennius salei as a model for evolutionary
developmental studies. Together with Diethard
Tautz he moved to the University of Cologne
in 1999 where he started as an independent
group leader at the Institute for Genetics. His
work focuses on the evolution of segmentation
and appendage formation using the spider as a
model.
Group members
Ralf Janßen, PhD Student
Nikola-Michael Prpic, PhD
Student
Michael Schoppmeier, PhD
Student
Evolution and Development in Arthropoda
We are using the spider Cupiennius salei as a model to study the evolution of developmental
processes. Spiders belong to the chelicerates, a basal arthropod group with a fossil record
that dates back to the Cambrian. This makes spiders an excellent model for evolutionary
studies. Our focus is on the understanding of fundamental developmental processes in
the spider, mainly segmentation and appendage formation, in order to permit evolutionary
comparisons. This comparison of developmental processes in the spider with those in
other arthropods, like the well studied insect Drosophila, will show us their degree of
conservation and divergence and eventually may allow us to define a molecular archetype
for the arthropods. This in itself will be a requirement for comparing different phyla and
will contribute to our understanding of the evolution of animal body plans.
7
Wim Damen
Segmentation in a spider
Fig. 1. Adult female specimen of the spider Cupiennius
salei. This female carries a cocoon that is filled with up
to thousand developing eggs.
Segmentation has been studied intensively in the
fly Drosophila. At the molecular level, a hierarchic
cascade of segmentation genes controls the
segmentation process. Comparative studies have
demonstrated differences as well as similarities of
segmentation genes in diverse arthropods. We are
using the spider Cupiennius salei (Fig. 1) as a
model system for the evolution of the molecular
basis of segmentation. Here we take advantage of
the basal position of the chelicerates within the
arthropods. We try to understand the segmentation
process in the spider by expression studies and
functional analyses (RNAi) of segmentation genes
known from Drosophila.
The formation of the segments in the spider also
shows similarities to the process of somitogenesis
in vertebrates. Somites are the segmental units of
the vertebrate embryo (see Diethard Tautz’s group).
The Notch/Delta signalling pathway is an important
component of the vertebrate somitogenesis
machinery, but is not involved in Drosophila
segmentation. However, the Notch-signalling
pathway is involved in segmentation of the spider,
showing a number of similarities to vertebrates (Fig.
2).
In contrast to Drosophila segmentation that is
derived, segmentation in the spider represents an
ancestral form of arthropod segmentation. The
8
involvement of Notch-signalling in both spider and
vertebrate segmentation therefore suggests a
common evolutionary origin of segmentation in
arthropods and vertebrates. Our strategy is to further elucidate the developmental process of spider
segmentation by the analysis of known components
of both Drosophila segmentation and vertebrate
somitogenesis. This data will shed more light onto
the evolutionary origin of the segmentation process.
Segmentation in a millipede
The millipedes that belong to the myriapods show
an aberrant mode of segmentation. The number of
dorsal and ventral segmental structures differs; one
dorsal segmental unit (tergite) covers two ventral
segmental units, e.g. two pairs of legs and two
sternites. The process of diplo-segmentation is
poorly understood. Using the millipede Glomeris
marginata as a model we try to elucidate the origin
of these dorso-ventral differences in millipede
segmentation by studying the molecular basis of
segmentation of Glomeris (Fig. 3).
Fig. 2. Notch and Delta genes are involved in spider
segmentation. RNAi for these genes results in segmental defects (A, B) as well as in disturbance of the striped
expression of the spider hairy gene in the growth zone
(C,D).
Wim Damen
Appendage formation in a spider
Fig. 4. Expression of the odd-skipped gene in the
developing spider leg. The gene is expressed in rings
that correspond to the future joints of the legs. Some
odd-skipped rings appear de novo, while other rings
form by splitting of rings.
Arthropod appendages display a high degree of
morphological diversity. This diversity is thought to
have evolved from one ancestral appendage during
arthropod evolution. An exciting question is how
such a morphological diversity of appendage types
in arthropods evolved. Changes in the genetic
regulation and interactions of genes during
appendage development as well as changes in the
function of proteins seem to contribute to this
diversity. The genetic basis of limb development
has been analysed in detail in the fly Drosophila;
however, limb formation from imaginal discs as in
Drosophila is a derived mode of limb development.
We use the spider Cupiennius salei as a model to
study appendage formation. Spider appendages
form as direct body outgrowths, representing the
ancestral way of arthropod appendage formation.
By performing gene expression studies and
functional studies using RNAi, we try to elucidate
the molecular basis of spider appendage formation
and try to explain how differences might have been
brought about during evolution. One intriguing
question here is how the future joints that demark
the podomeres (limb segments) become defined
(Fig 4), and how differences in the number and
position of the joints may contribute to
Selected Publications
Stollewerk, A., Schoppmeier, M., Damen, W.G.M.
(2003) Involvement of Notch and Delta genes
in spider segmentation. Nature 324, 863-865.
Prpic, N.-M., Janssen, R., Wigand, B., Klingler M.,
Damen W.G.M. (2003) Gene expression in spider
appendages reveals reversal of exd/hth spatial
specificity, altered leg gap gene dynamics, and
suggests divergent distal morphogen
signaling. Dev. Biol. (in press).
Damen, W.G.M., Saridaki, T., Averof, M. (2002)
Diverse adaptations of an ancestral gill: a
common evolutionary origin for wings,
breathing organs and spinnerets. Curr. Biol. 12,
1711-1716.
Damen, W.G.M. (2002) Parasegmental organization
of the spider embryo implies that the
parasegment is an evolutionary conserved
entity in arthropod embryogenesis.
Development 129, 1239-1250.
Fig. 3. Embryo of the millipede Glomeris marginata stained
with the nuclear dye DAPI and by an in situ hybridisation for engrailed mRNA (dark stripes) showing
discontinuous dorsal and ventral engrailed stripes.
Damen, W.G.M., Weller, M., Tautz, D. (2000) The
expression patterns of hairy, even-skipped, and
runt in the spider Cupiennius salei imply that
these genes were segmentation genes in a
basal arthropod. Proc. Natl. Acad. Sci. USA 97,
4515-4519
9
Ute Deichmann
Group Leader
Ute Deichmann received her PhD in Cologne
1991, where she worked at the dept. of Benno
Müller-Hill on the history of biology in Nazi
Germany. Her thesis was published by Campus
(1992), Fischer (1995) and in English translation
1996 (Harvard Univ. Press; title: Biologists under
Hitler). Then she worked on the history of science
(20th century) at the Institute of Genetics in
Cologne (dept. Müller-Hill) (chemistry and
biochemistry in Nazi Germany), at the Edelstein
Center for the History and Philosophy of Science
in Jerusalem (1994) and as International Edelstein
Fellow at the Beckman Center for the History of
Chemistry in Philadelphia and the Edelstein Center in Jerusalem (1996-1997) (scientific impact of
the expulsion of Jewish chemists and biochemists
from Nazi Germany). In 2000 she received her
Habilitation in Cologne and became Privatdozentin
at the Institute of Genetics, Cologne. From 1998
she has been a regular visiting professor (in the
history and philosophy of science) during spring
semesters at Ben Gurion University of the Negev,
Israel. In the fall of 2002 she was a research
fellow at the Max Planck Institute for History of
Science, Berlin.
Group members
Simone Wenkel, Graduate
Student
History of Biological and Chemical Sciences, 20th Century
We work on a number of different projects in the history of science, focusing on the
biological and chemical sciences in the 20th century. We review the history of experiments
and theories and analyse their interaction, and we examine political, social, and personal factors and their impact on the advancement of science. Areas of research include:
the reception of break through experiments in molecular biology, and the role of
experiments and theories in early protein research, case studies of scientific fraud, the
long term scientific impact of he National Socialist political ideology and policy of the
expulsion of Jewish scientists, the role of Jewish scientists in 19th and 20th century in
German academia.
10
Ute Deichmann
20th century history of biochemistry and
molecular biology, selected topics
We work on three topics
(1) The post WWII development of molecular
biology in Germany.
After the Second World War dynamic biochemistry, in particular intermediary metabolism,
and scientific fields which had been newly
developed in other countries, mainly the United
States, were hardly represented in Germany for
almost 20 years after the end of the war. Among
them are molecular biology and structural research
in proteins and nucleic acids. We have analysed
several factors as possible reasons for this decline
in the modern biological and biochemical sciences,
among them the expulsion and non-return of Jewish
scientists, the structure of German universities, the
international isolation and self-isolation of German
scientists reinforcing the conduct of traditional
genetic research at German universities, where
research until the 1960s was conducted almost
entirely in complex organisms. We want to expand
this research and analyse the reception in Germany
of major breakthroughs in molecular biology
elsewhere.
(2) Protein research at Kaiser Wilhelm Institutes (KWIs) and MPIs from 1930 to 1960 in
international comparison.
Starting with an overview on contents and results
of protein research in Germany from around 1930,
we analyse political and social factors before,
during, and after the Nazi era which influenced
directions and achievements in basic and applied
protein research until the 1960s. We focus on
research at the KWIs (from 1948 MPIs) for
Biochemistry (C. Neuberg until 1934, A. Butenandt
from 1935), Leather Research (M. Bergmann until
1933, W. Grassmann from 1934), and Cell
Physiology (O. Warburg). A first striking result is
the prevalence of questionable protein research at
the KWI for Biochemistry. Thus Butenandt and his
coworkers relied heavily on F. Kögl's claims of the
prevalence of d-amino acids in cancer tissues which
was later shown to be a fraud of Kögl's collaborator,
on L. Pauling's claim of being able to produce
antibodies in vitro, which soon after was shown to
be non-reproducible, and on E. Abderhalden's
claims of the existence of defence enzymes, already
shown to be non-existent. Scientific as well as nonscientific reasons for these and other phenomena
are discussed.
(3) The reception of Avery's et al.'s 1944
experiment on Pneumococcal transformation
by DNA.
In this nearly completed project we show, based
on a detailed analysis of the reception of Avery's et
al.'s 1944 paper, that G. Stent's and H. V. Wyatt's
claims of the prematurity does not apply to Avery's
et al.'s experiment on DNA as the transforming
principle. Avery's paper represents a discontinuity,
an unexpected discovery. As in other such cases, it
took scientists some time to fully understand its
meaning. Initial problems of transferring
transformation to other systems and prominent
criticism of its results nurtured skepticism. But on
the whole, Avery's results were immediately
appreciated and motivated new research on
transformation, the chemical nature of DNA's
biological specificity and bacteria genetics.
Nevertheless Avery's experiment was neglected by
many of the scientists working in the new fields of
biochemical genetics, genetic phage and TMV
research. This was not due to the fact, that, as Stent
put it, its implications could not be connected "by a
series of simple logical steps to canonical, or
generally accepted, knowledge". Rather, it was
because they were committed to finding solutions
to the question of the gene replication and genic
action by genetic and physical methods and clung
dogmatically to the assumption that proteins were
the sole carriers of biological specificity. We show
that dogmatism, occasionally combined with other
attitudes such as jealousy, explains the neglect of
Avery's findings more convincingly than their alleged
prematurity.
11
Ute Deichmann
Finding and analysis of cases of scientific
misconduct and fraud
This is a collaborative project with Ulrich Charpa
of the dept. of philosophy, University of Bochum.
Basing our analysis on reliabilism, i.e. an
epistemological approach which focuses on reliable
processes as the basis for scientific knowledge, we
compare cases of questionable practices in the
history of biomedical and chemical sciences, leading
to claims which turned out not to be reproducible.
Examples are E. Abderhalden, R. Goldschmidt, F.
Kögl, F. Moewus, and E. Waldschmidt-Leitz. We
show that the time which passed until these claims
were checked, and discovered as well as accepted
not to be reproducible largely depends on the
scientific reputation of that particular researcher,
the disciplinary relevance of his or her alleged new
discovery, the competencies of the scientific
community in that particular field, and social and
political factors, such as international scientific
exchange or isolation.
The influence of immigrants from Germany
on science in Mandate Palestine and Israel:
chemistry and biochemistry
This is a collaborative project with Anthony Travis,
Sidney Edelstein Center for the History and
Philosophy of Science, Jerusalem. German and
German-trained chemists and biochemists laid the
foundations of many scientific developments that
later took place in Israel. Here we review and
evaluate the careers of these immigrant scientists,
and their sciences, in a Middle Eastern context.
The introduction of chemistry at the Hebrew
University in the early 1920s was strongly influenced
by Andor Fodor, a student of E. Abderhalden’s in
Halle, who attempted to establish chemistry and
biochemistry on the basis of colloid chemistry. This
continued until the 1930s, despite the fact that as
biochemistry and polymer science developed the
belief that biological processes and large cellular
molecules such as proteins are of a colloidal nature
increasingly came under attack. The outcome of
committing a science so strongly to controversial
concepts was a poor record of research, and a
complacency that spilled over into institutional
12
politics during the first decade. This is an outstanding
example of an export of German science, even
though Fodor introduced a negative „founder
effect.“ The situation changed only from the mid
1930s, when a number of bright young chemists
were available from Germany due to the Nazi policy
of expulsion. Among them was Ladislaus Farkas,
a student of Karl-Friedrich Bonhoeffer’s. We show
how he secured a new grounding for chemistry in
Jerusalem through his work in theoretical physical
chemistry and, in his time at least, the initiation of
successful academic-industrial collaborations.
Another immigrant from Germany, Ernst David
Bergmann, transformed organic chemistry in Israel
and brought about its international standing.
Jews in German academia, 19th and 20th
century
This is a long term collaborative project with Ulrich
Charpa, University of Bochum, which aims at
documenting, rating, and explaining the role of Jews
in 19th and 20th century German-speaking
academia.
Based on primary as well as secondary sources,
such as calendars of universities, correspondences,
autobiographies, interviews conducted by others
and ourselves, scientists’ and scholars’ publications,
we want to provide a systematic overview of Jewish
participation in various disciplines and a
prosopography of all relevant persons, including
their biographies and scientific/scholarly
achievements.
Preliminary results by ourselves as well as an
analysis of existing publications show that many
features of Jewish accomplishments in various
disciplines cannot be explained by one major cause.
Various, sometimes overlapping factors such as
Jewish religious and secular traditions, academic
anti-Semitism, the state of development of
institutions and disciplines, emigration and forced
emigration, have to be taken in account. We shall
examine to what extent the relevant theses
(Schleiden, Veblen, Volkov, Hollinger and others)
are suitable to explain Jewish accomplishments in
the sciences and the humanities at different periods
of time.
Ute Deichmann
Our work concentrates on hitherto neglected facts
and possible explanatory factors, e.g. the uneven
distribution of Jews in different disciplines, and the
relationship between specific (philosophical,
respectively methodological) conceptions of science
and the humanities on one side and the promotion
or hindrance of Jewish scientists/scholars on the
other.
We collaborate with Anthony Travis, Sidney
Edelstein Center, Jerusalem, Dan Diner, Simon
Dubnow Institute Leipzig, and Raphael Gross, Leo
Baeck Institute London. In 2002 we organised two
international workshops (in Leipzig and Jerusalem)
Selected Publications
Deichmann, U. (2004) Early responses to Avery's et
al's 1944 paper on DNA as hereditary material.
Historical Studies in the Physical & Biological
Sciences 34, 207-233.
Charpa, U., Deichmann, U. (2004) Vertrauensvorschuß und wissenschafltiches Fehlhandeln
- eine reliabilistische Modellierung der Fälle
Abderhalden, Goldschmidt, Moewus und
Waldschmidt-Leitz. Berichte zur Wissenschaftsgeschichte 27, 187-204
Deichmann, U., Travis, A.S. (2004) A German
Influence on Science in Mandate Palestine and
Israel: Chemistry and Biochemistry. Israel
Studies 9.2, 34-70.
Deichmann, U. (2002) Emigration, isolation and the
slow start of molecular biology in Germany.
Studies in the History and Philosophy of
Biological and Biomedical Sciences 33, 433-455.
Deichmann, U. (2002) Chemists and biochemists
during the National Socialist era. Angew. Chem.,
Intl. Ed. 41, 3000-3018.
Deichmann, U. (2001) Flüchten, Mitmachen,
Vergessen. Chemiker und Biochemiker im
Nationalsozialismus. Weinheim: Wiley/VCH.
Deichmann, U. (2000) An unholy alliance. The Nazis
showed that 'politically responsible' science
risks losing its soul. Millenium-essay, Nature
405, 739.
Deichmann, U. (1999) The expulsion of Jewish
chemists and biochemists from academia in Nazi
Germany. Perspectives on Science 7, 1-86.
13
Walter Doerfler
Professor emeritus
M.D. Universität München Medical School
1959; Internshiopt Mercer Hospital Trenton, NJ
1959-1960; Approbation 1961; Facharzt Humangenetik 1996; Postdoctorate Max-Planck-Institut für Biochemie München 1961-1966; Assistant
Prof:, Associate Prof Rockefeller University
New York, NY, 1966-1971; Adjunct Prof.
Rockefeller Universisty until 1978; Guest Professer at University of Uppsala, Sweden 19711972; Professor, Institute for Genetics, University
of Cologne, emeritus.active 1998 - ; Guest Prof.
Universität Erlangen-Nürnberg 2002- ; Guest
Professor at Stanford 1978, 1993, Princeon 1986,
1999, Kawasaki Medical School 1988, Akad.
Nauk Mockba 1990, Uppsala 2002; Aronson
Prize 1981; Robert Koch Preis 1984; Leopoldina
1998
Group members
Holger Brondke, PhD Student
(Erlangen)
Andreas Dorn, PhD Student
(Erlangen)
Frauke Naumann, PhD Student
Beryl Schwarz-Herzke, PhD
Student (Erlangen)
Otilia Vieira Rocha, PhD Student
Dennis Webb, PhD Student
Gerd Wronka, PhD Student
Marianna Hösel, Postdoc (Erlangen)
Laurence Mangel, Postdoc
Sabrina Auerochs, Technician
(Erlangen)
Hanna Mansi-Wothke, Technician
Doris Renz, Technician
Birgit Schmitz, Technician
Ingeborg Bläser, Laboratory
Assistant
Monika Schmidt, Laboratory
Assistant
Petra Böhm, Secretary
Susanne Scheffler, Secretary
Sian Tjia, Department Manager
Molecular Virology and Medical Genetics
The research group working in the former Section Medical Genetics and
Virology of the Institute of Genetics has worked on the following problems. (i)
Early steps in the interaction of human adenovirus type 12 (Ad12) with permissive and non-permissive mammalian cells. (ii) In cells transgenic for Ad12
or for bacteriophage lambda DNA, methylation patterns are alteredRelevance
for viral oncogenesis. (iii) Ad12 oncogenesis in Syrian hamsters. (iv) On the
fate of food-ingested foreign DNA and of proteins in the gastrointestinal tract
of mice. (v) The CGGBP1 is a nuclear protein from human cells that binds
specifically to 5´-(CGG)n-3´ repeats. The overall interest of the group has
been the fate of foreign DNA in mammalian cells and its relevance for
oncogenesis as well as on the biological significance of the fifth nucleotide
and of DNA methylation.
14
Walter Doerfler
Early steps in the interaction of human
adenovirus type 12 (Ad12) with permissive
and non-permissive mammalian cells
Ad12 induces undifferentiated tumors in Syrian
hamsters (Mesocricetus auratus). We have
therefore analyzed the interaction of Ad12 with
hamster cells in detail. This interaction is abortive.
Viral DNA replication is totally blocked. This block
can be partly released by the constitutive expression
of E1 functions of Ad5, which does replicate in
hamster cells, or by the overexpression of the
preterminal protein (pTP) or the E1A protein of
Ad12 in Ad12-infected hamster cells. In
comparison to productively Ad2-infected hamster
or productively Ad12-infected human cells, the
amount of Ad12 DNA reaching the hamster cell
nucleus is about 1%. Hamster cells overexpressing
the human Coxsackie-Adenovirus Receptor (CAR)
take up considerably more Ad12 DNA into their
nuclei. Currently, we follow the fate of the parental
Ad12 virions in hamster or human cells by laser
scan microscopy using immunofluorescent (pTP)
and FISH (Ad12 DNA) detection methods.
Fig. 1. Binding of adenovirus type 12 virions (green) to
the surface of human HeLa cells. Ad12 virions were
visualized by immunofluorescence with an antibody
against protein IX of Ad12. Nuclear DNA was labeled
with propidium iodide (red).
Fig. 2. Real time PCR analysis of the adenovirus
DNA uptake and replication.
A. Hamster BHK21 cells were productively infected
with human adenovirus type 2 (Ad2) or abortively
infected with human adenovirus type 12. Nuclear
DNA was isolated at 4 h post infection (h p.i.),
and viral DNA was quantified by LightCycler PCR
technique. The nuclear amount of Ad12 virions in
the BHK21 x Ad12 abortive system was about 100
fold reduced compared to the amount of Ad2 in
the productive system.
B. BHK21 cells were transfected with the human CAR
gene (Coxackie and Adenovirus receptor)
In contrast to abortively Ad12-infected cells, in
hCAR-transfected and Ad12-superinfected
BHK21 cells, the concentrations of Ad12 DNA
were markedly increased at 24 h p.i. suggesting
viral DNA replication in these cells.
In cells transgenic for Ad12 or for
bacteriophage lambda DNA methylation
patterns are altered: relevance for viral
oncogenesis
In Ad12-transformed cells or in hamster cells
transgenic for the DNA of bacteriophage lambda,
alterations of the patterns of DNA methylation in
15
Walter Doerfler
different cellular genes have been documented by
restriction analyses and bisulfite genomic
sequencing. We pursue the possibility that
alterations in cellular DNA methylation and
transcription patterns are intimately associated with
the mechanism of adenoviral oncogenesis.
Ad12 oncogenesis in Syrian hamsters
The intramuscular injection of Ad12 into newborn
hamsters leads to tumor formation and extensive
intraperitoneal spreading of tumors. The tumor cells
express proteins characteristic for tumors of neuronal and of mesenchymal origins. Multiple copies
of Ad12 DNA are integrated in each tumor cell.
Only some of the early Ad12 genes are expressed
in the tumor cells. There is no specific chromosomal site for viral DNA integration, but all the cells
in one tumor carry the Ad12 integrates at the same
chromosomal site. The integrated viral DNA is
extensively de novo methylated. By using the DNA
array technology, we have analyzed cellular genes
which are transcribed in the tumor cells.
On the fate of food-ingested foreign DNA
and proteins in the gastrointestinal tract of
mice
This project has been pursued since 1988. Different types of test DNAs (M13, GFP, Rubisco, Ad2)
fed to mice can transiently persist in the
gastrointestinal tract (GIT) in small quantities and
in fragmented form. DNA fragments can also be
detected in the nuclei of cells in the intestinal wall,
in Peyer patches and in spleen, liver and kidney.
There is evidence that in very rare instances foreign
food-ingested DNA can be integrated into DNA
of spleen cells. Recently, we have shown that the
protein glutathion-S-transferase survives immunologically intact the passage through stomach and
small intestine in mice for maximally 30 min. It will
be interesting to study the biological and medical
implications of these findings.
16
The CGGBP1 a nuclear protein from human
cells that binds specifically to 5´-(CGG)n-3´
repeats
The characteristics of this protein have been
described in detail (Deissler et al., J. Biol. Chem.
272,16761-8,1997). We have now initiated studies
on the biological function of this protein which is
highly conserved in mammals and birds but lacks
similarities to known proteins. Constructs of the
CGGBP1 gene have been prepared for the knockout approach in mice. We are also performing
experiments in which this protein is overexpressed
in human cells and in which cellular transcription
patterns are subsequently analyzed by the DNA
array technique.
Plans and concepts for future research
The main emphasis in our research during the
following years will be on
(i) the consequences of foreign DNA insertion
into established mammalian genomes. We study
alterations in DNA methylation and transcription
patterns of the transgene and of the recipient
genome at and remote from the site of insertion of
the foreign DNA.
(ii) the persistence of foreign DNA and proteins
in mammalian organisms on the mechanism of
uptake via the gastrointestinal tract.
(iii) alterations of cellular transcription patterns
in adenovirus type 12-infected cells and in Ad12induced tumor cells. We pursue the notion that the
insertion of Ad12 DNA into the genome of the
transformed cell plays a decisive role in the
mechanism of oncogenesis via perturabtions of the
chromatin structure in the tumor cells.
(iv) the biological function of the CGG-binding
protein isolated from the nuclei of human cells. We
have adduced evidence for the possible role of this
protein as an inhibitor of mammalian cell promoters
rich in CGG motifs.
Walter Doerfler
Selected Publications
Heller, H., Kämmer, C., Wilgenbus, P., Doerfler, W.
(1995) Chromosomal insertion of foreign
(adenovirus type 12, plasmid or bacteriophage
lambda) DNA is associated with enhanced
methylation of cellular DNA segments. Proc.
Natl. Acad. Sci. USA 92, 5515-5519.
Schubbert, R., Renz, D., Schmitz, B., Doerfler, W.
(1997) Foreign (M13) DNA ingested by mice
reaches peripheral leukocytes, spleen and liver
via the interstinal wall mucosa and can be
covalently linked to mouse DNA. Proc. Natl.
Acad. Sci. USA 94, 961-966.
Doerfler, W., Hohlweg, U., Müller, K., Remus, R.,
Heller, H., Hertz, J. (2001) Foreign DNA
integration - perturbations of the genome oncogenesis. Ann. New York Acad. Sci. 945,
276-288.
Hösel, M., Webb, D., Schröer, J., Schmitz, B.,
Doerfler, W. (2001) Overexpression of the
adenovirus type 12 (Ad12) pTP or E1A gene
facilitates Ad12 DNA replication in nonpermissvie BHK21 hamster cells. J. Virol. 75,
10041-10053.
Hohlweg, U., Doerfler, W. (2001) On the fate of plant
or other foreign genes upon the uptake or after
intramuscular injection. Mol. Gen. Genom. 265,
225-233.
Müller, K., Heller, H., Doerfler, W. (2001) Foreign
DNA integration: genome-wide perturbations
of methylation and transcription in the recipient
genomes. J. Biol. Chem. 276, 14271-14278.
Schumacher, A., Arnhold, S., Addicks, K., Doerfler,
W. (2003) Staurosporine is a potent activator
of neuronal glial, and ‘CNS stem cell’-like
neurosphere differentiation in murine
embryonic stem cells. Mol. Cell. Neuroscience
23, 669-680.
Naumann, F., Remus R., Schmitz, B., Doerfler,W.
(2004) Gene structure and expression of the 5(CGG)n-3-binding protein (CGGBP1). Genomics
83,108-120.
17
Jürgen Dohmen
Professor
Jürgen Dohmen received his PhD with Cornelis
Hollenberg at the University of Düsseldorf in
1989, were he cloned amylase genes to generate a starch-degrading bakers’ yeast. He then
joined Alexander Varshavsky’s group at MIT
as an EMBO fellow. Here he started his studies
on protein degradation. Moving with the
Varshavsky lab to Caltech in 1992, he initiated
a screen for proteolysis-defective yeast
mutants. In 1994 he went back to Düsseldorf
as a group leader in a BMBF priority program.
Here he discovered a novel proteasome
maturation factor. In 2000 he moved to
Cologne.
Group members
Marcel Fröhlich, PhD Student
Cristoph Glanemann, PhD
Student
Markus London, PhD Student
Maria Miteva, PhD Student
Palani Murugan, PhD Student
Marion Schnellhardt, PhD
Student
Kristina Uzunova, PhD Student
Kerstin Göttsche, Technician
Molecular Biology of the Ubiquitin/Proteasome System, and SUMO Protein
Modification in Yeast
The ubiquitin/proteasome system selectively targets proteins for degradation in many
processes in eukaryotic cells. Naturally short-lived proteins, including many regulators,
as well as misfolded, damaged or otherwise abnormal proteins are marked for degradation
by the attachment of poly-ubiquitin chains. Poly-ubiquitylated proteins are recognised
and degraded by the 26S proteasome. One area of our research is concerned with the
regulation and biogenesis of this complex ~2 MDa protease. Other projects are focussing
on substrate recognition and selection in the ubiquitin system. The enzymatic system for
protein modification with SUMO (small ubiquitin related modifier) is similar to the ubiquitin
conjugation system. The essential SUMO system, however, has distinct function. Our
analysis of the genes encoding SUMO-activating enzyme and SUMO deconjugating
enzymes revealed that a critical balance of conjugating and deconjugating enzymes is
required for cell cycle progression. The identification of the underlying substrates and
how their function is controlled by the SUMO system is a subject of our current research.
18
Jürgen Dohmen
Fig. 1. Regulation of proteasome biogenesis. The core 20S proteasome is synthesised from α (blue) and β subunits
(green), which, together with a dedicated chaperone (Ump1) that underpins maturation of the proteasome,
assemble into a half-proteasome precursor complex. Upon dimerisation of such ~15S precursor complexes, the
active site β subunits mature by Ump1-assisted autocatalytic processing of N-terminal propeptides. The active
proteasome then degrades the enclosed Ump1 to form the 20S proteasome, which assembles with two 19S
activator complexes (brown) to yield the 26S proteasome. The latter is capable of recognising and degrading
ubiquitylated proteins. This process is, in part, thought to be mediated by interactions with enzymes of the
ubiquitin system (E2, E3). One substrate of the proteasome is the regulator Rpn4 that controls the transcription
of proteasome genes, thereby providing a regulatory feedback loop that controls proteasome function.
Biogenesis and regulation of the
proteasome
The 26S proteasome of eukaryotic cells is the main
protease system responsible for the degradation of
short-lived and abnormal proteins in the cytosol and
nucleus, as well as from the endoplasmic reticulum.
Most substrates of the proteasome are marked for
degradation by the attachment of poly-ubiquitin
chains that bind to 19S caps of the proteasome
(reviewed in Dohmen, 2000). As the ubiquitin
system, the proteasome is essential for viability in
eukaryotes. Among the substrates of the ubiquitin/
proteasome system are many regulatory proteins
including important regulators of the cell cycle and
cell proliferation or apoptosis. It is for this reason
that inhibitors that block the active sites of the
proteasome have received attention as potential
drugs to treat cancer or stroke.
The 26S proteasome of eukaryotic cells is a highly
complex protease consisting of at least 31 distinct
subunits, of which 14 form the 20S core particle
and 17 constitute the 19S regulatory particle (also
known as „19S cap“). The 20S core is a barrelshaped complex formed by four seven-membered
rings. The two outer rings are composed of
α subunits, and the two inner ones of β subunits
(Fig. 1). The proteolytically active β subunits are
synthesised as inactive precursors bearing
propeptides that are cleaved off autocatalytically
yielding the mature subunits with N-terminal
threonine residues.
Using the yeast Saccharomyces cerevisiae as
a model system, we are investigating aspects of
proteasome gene regulation, as well as of
proteasome assembly and maturation.
Our studies have led to the identification of a
19
Jürgen Dohmen
maturation factor, termed Ump1 (‘underpinning
maturation of proteasome’) that is assembled into
half-proteasome precursor complexes and assists
in the generation and maturation of 20S
proteasomes (Fig. 1). After Ump1-assisted
activation of the peptidase sites, the encased Ump1
is rapidly degraded (Ramos et al., 1998).
Recently we found that orthologues of Ump1
appear to be present in other eukaryotes as well,
including mammals (Burri et al. 2000). In our
current studies we are trying to unravel how Ump1
mediates its function in proteasome assembly and
maturation. In addition, we are trying to understand
the early steps in 20S proteasome precursor
assembly as well as the later steps that lead to the
formation of the larger 26S proteasome.
During our characterisation of mutants with
defects in proteasome assembly or function we
detected increased steady-state levels of
proteasome subunits. We found this increase to
be due to transcriptional upregulation of
proteasome subunit genes. We found the Rpn4
protein to be essential for this feedback regulation
(Fig. 1). Rpn4 was recently shown by the
laboratories of Alexander Varshavsky and Horst
Feldmann to be both a substrate of the proteasome
and a transcriptional activator. Our studies show
that Rpn4 itself is regulated both transcriptionally
and at the level of protein stability (M. London, J.
Dohmen, unpublished results). Our goal is to understand how these mechanisms are integrated to
adjust proteasome activity to a cell’s physiological
needs.
Ubiquitin-related protein modifier SUMO
Posttranslational modifications of proteins are used
in many cellular processes to regulate protein
function, e.g. by modifying a protein’s ability to
interact with other proteins or by altering its stability
or subcellular localisation. In recent years, a number
of distinct proteins have been identified that are
more or less similar to ubiquitin and like it are
conjugated to other proteins. SUMO („Small
Ubiquitin-related Modifier“) is the modifier that,
since its discovery in 1996, has received most of
the attention, because of its intriguing and essential
functions.
20
The enzymes involved in conjugation of SUMO
to proteins („sumoylation“) were first discovered
in yeast and found to be similar to those of the
ubiquitin system (Fig. 2). Like ubiquitin, SUMO
undergoes ATP-dependent activation by a specific
activating enzyme composed of Uba2 and Aos1
(Johnson et al. 1997). To study the essential function
of SUMO conjugation, we isolated conditional
uba2 mutants that are temperature-sensitive for
growth. In these mutants conjugation of SUMO to
other proteins is drastically reduced (Johnson et
al. 1997; Schwienhorst et al. 2000). At the nonpermissive temperature, uba2-ts mutant cells
accumulate in the G2/M phase of the cell cycle with
undivided nucleus and a short spindle
(Schwienhorst et al. 2000). Our phenotypic
analysis of uba2 mutants, in addition, led to the
discovery that SUMO conjugation is required for
import of proteins into the yeast nucleus (Stade et
al. 2002).
In a screen for spontaneous suppressors of the
temperature-sensitive growth phenotype of one
such uba2-ts mutant, we isolated a strain with a
null mutation in a gene of hitherto unknown function.
This gene encodes a protein with similarities to
Ulp1, a dual function protease that processes the
SUMO precursor and deconjugates SUMO from
its substrates. The novel protein was therefore
termed Ulp2. Inactivation of ULP2 in a strain
expressing wild-type SUMO-activating enzyme
resulted in slow and temperature-sensitive growth
and accumulation of SUMO conjugates. Thus,
mutations in SUMO-activating enzyme and
mutations in Ulp2 suppress each other, indicating
that SUMO conjugation and deconjugation must
be in balance for cells to grow normally. Other
phenotypes of ulp2 mutants include a defect in cell
cycle progression, hypersensitivity to DNA damage,
and chromosome missegregation (Schwienhorst et
al. 2000).
Ulp2 itself appears to be cell cycle-regulated.
In a collaboration with the laboratory of Dr. Wolfgang Seufert (Universität Stuttgart) we discovered
that Ulp2 is phoshorylated in G2/M and
dephosporylated in the G1 phase of the cell division
cycle. We are currently trying to identify the critical
Jürgen Dohmen
substrates, the functions of which within the cell
cycle are controlled by sumoylation. In addition,
we want to understand the role of Ulp2 regulation
in controlling the SUMO modified state of proteins
in the cell cycle.
Selected Publications
Johnson, E.S., Schwienhorst, I., Dohmen, R. J.,
Blobel G. (1997) The ubiquitin-like protein
Smt3p is activated for conjugation to other
proteins by an Aos1p/Uba2p heterodimer.
EMBO J. 16, 5509-5519.
Ramos, P.C., Höckendorff, J., Johnson, E.,
Varshavsky, A., Dohmen,R.J. (1998). Ump1p is
required for maturation of the 20S proteasome,
and becomes its substrate upon completion of
the assembly. Cell 20, 489-499.
Schwienhorst, I., Johnson, E.S., Dohmen, R.J. (2000)
SUMO conjugation and deconjugation. Mol.
Gen. Genet. 263, 771-786.
Burri, L., Höckendorff, J., Boehm U., Klamp, T.,
Dohmen, R.J., Lévy, F. (2000) Identification and
characterization of a mammalian protein
interacting with 20S proteasome precursors.
Proc. Natl. Acad. Sci. USA 97, 10348-10353.
Fig. 2. SUMO modification system in S. cerevisiae.
SUMO is synthesised as a precursor with a short C-terminal extension, which is cleaved off by a specific
processing protease termed Ulp1. For conjugation, the
C-terminus of mature SUMO needs first to be activated
by the SUMO activating enzyme (E1), a heterodimeric
protein composed of Uba2 and Aos1. Both subunits have
similarity to distinct regions of the ubiquitin-activating
enzyme. Activation is ATP-dependent, proceeds via a
SUMO-adenylate intermediate, and results in SUMO
being linked by a thioester to a cysteine residue in Uba2.
Activated SUMO is then transferred, in a
transesterification reaction, to a cysteine residue of
Ubc9, a conjugating enzyme (E2) specific for SUMO. In
conjunction with substrate recognizing ligases (E3
enzymes), Ubc9 conjugates SUMO to a variety of
substrate proteins. SUMO deconjugation is mediated
by two enzymes, Ulp1 and Ulp2. The activity of the SUMO
deconjugating enzyme Ulp2 is critical to counterbalance the conjugating enzymes in the regulation of
substrate modification within the cell cycle. Sumoylation
may act by altering subcellular location or function of
proteins, or may protect them against ubiquitinmediated proteolysis.
Verma, R., Chen, S., Feldman, R., Schieltz, D., Yates,
J., Dohmen, J., Deshaies, R.J. (2000) Proteasomal
proteomics: Identification of nucleotidesensitive proteasome-interacting proteins by
mass spectrometric analysis of affinity-purified
proteasomes. Mol. Biol. Cell 11, 3425-3439.
Dohmen, R.J. (2000) Primary Destruction signals.
In W. Hilt, and D.H. Wolf (eds.): Proteasomes:
the world of regulatory proteolysis.
Eurekah.com/ Landes Biosciences,
Georgetown, Texas, USA, pp. 186-198.
Stade, K., Vogel, F., Schwienhorst, I., Meusser, B.,
Volkwein, C., Nentwig, B., Dohmen, R.J.,
Sommer, T. (2002) A lack of SUMO conjugation
affects cNLS-dependent nuclear protein import
in yeast. J. Biol. Chem., 277, 49554-49561.
21
Jonathan Howard
Professor
Jonathan Howard received his PhD in Oxford in
1969, where he investigated the cellular origin of
antibody forming cells in rats under the supervision
of J. L. Gowans in the Sir William Dunn School of
Pathology. For his post-doctoral work he studied
lymphocytes reactive against major histocompatibility
antigens with Darcy Wilson at the University of
Pennsylvania. After 20 years in his own laboratory
at the Department of Immunology, The Babraham
Institute, Cambridge, working on monoclonal
antibodies and MHC immunogenetics, he joined the
Institute for Genetics in Cologne in 1994. Since then
his work has concentrated increasingly on the study
of novel mechanisms in natural immunity.
Group members
Cemaletti Bekpen, PhD Student
Julia Hunn, PhD Student
Natasa Papic, PhD Student
Iana Parvanova, PhD Student
Sascha Martens, PhD Student
Christoph Rohde, PhD Student
Katja Sabel, Diploma Student
Andreas Schmidt, Diploma Student
Stefan Weißhaar, Diploma Student
Steffi Könen-Waisman, Postdoc
Rita Lange, Technician
Claudia Poschner, Technician
Gaby Vopper,Technician
Novel Large GTPases as Cell-Autonomous Resistance Factors
For a long time the existence in mammals of complex pathogen recognition and control
mechanisms outside the domain of the classical lymphocyte-based immune system was not
seriously considered. Now it is clear that the classical immune system, for all its virtuosity,
depends for its effectiveness on the successful functioning of a raft of other mechanisms
which operate on entirely different principles. Novel receptor systems have been described
which recognise, not the unpredictable foreign structures seen by the classical immune
system, but rather recurrent molecular patterns characteristic of pathogens, while novel
effector systems are emerging which restrain and destroy pathogens in ways previously
not thought of. Our own programme is concentrated on a group of dedicated GTPases
which are essential for the survival of mice after infection with certain intracellular
pathogens. We are trying to find out what they do and how they do it.
22
Jonathan Howard
Interferons induce a resistant state in cells
The original description of interferon by Aleck
Isaacs in 1957 was of a soluble substance released
by virus-infected cells that induced resistance to
virus-infection in previously uninfected cells. Despite
nearly 60 years of work on this “viral” (or type I or
α- and β-) interferon. and on its later discovered
functional relative, “immune” (or Type II or γ-)
interferon, only minor superficial progress has been
made on its mechanism of action in pathogen
resistance. Most effort has been concentrated on
the now classical Jak-Stat signalling pathway
leading from the surface interferon receptors to
induced gene transcription, but the effector
mechanisms of pathogen resistance are far from
fully charted. A small group of effector mechanisms
were identified relatively early and have captured
most of the attention until recently. These were,
firstly, a group of gene products induced or activated
by interferons which recognise and destroy double-stranded RNA, and secondly, a small family of
large GTPases, the Mx proteins, induced only by
type I interferons, which contribute to cellular
resistance in man and mouse to influenza and a
number of other RNA viruses. The mode of action
of Mx proteins is essentially not known. About 10
years ago, several investigations began, including
one from our own lab, to understand better the
complexity of the cellular response to interferons.
We simultaneously embarked on a global literature
search for reports of genes regulated by interferons,
and to our surpise found over 200 different genes.
We have since extended the search and can now
identify over 700 genes where there is already a
literature report of their induction by interferon. In
1997, when we published the first version of this
list, the longest previously published list contained
fewer than 40 genes. These figures are now
generally confirmed by direct experiment using a
variety of modern methods. Thus the cellular
response to interferons is exceedingly complex, and
the induced cell is in a radically different state from
before. We have rationalised this complexity as a
series of programmes all of which individually
contribute to the resistant state, either of the cell or
of the animal.
Interferon-inducible GTPases
We made a subtractive library from fibroblasts
induced with interferon-γ with the initial intent of
looking for additional components of the class I
MHC antigen presentation apparatus. In the event,
this purpose was bypassed by the observation that
all the most abundant differentially expressed
cDNAs were members of (as it turned out) 3 different families of GTPases. With the example of
Mx proteins already on the table, the possibility
arose that further GTPase families might also play
a role in cell-autonomous pathogen resistance. After publishing a preliminary summary of the results
we have focused our work on a large family of 47
kDa GTPases which have turned out to be of great
interest from several points of view. The critical
observations were made, not by us, but by Greg
Taylor then at the NCI and now at Duke University,
who targeted first one and then two more of the
p47 GTPase genes in mice and established that all
three knock-outs have strong disease susceptibility
phenotypes. Indeed two of them were as vulnerable to the protozoal parasite Toxoplasma gondii
as animals with no interferon system. Taylor’s
results therefore showed that individual family
members are not redundant but rather contribute
individually to resistance.
Fig. 1. Unrooted phylogeny of the mouse and human
p47 GTPases based on amino acid sequence showing
the extensive sequence divergence within the family.
The single human member of the gene family, hCINEMA,
is almost identical to its mouse homologue, mCINEMA.
23
Jonathan Howard
The p47 GTPase family
With the completion of the mouse genome it has
been possible to show that the p47 GTPase family
has 23 members in this species. Of these, 5 are
pseudogenes by a variety of criteria, while the
remainder appear to be in working order. From an
analysis of their promoters we have been able to
show that all except one of the mouse genes are
likely to be inducible by interferons and have
confirmed this experimentally for 16 genes. The 22
p47 genes of the mouse are a closely knit family:
only a single gene, which we designate PAPER,
shows homology to the p47 group but is not itself a
p47 GTPase. Phylogenetic studies have failed to
find p47 GTPases outside the vertebrates but they
are present in fish. Within the mammals, the p47
GTPases show a very remarkable distribution:
while mice have 23 p47 GTPase genes, only a single
full-length p47 GTPase gene and a p47 GTPase
gene fragment are present in the human genome.
Furthermore, the only gene present in man is the
single p47 GTPase that in the mouse is not induced
by interferons, and conserved at the level of 90%
between man and mouse. There is no reason to
believe that this gene, which we designate
CINEMA, is associated with pathogen resistance.
Thus the entire p47 GTPase resistance mechanism
is absent from the human species.
Biochemistry of IIGP1
IIGP-1 is a strongly interferon induced p47 GTPase
with a significant resting level in some organs,
especially the liver and heart. We have focused
much of our recent work on this member of the
family. The protein is a slow GTPase with a basal
turnover of about 0.1 per minute. The enzymatic
activity shows cooperativity, rising to a maximum
of 2 per minute under ideal conditions. The
enhanced enzymatic activity at high protein
concentrations is accompanied by GTP-dependent
oligomerisation. The oligomers resolve with time
as the GTP is hydrolysed. This behaviour recalls
the behaviour of the dynamin large GTPases,
including the anti-viral interferon-inducible Mx
protein referred to above, as well as the interferoninducible p65 kDa GTPases. These anomalous
large GTPases have another feature in common,
24
namely that the classical regulatory apparatus of
GAP and GEF proteins familiar from the signalling
GTPases, among others, is missing. No regulatory
protein has yet been described for any of the large
GTPases. A central problem with these enzymes is
therefore to understand how their self-activated
GTPase activity is regulated. When and where are
these proteins functional at basal rate and when
and where do they oligomerise and function at high
rate? In view of the high protein concentration in
the cell (2.2 x 106 molecules per induced fibroblast)
and the high concentration of cellular GTP, it is probable that IIGP-1 in the cell is close to the
oligomerisation state.
IIGP1, as probably most if not all the p47
GTPases, is associated with internal cellular
membrane compartments. In fibroblasts the protein
is on the ER membrane. Other p47s show a
preference for the Golgi. The protein is targeted to
membranes via an N-terminal myristoyl
modification, but the association with membranes
is strong and independent of the lipid modification.
Another p47, LRG-47, has no lipid modification
but associates very strongly with cellular
membranes. It presently seems likely that this is
due to a membrane integrating amphipathic helix
which we are presently trying to locate. The
association of p47 GTPases with cellular
membranes seems consonant with their function in
limiting infection by intracellular pathogens, but does
not in itself provide direct clues to a mechanism of
action.
Structure of IIGP1.
In a collaboration with the MPI-Molekulare Physiologie in Dortmund we have determined the crystal
structure of IIGP-1. The protein crystallised as a
simple dimer with or without nucleotide, with an
extensive interface. Mutations in the dimer interface
were shown to destroy cooperative enzymatic
activity and block oligomerisation. We therefore
believe that the dimer is a necessary building block
for the GTP-dependent oligomers with enhanced
enzymatic activity. Present experiments are directed
towards establishing whether IIGP1 self-associates
in the cell, and if so under what conditions.
Jonathan Howard
Functional analysis of the p47 GTPases.
We have made mice with a targeted conditional
deletion of IIGP1. These mice will be assayed for
susceptibility to several intracellular pathogens such
as Toxoplasma gondii. We have prepared mouse
fibroblast clones that stably express individual p47
GTPases under the control of a synthetic
progestogen. In the case of IIGP1 the level of
induction is almost identical to that achieved by
gamma-interferon. In these cells the p47 GTPases
are expressed alone and in the absence of the 500
or more proteins induced by gamma interferon.
These cells will be tested in a variety of pathogen
systems for resistance.
Finally we have been able to reduce significantly
the expression of LRG-47, IGTP and IIGP1 in
interferon-induced cells by transient RNAi and
these cells are now ready to test in a Toxoplasma
infection correlated with p47 levels.
Future work
The p47 GTPases are essential genes in mouse.
They are highly individualised and divergent in
sequence, they have different subcellular
distributions, and are non-redundant in their
contribution to pathogen resistance. Nevertheless
Fig. 2. IIGP1 detected by a specific antiserum in gamma
interferon-induced mouse 3T3 fibroblasts (left) and in
fibroblasts engineered to express comparable amounts
of IIGP1 on induction by the synthetic progestogen,
Mifepristone (right)
Fig. 3. LRG-47 expressed in gamma interferon-induced
„resting“ fibroblasts. The cell margins are not visible
and the GTPase is restricted to intracellular membrane
compartments largely coincident with the Golgi (top),
and in fibroblasts ingesting collagen-coated latex beads,
where the LRG-47 is concentrated in surface regions
associated with phagocytic cups and membrane ruffles
rich in filamentous actin (bottom).
the entire resistance mechanism is absent from
humans and it is unclear what mechanisms in our
species replace the p47 GTPases in, for example,
Toxoplasma infection. Our future work will be
devoted to a detailed analysis of the cellular
biochemistry of individual p47 GTPases, of the
proteins that control their enzymatic activity, and
of their intracellular behaviour during infection. We
have already shown that LRG-47, which in the
resting interferon-induced cells (if you can call a
cell resting that has just transcribed 500 new
genes!) is associated with the Golgi and the ER,
quickly relocalises to phagocytic cups and actinrich membrane ruffles when the cells are invited to
phagocytose collagen-coated latex beads. In
collaboration with JP Gorvel of the CIML, Marseille, we have been setting up to examine the
interaction between Brucella abortus and the p47
GTPases. What we are seeking is an accessible
cellular model of resistance which we can
manipulate in vitro with our battery of techniques
25
Jonathan Howard
and mutants. The p47 GTPases appear to be able
to exploit some Achilles heel in the ecology of a
number of intracellular pathogens (though not yet
of viruses). It will be of the greatest interest to discover whether this Achilles heel is already being
successfully exploited by other resistance
mechanisms in man, or whether the p47 GTPases
may be pointing us to new approaches to the control
of intracellular pathogens.
Selected Publications
Boehm, U., Klamp, T., Groot, M., Howard, J. C. (1997)
Cellular responses to interferon-(. Annu. Rev.
Immunol. 15, 749-795.
Klamp, T., Boehm, U., Groot, M., Howard, J. C. (1997)
A list of genes regulated by interferon gamma,
http://wwwannurevorg/sup/materialhtml.
Boehm, U., Guethlein, L., Klamp, T., Ozbek, K.,
Schaub, A., Fütterer, A., Pfeffer, K., Howard, J.
C. (1998) Two families of GTPases dominate
the complex cellular response to interferon-(. J.
Immunol. 161, 6715-6723.
Klamp, T., Boehm, U., Schenk, D., Pfeffer, K.,
Howard, J. C. (2003) A giant GTPase, VLIG-1, is
inducible by interferons. .J Immunol., in press.
Uthaiah, R., Praefcke, G. J. M., Howard, J. C.,
Herrmann, C. (2003) IIGP-1, an interferon-g
inducible 47 kDa GTPase of the Mouse, is a
slow GTPase showing co-operative enzymatic
activity and GTP-dependent multimerisation.
J. Biol. Chem. in press.
26
27
Börries Kemper
Professor
Börries Kemper received his PhD at the
institute for genetics at the university of
Cologne in 1969, where he worked on mapping
IS mutations in the galactose operon of
Escherichia coli. He went then as a postdoc to
Jerry Hurwitz at the Albert Einstein College
of Yeshiva University in New York City where
he worked on isolation and characterization of
endonucleases of bacteriophage T4. He
returned to Cologne in 1972 to become
Hochschulassistent in Peter Starlingers
department. He fulfilled the Habilitation in 1981,
became Bayer-Stiftungsprofessor in 1985 and
C3 Universitätsprofessor in 1988
Group members
Klaus Neef, PhD Student
Ulrich Rass, PhD Student
Vera Illner-Bräutigam,
Technician
Proteins in Genetic Recombination and DNA-Repair
We are interested generally in DNA repair and specifically in genetic recombination. We
isolate and characterize DNA safeguarding enzymes from Prokaryotes, Eukaryotes and
Archaea and study their reactions with in vitro assembled target molecules in detail. DNAbranching is a natural event occurring during homologous genetic recombination, and
four-armed X-junctions (Holliday-structures) or three-armed Y-junctions comprise two of
the most common intermediates. Branched molecules require precise resolution by a
nuclease and repair by helper proteins timely before progeny cells devide or viruses
package their DNA into newly synthesized heads. DNA-debranching enzymes (X-solvases)
like Holliday-structure resolving endonuclease VII (endo VII) from bacteriophage T4,
mitochondrial cruciform cutting endonuclease 1 (CCE1) from yeast S. cerevisiae and
viral as well as cellular Holliday junction cleaving enzymes (HJC) from archaeal origins
are among our favourite enzymes.
28
Börries Kemper
overhangs, gaps, nicks, curved DNA and several
examples of branched DNAs. Attempting to identify
a target signal shared by all substrates an alteration
in the secondary structure of double-stranded DNA
(dsDNA) has been suggested. Furthermore, Endo
VII was shown to modulate the function of UvsX,
the T4 homolog of E. coli’s STP recA, in vitro. In
accordance with EndoVII’s pivotal role for DNA
packaging in vivo, Endo VII binds tightly to gp20
in vitro, the portal protein of the phage. Gp20 in
turn is also directly connected to gp17 which,
together with gp16 comprises the phage’s packaging
Fig. 1. Phage T4 seen in a gold shadowed electron
micrograph (with permission from Levine, A.J., Viruses
(1992), Freeman and Company, New York, New York).
Holliday-structure resolving enzymes
Branched DNAs are made during genetic
recombination when two homologous
chromosomes become intimately paired with each
other and strands of like polarity are mutually
exchanged. These branches are made by strand
transfer proteins (STP) and require precise
endonucleolytic resolution for a faithful distribution
of chromosomes to daughter cells or into newly
synthesized viral heads. The first enzyme shown to
suffice these requirements was endonuclease VII
(Endo VII) from phage T4 (Fig. 1). The enzyme is
the product of gene 49 which is responsible for
complete packaging of DNA into preformed heads
of the phage. Since then a substantial collection of
comparable enzymes were reported from the three
domains of life, eukarya, prokarya and archaea.
Althoug all nucleases share the ability to precisely
resolve Holliday-structures by pairs of cuts in
homologous strands located across the junction
(Fig. 2), the proteins share little or no amino acids
sequences with each other. Especially Endo VII is
remarkably different from all other resolvases
showing high selectivity for a broad range of
substrates, which includes all possible mispairings,
single-strand loops, single-strand 5'- and 3'-
Fig. 2. The electronmicrograph shows purified Endo VII
binding selectively to the junction of a Holliday structure
(left). An artistic view of the event (right).
29
Börries Kemper
machinery suggesting Endo VII also being part of
it. The crystal structure of Endo VII has recently
been established (Fig. 3). Until cocrystals of Endo
VII and Holliday-structures can be obtained in
sufficent amounts, the enzymes mode of cleaving
Holliday junctions can be deduced from detailed
kinetic experiments.
A collection of Holliday-structure resolving
enzymes has recently been isolated from Archaea
and two of their viruses SIRV1 and SIRV2. Their
cleavage pattern were identified with cruciform
DNA and found to vary markedly for each of the
enzymes. Whether these differences can be traced
to distinct aa sequences is presently under study
and suspected sequences become exchanged
between pairs of respective enzymes.
Cruciform DNA binding proteins
Besides the cruciform-DNA resolving enzymes we
are also interested in cruciform-DNA binding
proteins which do not show enzymatic resolution
of DNA constructs. These proteins comprise a large
group of unrelated examples obtained from a wide
variety of organisms. We have recently isolated
such a protein from the yeast S. cerevisiae (Crp1p)
and begun to characterize its function(s). The
protein has 465 amino acids (aa) and is coded by
ORF YHR146W. Crp1p is apparently susceptible
to proteolytic cleavage, being divided into two
halves of aa 1-160 and 161-465. The purified
protein, overexpressed in E. coli, exhibits selective
binding to cruciform DNA and with a lower
efficiency to Y-structures. It does neither react with
single-stranded DNA nor double-stranded DNA.
Using a two-hybrid system with Crp1p as pray,
two putative RNA-helicases could be identified as
binding partners. Both are involved in ribosomal
assembly and RNA processing. The role of Crp1p,
however, is still unknown. Knock-out mutants do
not show detectable sensitivity towards growth
conditions or mutagenic agents (X-ray, UV, EMS).
It seems therefore unlikely that the protein is
involved in one of the yeast repair and recombination
pathways.
30
Mutation detection
The ability of Endo VII to reliably detect all possible
mismatches in dsDNA has made the enzyme a potent tool in mutation detection technology.
Oligonucleotides deriving from given wildtype and
corresponding mutant sequences are hybridized in
vitro resulting in heteroduplexes. These molecules
become cleaved by Endo VII at the sites of
dissimilarities within the sequences. Qualitative as
well as quantitative measurements of mutations in
interesting DNA sequences can thus be performed.
Figure 3. Structure of the wild-type Endo VII dimer.
Ribbon plot representation in stereo. The bound zink
and calcium ions are shown as blue and yellow speres
(from Raaijmakers et al. (1999), !8:1447-1458).
Börries Kemper
Selected Publications
Kemper, B. (1997) Branched DNA resolving enzymes
(X-solvases). In: DNA Damage and Repair.
Biochemistry, Genetics and Cell Biology,
J.A.Nickoloff and M.Hoekstra, eds. (Totowa:
Humana Press), pp. 179-204.
Rass,U., Kemper,B. (2002) Crp1p, a new cruciform
DNA-binding Protein in the yeast
Saccharomyces cerevisiae. J. Mol. Biol. 323,
685-700.
Cotton, N.R.G., Youil, R., Kemper, B. Detection of
mutation by resolvase cleavage. Avitech
Birkenbihl, R.P., Kemper, B. (1998) Endonuclease
VII has two DNA-binding sites each composed
from one N- and one C-terminus provided by
different subunits of the protein dimer. EMBO
J. 17, 4527-4534.
Greger, B., Kemper, B. (1998) An apyrimidinic site
kinks DNA and triggers incision by
endonuclease VII of phage T4. Nucl. Acids Res.
26, 4432-4438.
Kupfer, C., Lee, S., Kemper, B. (1998) Binding of
endonuclease VII to cruciform DNA.
Visualization in the electron microscope. J. Biol.
Chem. 273, 31637-31639.
Golz, S., Kemper, B. (1999) Association of Hollidaystructure Resolving Endonuclease VII with
gp20 from the Packaging Machine of Phage
T4. J. Mol. Biol. 285, 1131-1144.
Raaijmakers, H., Vix, O., Golz, S., Kemper, B., Suck,
D. (1999) X-ray structure of T4 endonuclease
VII: a DNA junction resolvase with a novel fold
and unusual domain-swapped dimer
architecture. EMBO J. 18, 1447-1458.
Birkenbihl, R.P., Neef, K., Prangishvili, D., Kemper,
B. (2001) Holliday junction resolving enzymes
of archaeal viruses SIRV1 and SIRV2. J. Mol.
Biol. 309, 1067-1076.
Birkenbihl, R.P., Kemper, B. (2002) High affinity of
endonuclease VII for the Holliday structure
containing one nick ensures productive
resolution. J. Mol. Biol. 321, 21-28.
Neef, K., Birkenbihl, R.P., Kemper, B. (2002) Holliday
junction-resolving enzymes from eight
hyperthermophilic archaea differ in reactions
with cruciform DNA. Extremophiles 6, 359-367.
31
Thomas Klein
Group Leader
Thomas Klein has received his Ph.D. in
Cologne 1994, where he worked on the
function of the Drosophila gene klumpfuss
during adult neurogenesis. He then joined the
lab of William Chia at the University of
Singapore for 14 month before moving on to
the lab of Alfonso Martinez-Arias at the
University of Cambridge, UK. In 1999 he
moved to the University of Cologne as an
independent group leader. His work covers
several aspects of adult development of the
fruit fly Drosophila melanogaster, such as wing,
leg and head development as well as
neurogenesis.
Group members
Melanie Beer, PhD Student
Robert Jaekel, PhD Student
Markus Kaspar, PhD Student
Stefan Kölzer, Technician
Hannelore Wirges-Koch,
Technician
Pattern Formation and Neurogenesis During Adult Development of
Drosophila melanogaster
Pattern formation is the fundamental process, which ensures that an organism develops
into the highly stereotypic array of different cells and tissues that is characteristic for a
species. We are using the adult development of Drosophila to study the molecular
mechanisms that underly pattern formation in a cellular field. These mechanisms provide
a cell in a developing organ with positional information, which is used by it to differentiate
into the appropriate fate. In many cases the cells have to communicate with each other to
aquire the information about its position. This communication is mediated by signalling
pathways. One focus of our research is the function of one evolutionary conserved pathway,
the Notch pathway.This pathway is involved in an uncountable number of developmental
pathways and plays an important role in many human diseases, ranging from cancer to
stroke. Hence, it is of great importance to understand how this pathway works.
While the development of the wing was the focus of our research in the past, we have
recently also started to investigate aspects of the development of the leg and head and the
peripheral nervous system. In the center of our interest are the characterization of the
function of the genes defective ventriculus (dve), klumpfuss (klu), lethal (2) giant discs
(lgd) and the Notch pathway during these processes.
32
Thomas Klein
Pattern formation during adult development
In holometabolous insects, such as Drosophila
melannogaster, the development of most parts of
the imago occurs in so called imaginal discs. These
discs are sheets of epithelial cells that are defined
during embryogenesis and proliferate during the larval live to form the adult structures during the pupal
phase (see Fig. 1). Since imaginal discs consist of
only one cell layer, pattern formation occurs in a
two-dimensional plane, with help of the two existing
axes, the antero-posterior (A/P) and dorso-ventral (D/V) axes. From this follows that the third,
proximo-distal axis must be defined and subdivided
with help of the two existing axes.
Whereas a great deal of information has been
collected about the molecules that are required
during pattern formation of the A/P and D/V axes
of the Drosophila wing, little is known about the
molecular mechanisms that operate along the P/D
axis. Furthermore, although it is clear that pattern
formation is a prerequisite for the proliferation of
cells in a developing organ/organism, there is little
knowledge about how these processes are
connected. We have found that the gene defective
proventriculus (dve) is involved in both processes.
The loss of dve function causes the deletion of one
region of the P/D axis without affecting the rest.
Furthermore, the cell proliferation along the A/P axis
is disturbed. dve mutant flies also exhibit defects in
other adults structures: the central region of the head
capsule is missing and extra joints with opposite
orientation to the normal ones develop in each tarsal segment of the legs. We are investigating the
role of dve during these developmental processes.
Fig. 1. The wing phenotype of dve mutant flies. (A) The
proximal region of a wild type wing is subdivided in
tree parts. (B) The proximal part of this region is deleted
in dve mutants, indicating that dve is required for its
formation.
Fig. 2. The mutant phenotype of lethal (2) giant discs in
the wing imaginal disc. (A) A wild type wing imaginal
disc revealing the expression of the Notch target gene
wingless by antibody staining. The arrow points to the
small stripe of expression along the D/V boundary. This
reveals that the activity of Notch is restricted to this
small region. (B) The expression of wg is expanded in
lgd mutant wing imaginal discs, indicating that the
Notch pathway is activated is a much broader domain.
The Notch signalling pathway is on of the fundamental pathways used in a uncountable number
of developmental processes in all animals. The
activity of this pathway is tightly regulated during
adult development. Any relief of the restriction of
its activity leads to pattern defects and overproliferation of the cells of the imaginal discs in
Drosophila. We found that the tumour suppressor
gene lethal (2) giant discs (lgd) is involved in
the control of the activity of the Notch pathway
during adult development (see Fig. 2). We are
currently focussing on the identification of the
transcription unit of the gene.
Two ligands of the Notch receptor exist in Drosophila, encoded by the Delta (Dl) and Serrate
(Ser) genes. We have previously shown, that the
activity of the Notch receptor is dependent not
only on the presence of one of these ligands, but
also on the ratio of the concentration of ligand
versus receptor. If the ratio is below a certain value,
Notch is activated, if above, the activation of the
receptor is suppressed in a cell autonomous
manner. This new type of regulation of the activity
of a signalling pathway is used to control and
33
Thomas Klein
restrict the activity of the Notch pathway during
later stages of wing development. The mechanism
of this regulation of the Notch receptor is not
understood. We have introduced several vertebrate
orthologs of the Dl ligands (called Delta-like (Dll))
and found that these ligands have different abilities
to regulate Notch: Dll-1 activates Notch at all
concentrations tested. In contrast Dll-4 seems
always suppress the activation cell autonomously.
We are currently using these differences to
determine the domain within the Dl molecules that
are responsible for the differences between Dll-1
and -4. This is done by domain swopping between
the molecules. By identifying the responsible region
in the Delta genes, we hope to get further insight in
the mechanism of the concentration dependent
control of Notch activity by its ligands.
The development of the bristle sense organ
of Drosophila
Two types of bristles, which can be discriminated
by their size, cover the body of the fly. The large
bristles are called macro- and the small bristles
microchaete (Fig. 3 and 4A). Both types of bristles
are part of the adult peripheral nervous system and
required for the perception of mechanic stimuli. The
macrochaete are arranged in a precise stereotypic
Fig. 3. The bristle sense organ of Drosophila. It consists
of four cells of whomm three are visible in the picture.
34
Fig. 4. Over-expression of klumpfuss leads to the
formation of supernurmary bristles. (A) the bristles of
the notum of a wild type fly ar arranged in a stereotypic
pattern. (B) The over-expression of klu dramatically
increases the number of bristles, indicating that much
more SOP have formed.
pattern that allows assigning names for each one
(Fig. 4A). Because of the simple composition and
accessibility, the bristles have become a classical
model system to study pattern formation and
neurogenesis. Many of the results obtained during
investigation of this system were extremely valuable
for the subsequent investigation of mammalian
neurogenesis.
Both types of bristle organs consist of four cells
(Fig. 3) that arose from one precursor cell, the
sensory organ precursor (SOP). This cell is selected
from a group of cells, which are competent to
develop as SOP because they express the genes
of the achaete-scute complex (AS-C). These genes
are called proneural genes and they are expressed
in groups of cells, within the imaginal discs, called
proneural clusters. The selection of the SOP out of
the proneural cluster is mediated by the Notch
signalling cascade in a process called lateral
inhibition. We have found that the one of the core
members of the Notch signalling pathway, the
transcription factor Suppressor of Hairless (Su(H))
is not only required to prevent SOP formation, but
also to promote development of the SOP, once it
is determined. This second function is independent
of the rest of the Notch pathway. We are now
investigating this new function of Su(H) in detail.
Our studies also suggest that the role of the Notch
pathway is not to select the SOP out of the proneural cluster as prviously thought, but to prevent
Thomas Klein
SOP formation until a cell is determined by other
patterning processes of unexpected precision.
Another project in the lab investigates the function
of the klumpfuss (klu) gene during neurogenesis. klu
encodes a zinc-finger protein of the EGR-family. Its
closest relative is the Wilms tumour suppressor gene.
Our results show that Klu is a transcriptional repressor
that seems to positively regulate the threshold for the
proneural activity required to coax a cell to develop
as a SOP (Fig. 4).
Selected Publications
Klein, T., Martinez-Arias, A. (1999) The Vestigial
gene product provides a molecular context for
the interpretation of signals during the
development of the wing in Drosophila.
Development 126, 913-925.
Bishop, S.A., Klein, T., Martinez-Arias, A., Couso,
J.P. (1999) Composite signalling from Serrate
and Delta establishes leg segments in
Drosophila through Notch. Development 126,
2993-3003.
Brennan, K., Klein, T., Wilder, E., Martinez-Arias, A.
(1999) Wingless modulates the effects of
dominant negative Notch molecules in the
developing wing of Drosophila. Implications
for Notch and Wingless signalling. Dev. Biol.
216, 210-229.
Lawrence, N*., Klein, T*., Brennan, K*, Martinez
Arias, A. (2000) Structural requirements for
Notch signalling with Delta and Serrate during
the development and patterning of the wing
disc of Drosophila. Development 127, 31853195, (*gleicher Beitrag der Autoren).
Klein,T., Seugnet, L., Haenlin, M., Martinez-Arias,
A. (2000) Two different activities of Suppressor
of Hairless during wing development in
Drosophila. Development 127, 3553-3579.
Klein, T. (2001) Wing disc development in the fly:
The early stages. Curr. Opin. Gen.& Dev. 11,
470-475.
Klein, T. (2002) kuzbanian is required cell
autonomously during Notch signalling in the
Drosophila wing. Dev. Genes Evol. 212, 251255.
Klein, T. (2003) The tumour suppressor gene
l(2)giant discs is required to restrict the activity
of Notch to the dorso-ventral boundary during
Drosophila wing development. Dev. Biol. 255,
313-333.
Kölzer, S. und Klein, T. (2003) A Notch independent
function of Suppressor of Hairless during the
development of the bristle sensory organ
precursor cell of Drosophila. Development 130,
1973-1988.
35
Michael Knittler
Group Leader
Michael Knittler received his PhD at the
University of Cologne, 1994, where he
investigated at the Institute of Genetics in the
lab of I. G. Haas the function of ER-localized
chaperones in folding and assembly of antibody
molecules. During his PhD thesis he moved to
the Biochemiezentrum Heidelberg to work on
ER-associated protein degradation. He then
joined as a postdoc the lab of Prof. Howard
at the University of Cologne where he began
his studies on MHC class I-mediated antigen
presentation. In 2000 he started as independent
group leader in the Dept. of Cell Genetics. His
research interest is focussed on the
biochemistry and function of the ER-localized
ABC-transporter TAP which delivers antigenic
peptides for loading onto MHC class I
molecules.
Group members
Ralf Max Leonhardt, PhD
Student
Cell Biology of Antigen Presentation
Goal of our research is to understand the intracellular process of MHC class I-restricted
antigen presentation. We are especially interested in the function and mechanism of the
peptide transporter TAP, which is a member of the ATP-binding-cassette (ABC)
transporter family. The transporter is localized in the membrane of the endoplasmic
reticulum (ER) and is a critical part of the antigen processing machinery of vertebrates.
TAP transports short peptides, generated in the cytosol by the proteasome, into the ER
lumen where they load onto MHC class I molecules. TAP forms a transient loading
complex with newly-formed MHC class I molecules. The release of MHC class I from
the complex is induced by peptide binding and the dissociation coincides with the export
of loaded MHC class I molecules from the ER into the secretory pathway. Cytotoxic T
lymphocytes identify and eliminate cells harbouring pathogens by monitoring the peptideMHC class I molecule complex at the cell surface. Although much has been learned
about the MHC class I antigen presentation pathway, little is known about the central
processes involved in peptide translocation by TAP. We use genetic and biochemical
approaches to elucidate the peptide transport process of TAP and the critical function
of the loading complex in the assembly and maturation of MHC class I molecules.
36
Michael Knittler
Formation of the TAP-MHC loading
complex
TAP consists of the two polypeptide chains, TAP1
and TAP2, together forming a heterodimeric
transporter. Furthermore, TAP is an integral part
of a large complex of multiple proteins that is
optimized for loading of translocated peptides onto
MHC class I molecules (TAP-MHC loading
complex). Initially stabilized by ER-resident
chaperones calnexin and calreticulin, newly
synthesized MHC class I molecules dock onto the
TAP heterodimer via the dedicated chaperone
tapasin (Fig. 1). The established stochiometry
between TAP, tapasin and MHC class I molecules
is 1:4:4.
Although it has been demonstrated by different
research groups that tapasin plays an important role
in the in vivo stabilization of TAP and the physical
interaction between TAP and MHC class I
molecules, nearly nothing is known about the
molecular basis and structural requirements of the
TAP-MHC loading complex formation. We are
now addressing this question by using different
biochemical and molecular approaches.
Hydrophobicity analysis and sequence alignments
of TAP with related ABC transporter proteins point
Fig. 1. The TAP-MHC loading complex. Each of the two
subunits of TAP interacts with two tapasins which act as
a molecular bridge for the MHC class I molecules. The
premature and unloaded conformation of MHC class I
molecules in the loading complex is stabilized by the
ER-localized chaperons calreticulin and ER60.
to a 2 x 6 transmembrane helix model of TAP,
extended by additional transmembrane helices
predicted for the hydrophobic N-terminal
subdomains. In view of the formation of the TAPMHC loading complex, our research project is
concentrated on the structural and functional role
of the N-terminal extensions in TAP1 and TAP2.
The results of our work will help to understand the
process of the TAP-MHC loading complex
formation and the function of the two TAP subunits
in peptide loading of MHC class I molecules.
Distinct functional properties of TAP1 and
TAP2 in the peptide transport cycle
Both subunits of TAP contain an N-terminal
transmembrane domain (TMD) and a C-terminal
cytosolic nucleotide binding domain (NBD). The
TMDs are involved in peptide binding and
translocation whereas the NBDs energize the
transport by nucleotide hydrolysis. Our previous
experiments on TAP revealed that the two highly
conserved NBDs (70% sequence homology
between TAP1 and TAP2) seem to serve different
functions during the peptide transport. Thus, in the
resting state of the transporter, TAP1 binds ATP
much more efficiently than does TAP2. Furthermore, homologous mutations in the NBDs of the
two TAP subunits have radically different effects
on peptide transport activity. We could recently
show that distinct nucleotide binding and functional
non-equivalence of TAP1 and TAP2 is an intrinsic
property of their NBDs. Our current view is that
the two TAP-NBDs work as non-equivalent
modules and control different steps of the peptide
transport cycle. We propose that both NBDs are
catalytically active in an alternating and interdependent manner. ATP binding to TAP1 seems to be
the initial step in energizing the transport process
while ADP is bound to TAP2 (Fig. 2). At present,
our research interest is focussed on the identification
of amino acid residues and/or domain segments that
are responsible for the biochemical and functional
asymmetry of the two TAP-NBDs. The results of
our studies will provide insights into the structural
and mechanistic principles of the peptide transport
cycle of the ABC transporter TAP.
37
Michael Knittler
Fig. 2. Model of the peptide translocation by TAP. In the
resting state of TAP (top), TAP1 is competent for ATP
binding whereas TAP2 (in red) binds ADP. Peptide
binding triggers ATP hydrolysis in NBD1 and
translocation by the transporter. In the subsequent step
(bottom), conformational changes in TAP2 allow ATP
binding in NBD2. The cycle is completed and reinitiated
when ATP hydrolysis in NBD2 allows the recharging of
NBD1 with ATP .
Conformational interplay between the NBDs
and TMDs of TAP
The substrate transport of TAP and all other ABC
transporters is strictly ATP-dependent. A single TAP
complex hydrolyses about five molecules of ATP
to transport two to three peptides. It is believed
that the NBDs interact with the TMDs in order to
transmit conformational changes resulting from the
hydrolysis of ATP. The NBDs contain highly
conserved sequence motifs that are involved in ATP
binding and hydrolysis, including the Walker A, B
and signature motifs (ATP-binding-cassette) (Fig.3).
The so-called helical domain that connects the Walker A motif with the signature motif and comprises
around 100 amino acid residues has been suggested
as a candidate to transduce structural signals to the
TMDs during peptide translocation. The helical
domain is thought to change its conformational
properties upon ATP hydrolysis and subsequently
puts the TMDs into motion. Results from our
previous studies on the functional exchange of TAP
domains suggest that the nucleotide binding states
38
of the NBDs control the process of peptide binding
to the TMDs during the transport cycle. However,
at the structural level, interactions between NBDs
and TMDs of TAP are not understood. To
investigate the molecular basis of the conformational
interplay between the different TAP-domains, we
have recently constructed a variety of mutant TAP
variants. These mutant transporters will give us further information about the nucleotide-dependent
mechanism of peptide uptake and translocation. As
different ABC transporters share sequence identity
and a common domain organization, the results of
our work may also apply to other members of the
ABC transporter family.
Fig. 3. Molecular structure of the NBD1 of human TAP
(R., Gaudet, and D., Wiley (2001) EMBO J, 20, 4972).
The polypeptide chain is represented as a ribbon diagram. The helical domain and the conserved sequence
motifs of the ATP-binding-cassette (Walker A, Walker B
and signature motif) are indicated.
Michael Knittler
Selected Publications
Knittler, M.R., Gülow, K., Seelig, A., Howard, J.C.
(1998) MHC class I molecules compete in the
endoplasmic reticulum for access to transporter
associated with antigen processing. J. Immunol.
161, 5967-5977.
Knittler, M.R., Alberts P., Deverson, E.V., Howard,
J.C. (1999) Nucleotide binding by TAP mediates
association with peptide and release of
assembled MHC class I molecules. Curr. Biol.
9, 999-1008.
Alberts, P., Daumke, O., Deverson, E.V., Howard,
J.C., Knittler M.R. (2001) Distinct functional
properties of the TAP subunits coordinatethe
nucleotide-dependent transport cycle. Curr.
Biol. 11, 242-252.
Daumke, O., Knittler, M.R. (2001) Functional
asymmetry of the ATP-binding-cassettes of
ABC transporter TAP is determined by intrinsic
properties of the nucleotide binding domains.
Eur. J. Biochem. 268, 4776-4786.
Leonhardt, R.M., Knittler, M.R. (2003) Erste-KlasseTicket" für Antigene: Zelluläre Stationen der
MHC Klasse I vermittelten. Antigenpräsentation. BioSpektrum 1, 23-26.
Bouabe, H., Knittler, M.R. (2003) The distinct
nucleotide binding states of the transporter
associated with antigen processing (TAP) are
regulated by the non-homologous C-terminal
tails of TAP1 and TAP2. Eur. J. Biochem., in
press.
39
Sigrun Korsching
Professor
Sigrun Korsching received her Ph. D. 1984 in
Munich. In the lab of H. Thoenen at the MPI
she developed a quantification method for nerve growth factor, enabling analysis of
biosynthesis and axonal transport. During a
postdoc period at the Caltech she studied LIF
in the group of P. Patterson. In 1988 she
became independent group leader at the MPI
in Tübingen in F. Bonhoeffers department, and
1995 she joined the Dept. of Genetics in
Cologne as C3 professor. Since 1991 she works
on information processing and development of
the olfactory nervous system using zebrafish
and mouse as model systems.
Group members
Hans Fried, PhD Student,
Aswani Kotagiri Kumar, PhD
Student
Sunil Kumar, PhD Student
Mehmet Saltürk, Technician
Christina Sterner, Technician
Development and Function of the Olfactory Nervous System
More chemicals can be smelled than there are odorant receptors for them, necessitating a
combinatorial representation by somewhat broadly tuned receptors. To understand the
perception of odor quality, it is essential to establish the ligand spectra of individual
odorant receptors, the nature of the receptor repertoires that are activated by particular
odorants, as well as the modification of the odor response of olfactory receptor neurons
by the circuitry of the olfactory bulb. The transition from presumably stochastic expression
of odorant receptor genes in scattered olfactory receptor neurons within the sensory
epithelium to an ordered, chemotopic map in the olfactory bulb requires sophisticated
axonal targeting of olfactory receptor neurons. We employ the model systems zebrafish
and mouse to study these problems using an array of molecularbiological and physiological
methods.
40
Sigrun Korsching
Fig. 1. Calcium Imaging shows odorant receptors with
divergent response properties are recruited in response
to a simple odorant – an amino acid.
The lateral, amino acid-responsive region of an adult
olfactory bulb is depicted. The bottom row shows
difference images, i.e. responses specific for 2aminohexanoic acid (2-aha, left panel) and specific
for lysine (lys, right panel). Scale bar, 100 µm.
Deducing properties of odorant receptor
repertoires
The response properties of many odorant receptors
can be visualized simultaneously by optical imaging
of neuronal activity in the olfactory bulb, since
olfactory receptor neurons expressing the same
odorant receptor converge onto common neuropil
structures in the olfactory bulb, so-called glomeruli.
We have introduced a calcium-sensitive dye
selectively in the presynaptic compartment of
glomeruli and measured calcium changes in
response to a series of naturally occurring amino
acids and some structurally related compounds. We
report that the amino acid head region is required
to elicit a response, and that different subgroups of
receptors are activated by amino acids of differing
chain length and functional groups.
We conclude that odorant detection requires
several receptors even for relatively simple
odorants such as amino acids. Furthermore, we
find that individual odorant receptors require the
presence of some molecular features, the absence
of others, and tolerate still other molecular features.
Thus, odorant receptors appear not to be simple
‘feature detectors’ but to detect particular
combinations of molecular features (odotopes).
In a second project we have analyzed the tuning
properties of a major mammalian odorant receptor
population using the same method of high resolution
calcium imaging. We show that eighty different
odorant receptors projecting to the dorsal olfactory
bulb of mouse respond to high concentrations of
aldehydes with limited specificity. Different
ensembles of about 10 to 20 receptors encode any
particular aldehyde at low stimulus concentrations
with high specificity. Pronounced differences in
affinity were observed within the aldehyde receptor
repertoire.
Regulated expression of zebrafish odorant
receptors
Olfactory receptor neurons select a single odorant
receptor gene for expression out of a large gene
family. To investigate mechanisms for this extreme
selectivity, we have determined by quantitative in
situ hybridization the developmental expression
dynamics of a representative subset of the zebrafish
odorant receptor repertoire. Features of the
expression pattern include distinct, but broadly
overlapping expression domains, and particular
ontogenetic onsets of expression for different
odorant receptors.
Statistical analysis of the expression frequencies
supports a model in which the final choice of an
individual odorant receptor gene occurs
stochastically from within a group of genes sharing
a deterministically defined onset of expression.
In the second project we have analysed the
expression pattern conferred by short promoter
regions of two zebrafish odorant receptor genes.
We report that regions of around one kb suffice to
direct expression of a marker protein to olfactory
receptor neurons, with the normalized frequency
of expression being similar for the transgene and
the endogenous gene. The time of onset of
expression is mimicked by one, but delayed for
the other transgene, indicating that not all regulatory
elements reside within the promoter regions
examined. Axon terminals of transgenic olfactory
receptor neurons terminate in a subregion of the
area covered by all receptor neuron terminals,
consistent with expression of the construct restricted
to a subgroup of olfactory receptor neurons.
41
Sigrun Korsching
Fig. 2. In situ hybridization with an odorant receptor
probe. Left panel, sections of zebrafish olfactory
epithelia (OE) 5 days post fertilization were hybridized
with a ZOR1 probe and show several labelled olfactory
receptor neurons (ORNs) within the OE. Right panel,
scanning electron micrograph of a zebrafish head, from
anterior. The arrow indicates the olfactory pit. Scale
bars, left 10 µm, right 100 µm.
Mapping spatial expression patterns in the
olfactory bulb
The invariant chemotopic pattern of odor responses
in the olfactory bulb - reported in our earlier studies
- implies the existence of positional cues or guidance
molecules within the olfactory bulb. In search of
such guidance molecules we constructed a differential library by subtractive hybridization using
functionally divergent subregions of the olfactory
bulb as tissue sources. We have identified several
novel genes with highly restricted patterns of
expression, and associated with particular neuronal subpopulations (e.g. zPQRFamide) or neural
differentiation stages (e.g. beta 1 tubulin).
In another approach we have analysed the
expression patterns of axonal guidance molecules
characterized in other neuronal systems, focusing
on the eph/ephrin gene families. Many of the eph
and ephrin genes are expressed in a layer-specific
manner. We observe expression restricted e.g. to
the inner cell layer of the olfactory bulb, or to the
glomerular layer, or to the sensory domain of the
olfactory epithelium, etc. Often we find complementary expression patterns of an ephrin and its
cognate eph receptors. These patterns of
expression suggest a role for eph/rins in the
formation and maintenance of neuronal layers.
42
Genetic dissection of the olfactory nervous
system
Understanding the circuitry of the nervous system
would be greatly facilitated by specifically
addressing particular neuronal subpopulations using
a genetic approach.
Zebrafish have definite advantages for such
genetic manipulations, because large numbers of
progeny can be obtained, transgenes can be
introduced with ease in early embryos in great
numbers and development is very fast compared
to that of higher vertebrates. Embryos are transparent, allowing the visualization of individual cells
and their projections in living animals.
Olfactory marker protein (OMP) is a reliable
marker for olfactory receptor neurons in several
other vertebrates. We have cloned and
characterized zOMP, the zebrafish homologue. The
5’-flanking region strongly and specifically drives
reporter gene expression in olfactory receptor
neurons and thus constitutes a powerful tool to
selectively introduce a wide variety of genetic
modifications into olfactory receptor neurons.
Fig. 3. One of the medial-enriched genes is expressed in
terminal nerve neurons
(A) Whole mount in situ hybridization with zfPQRF in
the two olfactory bulbs of a mature zebrafish shows
labeled cells at the entry region of the olfactory nerve,
and along the olfactory bulb.
(B) Enlargement from panel A. Individual neurons, with
labeled somata and spared nuclei are visible. Scale bars
equal 100 µm in panel A, and 25 µm in panel B.
Sigrun Korsching
We have begun to test various genetic calcium
indicators using the zOMP promoter in transient
transfection experiments of zebrafish embryos.
Preliminary experiments suggest the suitability of
some indicators for measuring odorant-induced
neuronal activity.
We are currently characterizing promoters for
genes that are expressed specifically in mitral and
granule cells, the main components of the olfactory
circuitry besides the receptor neurons. Once such
promoters are obtained, they will be used to drive
calcium indicator gene expression specifically in the
respective cell population. Thus it will be possible
to analyse the neuronal representation of odors
separately at each level of olfactory information
processing.
Selected Publications
Friedrich, R.W., Korsching, S.I. (1998) Chemotopic,
combinatorial, and noncombinatorial odorant
representations in the olfactory bulb revealed
using a voltage-sensitive axon tracer, J.
Neurosci. 18, 9977-9988.
Fuss, S.H., Korsching, S.I. (2001) Odorant feature
detection: activity mapping of structure
response relationships in the zebrafish
olfactory bulb. J. Neurosci. 21, 8396-8407.
Fried, H.-U., Fuss, S.H., Korsching, S.I. (2002)
Selective imaging of presynaptic activity in the
mouse olfactory bulb shows concentrationand structure dependence of odor responses
in identified glomeruli. Proc. Natl. Acad. Sci.
USA 99, 3222-3227.
Çelik, A., Fuss, S.H., Korsching, S.I. (2002) Selective
targeting of zebrafish olfactory receptor
neurons by the endogenous omp promoter. Eur.
J. Neurosci. 15, 798-806.
Oehlmann, V.D., Korte, H., Sterner, C., Korsching,
S.I. (2002) A neuropeptide FF-related gene is
expressed selectively in neurons of the terminal
nerve in Danio rerio. Mech. Dev. 117, 357-361.
Korsching, S.I. (2002) Olfactory maps and odor
images, Curr. Opin. Neurobiol. 12, 387-392.
Argo, S., Weth, F., Korsching, S.I. (2003) Analysis
of penetrance and expressivity during
ontogenesis supports a stochastic choice of
zebrafish odorant receptors from predetermined groups of receptor genes. Eur. J.
Neurosci. 17, 833-843.
Fig. 4. In vivo reporter gene expression in zebrafish
embryos, using EYFP under OMP promoter control.
Three fluorescent olfactory receptor neurons with
dendrites and single axons are visible in a 72 h embryo.
Scale bar, 25 µm
Oehlmann, V.D., Berger, S., Sterner, C. Korsching,
S.I. (2003) Zebrafish beta tubulin 1 expression
is limited to the nervous system throughout
development, and in the adult brain is restricted
to a subset of proliferative regions. Mech. Dev.,
in press.
43
Thomas Langer
Professor
Thomas Langer received his PhD at the
University of Munich in 1993, where he worked
on chaperone-mediated protein folding in the
group of Franz-Ulrich Hartl. The experimental work for his doctoral thesis was mostly
performed at the Memorial Sloan-Kettering
Cancer Institute, New York. As research
associate and group leader he worked at the
Adolf Butenandt Institute in Munich where his
research focused on functions of ATPdependent proteases in mitochondria. Since
October 2000 he is full professor at the Institute of Genetics.
Group members
Tanja Engmann, PhD Student
Dominik Galluhn, PhD Student
Martin Graef, PhD Student
Melanie Kambacheld, PhD
Student
Mirko Koppen, PhD Student
Metodi Metodiev, PhD Student
Mark Nolden, PhD Student
Stephanie Wurth, PhD Student
Isabel Arnold, Postdoc
Mafalda Escobar-Henriques,
Postdoc
Takashi Tatsuta, Postdoc
Daniela Tils, Technician
Gudrun Zimmer, Technician
Brigitte Kisters-Woike,
Department Manager
Proteolysis in Mitochondria
Proteolysis has been recognised as a major mechanism to regulate cell functions. We are
interested in the proteolytic system of mitochondria and try to understand its roles in
maintaining mitochondrial activity and ensuring quality control of mitochondrial proteins.
One focus of our research concerns the turnover of membrane proteins, which we analyse
with respect to its physiological roles and molecular mechanisms both in the mitochondrial
inner and outer membranes. Proteolysis of mitochondrial proteins results in the formation
of peptides and amino acids, which are released from the organelle. We characterise the
export of peptides from mitochondria and examine potential functions of the released
peptides with respect to the coordination of nuclear and mitochondrial gene expression.
The yeast Saccharomyces cerevisiae serves as the main model organism in our studies.
However, the parallel analysis of some of the conserved components in mammalian cells
should reveal their possibly different roles in a multicellular organism and provide insights
into the molecular basis of neurodegeneration associated with mutations in the mitochondrial proteolytic system in humans.
44
Thomas Langer
Fig. 1. Electron tomography of a mitochondrion
from N.crassa (courtesy of T.Frey, San Diego)
The maintenance of cell functions requires not only
ongoing synthesis of proteins at cytosolic
ribosomes but also depends on constant proteolysis
of short-lived regulatory and misfolded proteins.
Impairment of proteolytic processes results in a
severe disturbance of cellular homeostasis and cell
death and has been recognised as an important
disease mechanism in a variety of neuroProhibitini-AAAprotease
protease
Prohibitin - degenerative
i-AAA
disorders in human. Mitochondria
complex
complex harbour a conserved proteolytic system with essential functions in organellar biogenesis and
IMS
therefore offer the possibility to study general
principles of proteolytic control on a subcellular
level. Our research has focused over recent years
Matrix
on the turnover of membrane proteins, in particular
in the inner membrane of mitochondria. Several
m-AAA
protease protein complexes have been
protease
m-AAA
membrane-bound
identified which ensure the quality control of inner
membrane proteins and regulate mitochondrial
biogenesis (Fig. 2). We analyse the molecular
function of these components and try to understand
consequences of an impaired protein quality control
for cellular activities.
Control of mitochondrial biogenesis by AAA
proteases
AAA proteases exert key functions in the
maintenance of the integrity of the inner membrane.
They constitute a unique class of ATP-dependent
proteases as they are embedded in membranes and
recognise membrane proteins as their substrates.
Two large proteolytic complexes are present in
mitochondria which were termed m- and i-AAA
proteases to indicate their different topology in the
inner membrane: the m-AAA protease is active on
the matrix, the i-AAA protease on the
intermembrane side of the membrane. Both
proteases are built up of highly homologous subunits
with sequence identities of >40% between the
bacterial, yeast, plant and human members. The
m-AAA protease is a hetero-oligomeric complex
of Yta10 and Yta12 subunits in yeast and of
AFG3L2 and paraplegin subunits in human,
whereas Yme1 subunits most likely form homooligomeric i-AAA proteases in mitochondria of
both organisms.
Inactivation of AAA proteases causes severe
phenotypes in various organisms including
neurodegeneration in humans. Yeast cells lacking
one AAA protease exhibit pleiotropic defects,
whereas inactivation of both AAA proteases is
lethal. The molecular basis of defects associated
with AAA protease mutants is in most cases not
understood. AAA proteases constitute a
membrane-embedded quality control system and
degrade misfolded and unassembled polypeptides.
It is therefore conceivable that non-native
polypeptides accumulating in the absence of AAA
proteases impair cellular functions. This might
explain the progressive nature of the neuro-
Fig. 2. AAA proteases of the inner membrane of
mitochondria. The prefixes indicate the sub-mitochondrial localisation of the catalytic sites. The m-AAA
protease is part of a supercomplex with prohibitins. See
text for detail. IMS: intermembrane space.
45
Thomas Langer
degenerative disorder in humans, which is
associated with the inactivation of the m-AAA
protease subunit paraplegin. Our current
understanding of AAA proteases is generally limited
by the fact that short-lived mitochondrial substrate
proteins with potential regulatory functions have not
been identified. We therefore analyse currently the
mitochondrial proteomes in yeast and mouse and
pursue genetic approaches to identify substrate
proteins of mitochondrial AAA proteases in yeast
and mammalian mitochondria.
AAA proteases – molecular machines for
degrading membrane proteins
The turnover of membrane proteins has been
puzzling for a long time as the hydrophobic
environment of the lipid bilayer was thought to
preclude the cleavage of membrane-embedded
protein segments. We exploit the quality control
function of AAA proteases and analyse the stability
of a variety of model substrate proteins in isolated
mitochondria to gain new insights into the
mechanism of membrane protein degradation.
To maintain the specificity of proteolysis is critical
to avoid cell damage. The substrate specificity of
AAA proteases is ensured by the AAA domain,
which exerts chaperone-like activity and specifically
binds unfolded solvent-exposed domains of mem-
Fig. 3. Structural model of the AAA domains of the i-AAA
protease subunit Yme1 of S. cere-visiae based on the
crystal structure of the related ATPase p97. Helices
involved in substrate bind-ing are shown in red.
Conserved Y354 located in a loop exposed to the central
pore is shown in green.
46
brane proteins. Next to nothing is known, how
substrates are bound by AAA proteases or AAA
proteins in general. Using the available crystal
structures of a number of AAA proteins we have
generated a hexameric ring model for the AAA
domains of the i-AAA protease of yeast
mitochondria (Fig. 3). An ongoing structurefunction analysis should identify regions involved in
substrate binding and examine a potential transport
of substrate polypeptides through the central pore
into a sequestered proteolytic microcompartment.
Membrane proteins are dislocated from the
membrane bilayer to allow proteolysis to occur in
a hydrophilic environment. Depending on the
membrane topology of the substrate, dislocation
and proteolysis can be mediated from both
membrane sides by either AAA protease, which
exert overlapping substrate specificities. The
dislocation of polypeptides from the mem-brane is
reminiscent of ER-membrane proteins, which are
translocated in a retrograde manner via the Sec61channel into the cytosol and degraded by the 26S
proteasome. The mechanisms of dislocation,
however, appear to be different in mitochondria,
as proteolysis by AAA proteases does not depend
on protein translocases in the inner membrane.
Recent experiments assigned a crucial role in this
process to transmembrane segments of AAA
protease subunits, which may form a pore-like
structure in the membrane facilitating dislocation.
Thus, despite striking similarities considerable
mechanistic differences become apparent in the
degradation of proteins in different cellular
membranes.
Prohibitins - regulation of proteolysis and
more?
Studies on AAA proteases took yet another twist
when the yeast m-AAA protease was identified as
part of a supercomplex in the inner membrane with
a native molecular mass larger than ~2000 kDa. It
assembles with prohibitins which form a multimeric
protein complex composed of Phb1 and Phb2
subunits. Prohibitins comprise a ubiquitous, highly
conserved protein family in eukaryotic cells with
Thomas Langer
two homologous proteins forming a hetero-oligomeric complex in the mitochondrial inner membrane
in various organisms.
Deletion of prohibitin genes in yeast caused an
accelerated degradation of non-assembled
membrane proteins by the m-AAA protease
pointing to a regulatory function of prohibitins
during membrane protein turnover by the m-AAA
protease. This notion is also supported by genetic
experiments revealing a dramatic synthetic growth
phenotype of cells lacking both the m-AAA
protease and prohibitins. The activity of prohibitins
on the molecular level, however, is still unclear.
Prohibitins could interfere with proteolysis by
exerting chaperone activity and direct binding to
substrate proteins. Alternatively, prohibitins may
affect the turnover of membrane proteins by
modulating the enzymatic activity of the m-AAA
protease. Experiments to distinguish between these
possibilities are currently in progress.
The identification of prohibitins as regulators of
AAA-proteases raises the question of whether
cellular activities previously attributed to prohibitins
are linked to the degradation of mitochondrial inner membrane proteins. In mammalian cells, prohibitin has been proposed to regulate cell cycle
progression and senescence because it inhibits cell
proliferation of fibroblasts and is expressed at
decreased levels in resting liver cells. A physical
interaction of mammalian prohibitin with the retinoblastoma tumour suppressor protein has been
described, but the physiological significance of this
finding remains unclear considering the
mitochondrial localisation of prohibitins. Yeast cells
lacking prohibitins grow normally but show a
decreased replicative life span and aberrant
mitochondrial morphology in senescent cells. In
multicellular organisms like mouse or C. elegans,
on the other hand, inactivation of prohibitins was
reported to cause an embryonic lethal phenotype.
It is presently unclear whether this results from a
deregulation of an AAA protease or the loss of
another, yet unknown function of prohibitins. To
characterise the role of prohibitins in mammals we
are currently generating a mouse strain allowing the
conditional inactivation of prohibitin. These studies
should complement our efforts on the
characterisation of mammalian AAA proteases and
their function in mitochondrial biogenesis.
Peptide export from mitochondria
Early studies revealed that mitochondrial proteins
can be completely degraded to amino acid residues
within the organelle. The identification of peptides
derived from mitochondrially-encoded proteins in
association with MHCI molecules on the cell surface
of mammalian cells prompted us to study the fate
of proteolytic products generated by AAA
proteases. Proteolysis of non-assembled mitochondrially-encoded respiratory chain subunits was
found to result in the formation of a heterogeneous
spectrum of peptides and free amino acid residues
within mitochondria. Strikingly, these proteolytic
products were released from the organelle. Two
pathways for the efflux of peptides composed of
more than approximately 10 amino acid residues
can be distinguished which converge in the
intermembrane space (Fig. 4): First, peptides
generated by the m-AAA protease in the matrix
space are actively transported across the inner
membrane by the mitochondrial ABC transporter
Mdl1. Mdl1 is highly homologous to the transporter
associated with antigen presentation (TAP) in higher
Fig. 4. Pathways of peptide export from mitochon-dria.
See text for details. OM: mitochondrial outer membrane;
IMS: intermembrane space; IM: inner membrane
47
Thomas Langer
eukaryotic cells which transports antigenic peptides
from the cytosol into the lumen of the ER. Second,
proteolysis of inner membrane proteins by the iAAA protease results in the release of peptides
directly in the intermembrane space. Peptides could
then cross the mitochondrial outer membrane by
passive diffusion, most likely either through porins
or the TOM-complex, the general protein import
pore of the outer membrane.
The physiological role of peptides exported from
mitochondria remains to be elucidated. The
presence of homologous ABC transporters in
mammalian mitochondria suggests that similar
pathways exist also in mitochondria of higher
eukaryotic cells, which will be examined in future
experiments. It is an attractive hypothesis that
mitochondrially encoded minor histocompatibility
antigens are generated by AAA proteases within
mitochondria and then released to the cytosol, from
where they enter the conventional class I antigen
presentation pathway. While it is conceivable that
exported mitochondrial peptides are exploited by
the immune system for immune surveillance in
mammals, our results in yeast point to additional
cellular functions of mitochondrial peptide export.
These may include signalling mechanisms to allow
coordination of mitochondrial and nuclear gene
expression, including stress specific pathways. Understanding the cellular role of mitochondrial
peptide export for cellular homeostasis is a central
question of our current research activities.
Selected Publications
Arlt, H., Steglich, G., Perryman, R., Guiard, B.,
Neupert, W., Langer, T. (1998) The formation of
respiratory chain complexes in mitochondria is
under the proteolytic control of the m-AAAprotease. EMBO J. 17, 4837-4847.
Leonhard, K., Stiegler, A., Neupert, W., Langer, T.
(1999) Chaperone-like activity of the AAAdomain of the Yme1p AAA-protease. Nature
398, 348-351.
Leonhard, K., Guiard, B., Pellecchia, G., Tzagoloff,
A., Neupert, W., Langer, T. (2000) Membrane
protein degradation by AAA proteases in
mitochondria: extraction of substrates from
either membrane surface. Mol. Cell 5, 629-638.
Langer, T. (2000) AAA proteases - cellular machines
for degrading membrane proteins. Trends
Biochem. Sci. 25, 247-251.
Young, L., Leonhard, K., Tatsuta, T., Trowsdale, J.,
Langer, T. (2001) Role of the ABC-transporter
Mdl1 in peptide export from mitochondria.
Science 291, 2135-2138.
Arnold, I., Langer, T. (2002) Mem-brane protein
degradation by AAA proteases in
mitochondria. Biochim. Biophys. Acta 1592, 8996.
Käser, M., Kambacheld, M., Kisters-Woike, B.,
Langer, T. (2003) Oma1, a novel membranebound metallopeptidase in mitochondria with
activities overlapping with the m-AAA
protease. J. Biol. Chem., in press.
Atorino, L., Silvestri, L., Koppen, M., Cassina, L.,
Ballabio, A., Marconi, R., Langer, T., Casari, G.
Loss of m-AAA protease in mitochondria
causes complex I deficiency and increased
sensitivity to oxidative stress in hereditary
spastic paraplegia. J. Cell Biol., in press.
48
49
Maria Leptin
Professor
Maria Leptin received her Ph.D. in 1983 for work
on B cell activation carried out at the Basel Institute
for Immunology under the supervision of F.
Melchers. She switched to the study of development
in Drosophila when she joined the lab of M. Wilcox
at the LMB in Cambridge, UK, for her postdoctoral
work on Drosophila integrins. After a research visit
at the lab of Pat O'Farrell at UCSF, where she began
her work on gastrulation, she spent the years from
1989 to 1994 as a group leader at the Max Planck
Institut in Tübingen. She has been at the Institute of
Genetics since 1994
Group members
Magdalena Baer, Graduate
Student
Andreas Bilstein, Graduate
Student
Agnes Csiszar, Graduate Student
Verena Kölsch, Graduate
Student
Sam Jacob Mathew, Graduate
Student
Min-Yan Zhu, Graduate Student
Thomas Seher, Postdoc
Robert Wilson, Postdoc
Juliane Hancke, Technician
Lisa Vogelsang, Technician
Klaus Reiners, Department
Manager
Mechanisms of Morphogenesis
Morphogenesis, the shaping of structures in a developing organism, is an aspect of
differentiation that, like other differentiation events, is controlled by transcription factors
and involves a variety of subcellular effector mechanisms. The first major morphogenetic
event in the Drosophila embryo, an epithelial folding that leads to the invagination of the
mesoderm, depends on two transcription factors, Snail and Twist. Many of their target
genes are known, but those responsible for mediating the cellular changes during the
invagination of the mesoderm have not been identified. One aim of our work is to establish
the hierarchy of regulatory events downstream of Twist and Snail to the effector molecules
that mediate morphogenesis.
A different type of morphogenetic process, the branching of cells, occurs during the
development of the tracheal system which delivers oxygen to all cells of the body. The
branched network of the tracheal system in the embryo initially develops according to a
stereotypic programme. Later, during larval life, tracheal cells respond to the need for
oxygen in the surrounding tissue by sending out long protrusions towards oxygen-starved
cells. Both the embryonic and the larval events are controlled by the Drosophila FGFhomolog, Branchless. We are investigating how the subcellular events downstream of
FGF signalling contribute to branch formation and what other mechanisms are involved
in the localized outgrowth of branches.
50
Maria Leptin
Genetic hierarchy of early mesoderm
development
Many transcription factors are known that are
responsible for the differentation and
morphogenesis of organs or regions of developing
organisms. However, in no case have the genetic
cascades that control morphogenesis downstream
of the transcription factors been fully elucidated.
We have been studying the formation of the ventral
furrow and the subsequent invagination of the
mesoderm in the Drosophila embryo as an example
of morphogenesis in which the first steps of the
genetic regulatory cascde are extremely well
understood. The mesoderm arises from a region
covering most of the ventral side of the embryo
which is set up under the control of the dorso-ventral patterning system of the embryo. The
accumulation of high levels of the transcription
factor Dorsal in nuclei on the ventral side of the
embryo leads to the transcription of two genes,
snail and twist, which together define the
mesoderm primordium and are essential for all
aspects of its differentiation and morphogenesis.
Snail, a zinc-finger protein, acts mainly as a
repressor of ectodermal genes in the region of the
prospective mesoderm. Twist, a bHLH-protein,
acts as an activator for mesodermal genes. Loss of
either factor leads to specific defects in mesoderm
development, loss of both to the absence of any
mesodermal differentiation or morphogenesis.
No single gene has been found whose loss leads
to phenotypes resembling those of either twist or
snail. However, loss of function of one known
target gene of Snail and Twist, folded gastrulation, causes moderate defects in mesoderm
invagination. We have identified four regions in
the genome whose loss results in similarly subtle
and transient mutant phenotypes.
For two of these, the genes responsible for the
loss of function phenotypes have been identified.
Both are expressed in the mesoderm primordium
and are involved in controlling the cell division cycle
in the mesoderm. In their absence, premature
mitoses interfere with the cell shape changes that
drive the invagination. We are in the process of
identifying the remaining two genes.
Fig. 1. The top panel shows a cross section through an
embryo that has just begun to gastrulate. The ventral
cells express the transcription factor Twist (visualized
in brown), which controls the differentiation and
morphogenesis of the mesoderm. The bottom panels
show possible regulatory cascades downstream of the
mesodermal transcriptional regulators Twist and Snail.
The embryo is approx. 0.2 mm in diameter. A: the simplest
possible cascade: one single gene downstream of Snail
and Twist is responsible for the morphogenetic activity.
B. a more likely scenario, consistent with our results:
the main genes controlling ventral furrow formation
downstream of Twist are snail and fog, with the Snail
acting primarily to prevenet ectoderm-specific gene
expression in the mesoderm primodium.
51
Maria Leptin
In a separate approach, we have analysed the
genetic cascade immediately downstream of Twist
by testing which of its targets are sufficient to mediate aspects of the morphogenetic activities that
depend on Twist. We have shown that in twist
mutant embryos the ability of cells to undergo cell
shape changes is restored when Snail and Fog are
expressed in the mesoderm, but with a considerable
delay compared to the wildtype. Thus, Twist acts
mainly through its targets snail and fog to induce
cell shape changes, but other targets are necessary
for the timing of these changes.
FGF signalling mechanisms in the tracheal system
The development of the tracheal system depends
on the activity of the Drosophila FGF-receptor
Breathless. FGF directs the migrating cells in the
embryo and the extension of fine branches of individual tracheal cells in the larva towards cells
expressing the FGF-homolog Branchless. FGF
receptors are receptor tyrosine kinases (RTK) that
activate the MAPK cascade via the small GTPase
Ras. However, unlike other RTKs, FGF receptors
do not interact directly with the Grb2/sos complex
that activates Ras. We have found a cytoplasmic
molecule, Dof, that is essential for the transmission
of the FGF signal. Dof acts downstream of the
FGF receptor (hence the name) and upstream of
the Ras and the MAPK cascade. It does not
resemble other adaptor or linker proteins in that it
lacks structural domains, such as SH2 or PTB
domains, typically found in molecules involved in
signal transduction. Dof’s potential protein
interaction domains are a coiled coil region and an
ankyrin repeat. Neither of these are essential for
function, but we have recently found that a new
protein interaction motif, the DBB domain, shared
by Dof with two vertebrate signalling proteins from
B lymphocytes (BANK and BCAP) is required
for Dof’s interaction with the FGF receptor and
for Dof function in vivo.
Dof interacts with a large number of proteins,
including the SUMOylation enzymes Ubc9/semi and
PIASy/Su(var)2-10. Dof’s DBB domain is also
required for this interaction, and it indeed contains
a consensus site for SUMOylation, whose
modification results in loss of biological function of
52
Fig. 2. Top: a nearly mature embryo in which the
tracheal system has been stained. Bottom: a tracheal cell in the third instar larva, visualized by its
GFP expression. The scale bar represents 0.1 mm for
both images.
the protein. Together these findings are a strong
indication that SUMOylation of Dof affects FGF
signal transmission. In addition, Dof is
phosphorylated upon receptor activation, and may
be proteolytically cleaved. How these
posttranslational modifications affect the activation
of Dof, the recruitment of other proteins, and the
transmission of the signal from the receptor to its
cellular targets is currently being investigated.
Genetic screens are being used to identify other
components involved in tracheal branch
outgrowth.
Maria Leptin
Selected Publications
Vincent, S., Wilson, R., Coelho, C., Affolter, M.,
Leptin, M. (1998) Drosophila dof, a protein
required specifically for FGF signalling.
Molecular Cell 2, 515-525.
Leptin, M. (1999) Gastrulation in Drosophila: The
logic and the cellular mechanisms. EMBO J. 18,
3187-3192.
Irion, U., Leptin, M. (1999) Developmental and cell
biological functions of the Drosophila DEADbox protein. Abstrakt Current Biology 9, 13731381.
Wilson, R., Leptin, M. (2000) FGF-receptor
dependent morphogenesis of the Drosophila
mesoderm. Phil. Trans. R. Soc. 355, 891-895.
Seher, T., Leptin, M. (2000) Tribbles, a cell cycle
brake that coordinates proliferation and
morphogenesis during Drosophila gastrulation.
Current Biology 10, 623-629.
Frasch, M., Leptin, M. (2000) Mergers and
acquisitions: unequal partnerships in
Drosophila myoblast fusion.. Cell 102, 1-3.
Battersby, A., Csiszar, A., Leptin, M., Wilson, R.
(2003) Isolation of proteins that interact with
the signal transduction molecule Dof and
identification of a functional domain conserved
between Dof and vertebrate BCAP. J. Mol. Biol.
329, 479-493.
53
Benno Müller-Hill
Professor emeritus
Benno Müller-Hill received his PhD 1962 in
the laboratory of Chemistry at the University
of Freiburg i.B. He worked then as a postdoc
with Howard Rickenberg in the Department
of Microbiology of Indiana University in
Bloomington. He moved from there to the
Watson-Gilbert lab in the Biolabs of Harvard
University. There in 1966 he isolated with
Wally Gilbert the first transcription factor, Lac
repressor of E.coli. In 1968 he became full
professor at the Genetics Institute of Cologne
University. There much of his work went into
the lac system of E.coli. He formally retired in
1998.
Group members
Steffi Spott, Graduate Student
Laurence Mangel, Postdoc
Christoph Rudolph, Postdoc
Gerd Wronka, Postdoc
From Lac Repressor to the History of Genetics
Lac repressor and lac operator have been in the center of my interest since 1960 when
they were predicted by Francois Jacob and Jacques Monod. In 1965 I isolated two nonsense mutants in the lac I gene and thus proved that Lac repressor is a protein. One year
later Wally Gilbert and I isolated Lac repressor. In 1968 I isolated a mutant strain which
overproduced Lac repressor. In Cologne we isolated 11 g Lac repressor and sequenced it
1972. In 1990 we demonstrated the function of the auxiliary operators O2 and O3:
tetrameric Lac repressor makes loops with O1 and either O2 or O3. We showed which
amino acid recognizes which base of the lac operator. 2000 we changed the specificity of
dimerisation. Again 2000 we isolated a Lac repressor mutant, K84L, which is fourty (!)
degrees more stable than wild type repressor. Thus in the last three years of my activity in
a lab I concentrated on one object: Lac Repressor. In particular, we tried to make out of
Lac repressor a ß-galactosidase. So far we failed.
This is not all. In the last twenty years I have been interested in the (bad) history of human
genetics, particularly in Germany. In particular I investigated the connection of Mengele
- von Verschuer - Hillmann - Butenandt.
54
Benno Müller-Hill
Trying to understand Lac repressor
Lac repressor is one of the best analysed proteins.
More than 4000 mutants have been analysed and
a X ray structure is available. Does one understand
Lac repressor? One understands it, if one can make
testable predictions about mutant Lac repressors.
We tried three experiments and failed so far.
1. We tried to make a pair of LacR mutants which
would form specific heterodimers i.e. mutants
which make no homodimers but when mixed
together functional heterodimers. We used
exchanges in residue 251 and 278, - sofar without
success.
2. We tried to get LacR mutants where lac
repressor works as an activator when bound
upstream of a weak operator. We constructed E.coli
strains which carried on the chromosome a lac
operon in which the Cap site, O3 and O2 were
destroyed in which a lac operator was introduced
at position 40 or 60. We then mutagenised a
plasmid carrying a lac I gene and selected for Lac+
colonies in this background. So far we did not find
any lac+ mutant in the lacI gene.
3. We tried to get a mutant in the lacI gene which
converts lac repressor into a ß-galactosidase. I
recall: Lac repressor binds methyl-ß-D-galactoside
tightly. Is it possible to get a mutant lac repressor
which hydrolyses methyl-ß-D-galactoside? We
used as a background a mutant E.coli strain which
carries deletion of the lac and of the ebg operon.
We tried nitrosoguanidine mutagenesis, without
success. We tried to construct LacR mutants shich
carry in all 25 Is positions Glutamic acid. We are
still in the midst of these experiments, which when
working seem to us possibly answering a fundamental question: What is the difference of a binding
protein and an enzyme?
Reflections on the past and future of human
genetics
Twenty years ago I discovered that history of human genetics in Germany 1933 - 1945 was not
written. Nobody had ever looked at the files of the
Deutsche Forschungsgemeinschaft (DFG) which
are conserved in the Bundesarchiv in Koblenz.
There I found evidence that the DFG had paid a
project of Otmar von Verschuer, then director of
the Kaiser Wilhelm-Institute of Anthropology, Human Genetics and Eugenics in Berlin-Dahlem, were
the former postdoc of Verschuer, Josef Mengele
collected „material“ in Auschwitz. A graduate
student of Adolf Butenandt, Günther Hillmann, was
involved in the project. How much did Butenandt
know about this? It took years until my report was
appreciated by the German scientific community.
January 2001 I got access to the Butenandt-Nachlass in the Max-Planck Archiv in Berlin. A report
on this appeared.
Selected Publications
Müller-Hill, B. (2002) What can be learned from the
lacR family of Escherichia coli? In: Signal
Switches, Regulons and Cascades. Ed. by D.
A. Hodgson and C. M. Thomas. Cambridge
University Press, pp. 143-154.
Oehler, S., Müller, J., Alberti, S. (2003) Induction of
the lac promoter in the absence of DNA loops.
In preparation.
Müller-Hill, B. (2002) Murderous Science: a
Conversation with Benno Müller-Hill. In Istvan
Hargittai: Candid Science II. Conversations
with Famous Biomedical Scientists. Imperial
College Press, London, pp. 114-129.
Müller-Hill, B. (2002) Human Behavioral Genetics past and present. J. Mol. Biol. 319, 927-929.
Müller-Hill, B. (2003) Erinnerung und Ausblendung:
Ein kritischer Blick in den Briefwechsel Adolf
Butenandts MPG Präsident 1960-1972.
Hist.Phil.Life Sciences, in press.
English version: Comprehensive Biochemistry 42,
548-579.
Müller-Hill, B. (2003) My happy days with Lac
repressor - in a dark world. In: Selected Topics
in the History of Biochemistry: Personal
Recollections VII (Comprehensive Biochemistry, vol. 42). Ed. by G. Semenza and A. J.
Turner. Elsevier Science B.V., pp. 447-499.
55
Karin Schnetz
Professor
Karin Schnetz received her PhD in Freiburg in 1988,
where she studied the Escherichia coli aryl-â,Dglucoside operon in the laboratory of Bodo Rak at
the Institute for Biology III, University Freiburg. As
a Post-Doc she began to work on silencing of the
bgl operon. This project was continued in the
laboratory of James C. Wang, Harvard University
(1992-1995), and later with an independent group in
Freiburg. In 1997 she joined the Institute for
Genetics, University of Cologne.
Group members
Girish Neelakanta, PhD
Student
Andreas Paukner, PhD
Student
Madhusudan Srinivasan, PhD
Student
Nagarajavel Vivekananthan,
PhD Student
Sandra Kühn, Technician
Doris Renz., Technician
Pleiotropic Regulation in E. coli
The adaptation of enterobacteriaceae to changes in their environment is controlled by a
network of pleiotropic regulators. A key regulator in this control is the histone-like nucleoid
structuring protein H-NS. Specific regulation by H-NS and also by other pleiotropic factors
is poorly understood. We use the E.coli ß-glucoside (bgl) operon to address the question
of pleiotropic control. Our goals are to understand the mechanism(s) of repression by HNS, and the mechanism of regulation by other pleiotropic factors, as well as the operon
specific regulatory control mechanisms that amplify and integrate the impact of pleiotropic
regulators. In a second line of experiments we analyze the bgl operon and its pleiotropic
control in commensal and pathogenic E.coli. This approach relates to the unanswered
question of what makes a strain a pathogen and to the mosaic organization of the E.coli
genome.
56
Karin Schnetz
Pleiotropic control of bgl
The response of E.coli to external stimuli, such as
stress conditions, nutritional up- or downshifts, and
temperature shifts as well as the adjustment of its
cellular physiology to permanent changes in the
external conditions are controlled by a network of
pleiotropic regulators. Each of these regulators
controls many genes, and the specific response to
certain stress conditions is frequently controlled by
combinations of varying subsets of the pleiotropic
regulators. In addition, the pleiotropic regulators
cross-regulate each other, which presumably is a
prerequisite for differences in fast and delayed
responses.
The E.coli bgl operon encoding the gene
products for the fermentation of aryl-β,Dglucosides is one example of a pleiotropically
controlled system. This operon is not expressed
under laboratory growth conditions (in vitro). It is
kept silent by the histone-like nucleoid structuring
protein H-NS. In addition to H-NS, other pleiotropic regulators contribute to bgl operon control.
Why the bgl operon is silent in vitro, why it is
subject to pleiotropic control, which conditions
induce it, and its biological function beyond aryl-β,
D-glucoside fermentation are open questions.
Repression by H-NS
The highly abundant H-NS protein represses ~5%
of the genes in E.coli. Many of these genes,
including several pathogen determinants, are
induced by environmental signals. It is assumed that
repression by H-NS is mediated by the weakly
specific binding of H-NS to AT-rich and bent DNA,
and subsequent oligomerization of H-NS on the
DNA resulting in repression of transcription
initiation.
Repression of the bgl operon by H-NS is
exceptionally efficient and occurs at two levels
(Fig. 1). An AT-rich and bent silencer sequence
located upstream of the promoter is essential for
the repression of transcription initiation by H-NS.
Mutations that destroy or disrupt the silencer lead
to activation of the operon. In addition to the
repression of the promoter, H-NS acts
downstream, where it induces polarity within the
first gene of the operon (Fig. 1). In general polarity
is the result of a pause in transcription elongation
allowing termination factor Rho to catch up with
RNA polymerase and consequently to terminate
Fig. 1. Pleiotropic regulation of the bgl operon and integration and amplification of multiple inputs by the specific
regulator BglG.
57
Karin Schnetz
transcription. Our current model is that H-NS acts
as a road-block to the elongating RNA polymerase,
and our main focus is to clarify the mechanism of
this novel mode of repression by H-NS.
Other pleiotropic regulators
In addition to H-NS the bgl operon is regulated
by other pleiotropic regulators. The stress sigma
factor RpoS and its co-activator Crl contribute to
repression of the catabolite gene regulator protein
(CRP)–dependent promoter. CRP binding is
antagonistically controlled by DNA bending protein
FIS. The pleiotropic transcription factor LeuO and
the conserved putative transcription factors YjjQ
and BglJ also regulate the bgl promoter. The
downstream repression by H-NS, is reduced by
the protease Lon and the RNA-binding protein Hfq.
Current projects concern the elucidation of the
molecular mechanisms of regulation by these
pleiotropic factors.
Amplification of multiple inputs
The complex pleiotropic regulation of the bgl
operon generates multiple inputs, none of which is
very strong. H-NS which reduces expression of
the operon ~100 fold represses the promoter
~3fold and further decreases expression ~7fold by
causing polarity of transcription. The other factors
regulate the operon 2 to 3fold. We have shown that
these rather moderate effects are integrated and
amplified due to the inherent mechanism of specific
control by the operon encoded positive regulator
BglG. BglG is an antiterminator protein, that is
controlled by the general sugar transport system,
PTS. BglG is encoded within the operon and thus
positively auto-regulates its expression. At low
transcription rates BglG is limiting, and the operon
is hardly at all expressed. However, once a
threshold is reached BglG efficiently stimulates
expression of the operon. By this mechanism moderate (2 to 3fold) changes of the transcription rate
can be amplified into high (50 to 100fold) changes
of the expression level of the operon.
58
The bgl operon in commensal and
pathogenic E.coli
The biological meaning of silencing of the bgl operon
is an open question. To determine whether bgl
operon silencing in the laboratory E.coli strain K12 resembles bgl operon control in commensal and
pathogenic E.coli strains we analyzed a collection
of commensal, septicemic, and uropathogenic
E.coli strains for the presence of the bgl operon,
for the sequence of the regulatory region, and for
their aryl-ß,D-glucoside phenotype. Close to 60%
of the strains carry an operon which is silent under
laboratory growth conditions as in K-12, while in
~15% of the strains silencing of the operon is less
strict (‘relaxed’). Interestingly, for one of these ‘relaxed’ phenotype strains, a septicemic E.coli, it was
reported that the bgl operon is expressed in vivo
(when this strain infects mouse liver). A quarter of
the strains we analyzed carries no or a nonfunctional bgl operon. Correspondingly, the bgl
operon is present in only two of the three strains of
which the genome sequence has been published
(in K-12 and the uropathogenic strain CFT073,
but not in the enterohaemmorrhagic strain O157).
The sequences of these strains revealed that the
E.coli genome has a mosaic structure. The three
strains share 2/3 of their genome, while 1/6 of the
genes are shared with only one of the other strains,
and the remainder of genes is unique in each strain.
In our analysis no E.coli strain carried an operon
which is expressed at high levels in vitro
demonstrating that bgl operon silencing is
conserved. Why the bgl operon is conserved in its
silenced state is unknown, and this question may
not be easily answered. Possibly non-silenced
expression of the operon is disadvantageous e.g.
due to toxic aryl-ß,D-glucosides, while its silencing
may be relieved specifically under certain
conditions.
Karin Schnetz
Selected Publications
Caramel, A., Schnetz, K. (1998) Lac and Lambda
repressor relieve silencing of the Escherichia
coli bgl promoter. Activation by alteration of a
repressing nucleoprotein complex. J. Mol. Biol.
284, 875-883.
Caramel, A., Schnetz, K. (2000) Antagonistic control
of the E. coli bgl promoter by FIS and CAP in
vitro. Mol. Microbiol. 36, 85-92
Dole, S., Klingen, Y., Nagarajavel, V., Schnetz, K.
Protease Lon and RNA-binding protein Hfq
counteract silencing of the E.coli bgl operon.
(submitted).
Dole, S., Kühn, S., Schnetz, K. (2002) Posttranscriptional enhancement of Escherichia coli
bgl operon silencing by limitation of BglGmediated antitermination at low transcription
rates. Mol. Microbiol. 43, 217-226.
Dole, S., Nagarajavel, V., Schnetz, K. The histonelike nucleoid structuring protein H-NS
represses the Escherichia coli bgl operon
downstream of the promoter (submitted).
59
Frank Sprenger
Group Leader
Frank Sprenger received his PhD in Tübingen,
1991, where he uncovered at the Max-PlanckInstitute in lab of C.Nüsslein-Volhard the
molecular nature of the torso gene and its
implication for the early patterning system in
Drosophila. He then joined the lab of
P.O'Farrell at the University of San Francisco.
Here, he began his studies on cell cycle control
using Drosophila as a model system. In 1996
he moved to the University of Cologne as an
independent group leader. His work has
focused on cell cycle regulation during Drosophila development, mainly on mechanisms that
ensure correct entry into and exit from mitosis.
Group members
Axel Dienemann, PhD
Student
Norma Zielke, PhD Student
Heidi Thelen, Technician
Cell Cycle Control in Drosophila
We are using Drosophila as a model system to study fundamental aspects of cell cycle
control and its integration with the developmental program. We are especially interested
in the mechanisms that control entry into and exit from mitosis. Cyclin destruction is a
necessary step to exit mitosis and the mechanisms involved are well understood for Btype cyclins. In contrast, little is known about the turnover of Cyclin A during the cell
cycle. Inhibitory molecules can also mediate Cdk1 inactivation during mitosis. In Drosophila, the rux gene product performs such a function. Rux is also required for meiosis
in males, a process we would like to understand from the cell cycle perspective. Mitotic
cyclin destruction is important for mitotic exit and the establishment of the G1-state,
but it must be prevented in other cell cycle stages to allow accumulation of mitotic
cyclins. We have identified the rca1 gene product as an essential inhibitor of cyclin
degradation and have started to analyze its requirements during the cell cycle and
during development. Overall; our results will extend the knowledge about proliferation
control and its regulation during development.
60
Frank Sprenger
Turnover of Cyclin A
Mitotic cyclins are required to produce mitosis
promoting factor, MPF, which is composed of the
kinase Cdk1, and a cyclin subunit. Exit from mitosis
requires MPF inactivation that is mediated by cyclin
degradation. Cyclin destruction is well understood
for B-type cyclins. These have a so-called
destruction-box (D-box) that is recognized by
adaptors of the Anaphase-Promoting-Complex/
Cyclosome (APC/C), an ubiquitin ligase that
catalyzes poly-ubiquitinylation of substrates and
thereby targets cyclins for destruction in the proteasome.
In contrast to Cyclin B, we do not understand
how Cyclin A is targeted for destruction during
metaphase (see Fig. 1), and how its turnover rate
is modified at other cell cycle stages. Cyclin A has
recognizable destruction elements, but destruction
could not be prevented when these destruction
elements were inactivated or deleted. The
destruction is also not affected when the spindle
checkpoint is activated. In such a situation, most
targets of the APC/C are stabilized. We are now
using a combination of molecular and biochemical
techniques to identify destruction elements in Drosophila Cyclin A and will try to transfer this
information into the vertebrate system as well where
Cyclin A destruction is also not understood.
Cyclin A subcellular localization
Cyclin A undergoes a dynamic subcellular
localization during the cell cycle. It is cytoplasmic
Fig 1. Cyclin A localization and degradation during
the cell cycle. Cyclin A is cytoplasmatic during
interphase, accumulates in the nucleus at prophase and
starts to be degraded during metaphase. Mitotic stages
are visualized by staining for phosphorylated histone
H3 (PH3) in blue, Cyclin A is in green.
during interphase, accumulates during prophase in
the nucleus and is homogenously distributed after
nuclear envelope breakdown. Treating cells with
the drug LMB, which inhibits nuclear export, results
in nuclear accumulation of Cyclin A during
interphase. This indicates that Cyclin A shuttles
between the nucleus and the cytoplasm and that
nuclear export prevails during interphase. This
behaviour is similar to that of human Cyclin B1.
We have started to address the importance of the
prophase accumulation of Drosophila Cyclin A by
constructing versions of Cyclin A that have an altered subcellular localization. Surprisingly, all
constructs, including a membrane-anchored
version, were able to induce mitosis. This indicates
that nuclear accumulation is not necessary for
mitosis and that Cyclin A can perform its necessary
function also in the cytoplasm.
Cell cycle function of Rux
Rux is a cyclin dependent kinase inhibitor (CKI).
This class of molecules binds to Cyclin/Cdk
complexes and inhibit their kinase activity. Rux
specifically inhibits Cdk1 activity. rux mutants are
male sterile and have rough eyes. During eye
development, Rux is required for the establishment
of a critical G1-phase. In mutant males, meiosis I
and II occur normally, but then a third meiotic
division is initiated that finally results in sterility.
The sterility and eye phenotypes are observed
when weak rux are alleles were used. Strong
mutations are semi-lethal indicating that the gene
product has extra functions external to eye
development and meiosis. One of these functions
can be observed already during cell cycle 14. Here,
the inhibitory effect of Rux on mitotic cyclins/Cdk1
complexes is apparently involved during exit from
mitosis. In rux mutants metaphase is significantly
longer (Fig. 2). This is the first mitotic function
ascribed to a CKI in a multicellular organism and
indicates the existence of a regulatory mechanism
for the metaphase to anaphase transition that might
also exist in other metazoans.
61
Frank Sprenger
development of an organism. So far, our results
have put important new players into cell cycle game.
Our future program will tell us how these players
are stirred throughout the standard as well as
modified cell cycles.
Fig. 2. Metaphase is prolonged in rux mutants. The
duration of mitotic stages (in sec) was determined using
live imaging using GFP-Histone transgenic flies (GFPHis). Metaphase is significantly prolonged in rux mutants
(blue bars) compared to normal embryos (green bars).
Restricting cyclin degradation
Cyclin degradation during the cell cycle is restricted
to mitotic stages and G1. Cyclin degradation is
initiated by the APC/C and the association with
adaptor molecules regulates its activity. We could
identify the Rca1 gene product as an inhibitor of
the APC/C necessary to prevent cyclin degradation
during G2. This function is required at different
developmental stages. During embryogenesis its
requirement are during the 16th cell cycle. In the
absence of rca1 function, cyclins are degraded
prematurely in G2 cells and fail to enter mitosis.
We could show on a genetic as well as biochemical
level that rca1 binds to one of the APC/C adaptors,
Fizzy-related, and can inhibit APC/C activty. This
function of rca1 is also required at later stages of
development where it is required for cell proloferation for example during imaginal disk
development (Fig. 3). We are currently investigating
how Rca1 can inhibit the APC/C and how its
activity is regulated during the cell cycle and during
development.
Future prospects
Studying cell cycle control in Drosophila complements cell cycle studies in other organisms. A good
collection of cell cycle genes, excellent cell biological
tools and a powerful combination of molecular tools
allow for an efficient research. We can also study
the integration integration of the cell cycle with
62
Fig. 3. In rca1 mutant cells, mitotic cyclins are degraded
prematurely and consequently cells fail to proliferate.
Wing imaginal discs are shown in which control clones
of cells (right) or rca1 mutant cells (left) were induced
by the flp-FRT technique. Clones are recognized by the
absence of the GFP marker. Sister clones contain a higher
GFP signal. The rca1 mutant cells have low Cyclin A
levels (in red) and are smaller than their sister clones.
Frank Sprenger
Selected Publications
Foley, E., O'Farrell, P.H., Sprenger, F. (1999) Rux is a
cyclin-dependent kinase inhibitor (CKI)
specific for mitotic cyclin-Cdk complexes. Curr.
Biol. 9, 1392-1402.
Foley, E., Sprenger, F. (2000) Cyclins: Growing pains
for Drosophila. Curr. Biol. 10, R665-R667.
Kaspar, M., Dienemann, A., Schulze, C., Sprenger,
F. (2001) Mitotic degradation of Cyclin A is
mediated by multiple and novel destruction
signals. Curr. Biol. 11, 685-690.
Foley, E., Sprenger, F. (2001) Rux contributes to
mitotic exit in Drosophila. Curr. Biol. 11, 151160.
Grosskortenhaus, R., Sprenger, F. (2002) Rca1
inhibits APC-Cdh1Fzr and is required to prevent
Cyclin degradation in G2. Developmental Cell
2, 29-40.
63
Diethard Tautz
Professor
Diethard Tautz received his PhD at the EMBL
Heidelberg in 1983, where he worked on
characterizing simple sequences (microsatellites). He went then as a postdoc first to
Cambridge, UK with Gabriel Dover at the
Dept. of Genetics to work on molecular
evolution. Afterwards he joined the laboratory
of Herbert Jäckle (MPI Tübingen) to work on
Drosophila segmentation - cloning and
characterization of hunchback. Together with
Herbert Jäckle he moved to Munich in 1988,
where he started as independent group leader
in the Dept. of Genetics. In 1991 he became
C3 Prof. at the Dept. of Zoology in Munich
and in 1998 he joined the Dept. of Genetics in
Cologne as C4 professor.
Group members
Manuel Aranda Lastra, PhD
Student
Carmen Czepe, PhD Student
Tomislav Domazet-Loso, PhD
Student
Sonja Ihle, PhD Student
Arne Nolte, PhD Student
Alexander Pozhitkov, PhD
Student
Niko Prpic, PhD Student
Joel Savard, PhD Student
Sunita Shankaran, PhD Student
Dirk Sieger, PhD Student
Sebastian Steinfartz, PhD
Student
Meike Thomas, PhD Student
Chriz Voolstra, PhD Student
Martin Gajewski, Postdoc
Röbbe Wünschiers, Postdoc
Susanne Krächter, Technician
Vladimir Simovic, Technician
Irene Steinfartz, Technician
Eva Siegmund, Secretary
Heidi Fußwinkel, Department
Manager
Molecular Evolution
The group works on a number of different projects in the general field of molecular
evolution. One type of projects deals with understanding the evolution of developmental
processes. The focus is on the developmental analysis of segmentation mechanisms both
in arthropods and in vertebrates in order to allow an evolutionary comparison of the
basic molecular principles of segmentation. The major model systems are the fly Drosophila, the beetle Tribolium and the zebrafish Danio rerio. The second type of projects
deals with understanding the evolution of adaptive traits in the context of speciation. We
follow several approaches that aim to identify genes involved in adaptations and to analyse
their fate in natural populations. The current model systems are Drosophila, Mus musculus,
Cottus and Salamandra.
64
Diethard Tautz
Segmentation in insects
The molecular basis of segmentation has been
intensively studied in Drosophila. A crucial
component are the pair-rule genes, which relay the
spatial information from the transcription factor
gradients of the gap genes to the segment-polarity
genes that generate the segmental borders. Because
the regulation of the pair-rule genes occurs via
stripe-specific enhancer elements in the syncytial
blastoderm of Drosophila, it was for some time
assumed that pair-rule genes are a relatively late
evolutionary invention. However, it is now clear that
they occur throughout the arthropods and their
function is not restricted to syncytial stages. We
are using the flour beetle Tribolium as a genetically
accessible model system that represents the
ancestral form of arthropod segmentation.
Using reportergene constructs in germline
transformation experiments, we can indeed show
that the enhancers driving individual pairrule stripes
are functional both in a syncytial as well as in an
cellular environment (Fig. 1). Further work will be
required to elucidate the molecular basis for this
conserved regulation.
However, these genes are part of a completely
different regulatory circuit, namely the Delta/Notch
signalling pathway. This would suggest that there is
no common evolutionary origin between arthropod
and vertebrate segmentation. On the other hand,
the somitogenesis process is not yet fully
understood. It appears that additional signalling
pathways are involved, which might turn out to show
more similarity to the arthropod pattern (compare
also results of Wim Damen´s group in the spider).
Our strategy is therefore to elucidate the different
molecular components that are involved in
generating the dynamic expression pattern of the
her1/her7 genes in the presomitic mesoderm of
zebrafish. This work involves mainly classical
developmental studies, such as gene knockout,
over-expression analysis and elucidation of
regulatory interactions using reportergene
constructs in transgenic lines. In addition, we try to
identify novel candidate genes by microarray
experiments (see „Microarray-Facility“).
Orphan gene evolution
Orphan genes are protein coding genes that have
no recognizable homologue in distantly related
species. However, they can readily be traced in
closely related species and their evolutionary
characteristics can therefore be analysed. For this
purpose we have done a comparative EST project
between D. melanogaster and D. yakuba. We
Fig. 1. Transgenic Tribolium embryo (left) and transgenic
Drosophila embryo (right) carrying the same
reportergene construct with regulatory sequences from
the Tribolium pair-rule gene hairy.The mechanism of
stripe generation is at least partly conserved between
these species.
Somitogenesis in zebrafish
Somite formation in vertebrates resembles
superficially the segmentation process in shortgerm
insects, such as Tribolium. Moreover, two
homologues of the pair-rule gene hairy are also
required in generating the somites, namely her1 and
her7 (Fig. 2).
Fig. 2. Expression of the bHLH genes her1 (left) and
her7 (middle) in the presomitic mesoderm of zebrafish,
as monitored by two-colour fluorescent in situ
hybridisation. Both genes are expressed in dynamically
cycling stripes, but are always co-expressed in the same
stripes (right).
65
Diethard Tautz
found that many orphans are fast evolving genes,
which could explain their divergence, However,
some orphans are evolving only very slow, as slow
as otherwise highly conserved genes. We propose
that these genes might have been fast evolving
genes which have become locked in an important
functional circuit that is particularly relevant for the
clade in which they are now evolving slowly (Fig.
3).
Selective sweeps in mouse
Identifying genes that are directly involved in
specific adaptations has so far only been possible
in very special circumstances. We have now started
to systematically search for such loci, by taking
advantage of the evolutionary signature that they
leave in the genomes of natural populations. Any
genomic region that is under positive selection
because of a recent or still ongoing adaptation will
loose polymorphism, because the advantageous
slow evolution due to
conservation of function
fast evolution due to
strong positive selection
acquisition of
clock-like slow
lineage specific function
evolution
fast evolution due to
reduced selective constraints
Fig. 4. Susanne Krächter (left) and Sonja Ihle (right)
during field expeditions to catch wild mice. The sampling
scheme is designed such that only unrelated individuals
are included from a sampling area of up to 50 km in
diameter. The mice are dissected in the field and only
the DNA is used at present. Samples were obtained from
Kazhachstan, Czech Republik, Bonn, Montpellier and
Cameroon.
allele is fixed on the expense of the neutral alleles
at the locus. Such regions can be identified with
the help of neutral markers, for example
microsatellites or SNPs. We are studying five wild
populations of the house mouse (Mus musculus
and Mus domesticus) from different areas of the
world with up to 50 unrelated individuals from each
population (Fig. 4). We plan to screen up to 3000
markers across the genome, to obtain a
comprehensive survey of genes involved in
adaptations. Candidate genes will be selected on
the basis of a significant population specific loss of
heterozygosity. We expect that this project will yield
new insights into the genetics of adaptation and
speciation.
initial gene duplication
Fig. 3. Model for the evolution of orphan genes. This
model assumes an ancestral duplication for the origin
of a new gene that can diverge more or less freely, because
of relaxed constraints (left), while the original gene
keeps its function and evolves slowly (right). When the
fast evolving gene becomes locked into a new pathway
in a particular subclade, we expect fast molecular
adaptation, followed by a phase of slow evolution. Slow
evolving orphan genes may therefore be excellent
indicators for genes involved in generating lineage
specific characters.
66
Speciation
Speciation creates evolutionary novelties and it is
therefaore necessary to better understand the
process of speciation. We are studying two model
systems which provide direct insights into ongoing
speciation processes (Fig. 5).
One are fire salamanders (Salamandra
salamandra) and the other is a fish species in the
rhine system - the bullhead (Cottus gobio). In both
cases we have identified very recent cases of
specific environmental adaptations which are likely
to have occurred in the context of speciation. We
Diethard Tautz
Selected Publications
Gajewski, M., Sieger, D., Alt, B., Leve, C., Hans, S.,
Wolff, C., Rohr, K., Tautz, D. (2003) Anterior
and posterior waves of cyclic her1 gene
expression are differentially regulated in the
presomitic mesoderm of zebrafish. Development
130, 4269-4278.
Domazet, T., Tautz, D. (2003). An evolutionary
analysis of orphan genes in Drosophila.
Genome Research (in press).
Tautz, D. (2000) Evolution of transcriptional
regulation. Curr. Opin. Gen. Dev. 10, 575-579.
Schröder R., Eckert, C, Wolff, C., Tautz, D. (2000)
Conserved and divergent aspects of terminal
patterning in the beetle Tribolium castaneum.
Proc. Natl. Acad. Sci. USA 97, 6591-6596.
Fig 5. Pictures of our model systems for studying
speciation processes. Top: Salamanders from the
Kottenforst near Bonn. Bottom: bullhead from
Rhine tributaries.
are studying the animals in the field, in hybrid zones
and under laboratory controlled conditions. The
work on Cottus involves in addition the generation
of an F2 backcross panel to map the genes causing
the specific adaptations. We expect that this work
will yield insights into the process of speciation and
the genetics of speciation genes.
Steinfartz, S., Veith, M., Tautz, D. (2000)
Mitochondrial sequence analysis of
Salamandra taxa suggests old splits of major
lineages and postglacial recolonizations of
Central Europe from distinct source
populations of S. salamandra. Molecular
Ecology 9, 397-410.
Englbrecht, C., Freyhof, J., Nolte, A., Rassmann,
K., Schliewen, U., Tautz, D. (2000)
Phylogeography of the bullhead Cottus gobio
(Pisces: Teleostei: Cottidae) suggests a prePleistocene origin of the major central european
populations. Molecular Ecology 9, 709-722.
67
Ari Waisman
Group Leader
Ari Waisman recieved his Ph.D. at the
Weizmann Institute for Science in Rehovot at
1994 where he worked with Edna Mozes on a
mouse model for SLE. He then did his first
postdoc with Lawrence Steinman, also in
Rehovot, where he worked on EAE, which is
the mouse model for multiple sclerosis. In this
project he protected mice from brain
inflammation by DNA vaccination. In late 1996
he moved to the Institute for Genetics in
Cologne where he joined the group of Klaus
Rajewsky to work on the T-cells in MS brains
and also to develop new mouse tools to explore
autoimmunity.
Group members
Friederike Fromme, PhD
Student
Nadine Hövelmeyer, PhD
Student
Jian Song, PhD Student
Thorsten Buch, Postdoc
Claudia Uthoff-Hachenberg,
Technician
Molecular Immunology
We are interested in the molecular mechanisms that lead to the induction and development
of brain inflammation. We use a disease model called experimental autoimune
encephalomyelitis (EAE) . EAE is considered the best model for the human disease multiple sclerosis (MS) which is an autoimmune disease in which cells of the immune system
invade the brain and induce demyelination and neuron degenaration that lead to gradual paralysis. We employ the method of conditional gene inactivation or induction using
the Cre-loxP system greatly developed in the institute by Klaus Rajewsky. Using conditional
gene deletion we are investigating different genes that are involved in apoptosis for their
role in development of EAE. For that, we use transgenic mouse strains in which the Crerecombinase (Cre) is experessed in the brain, in either oligodendrocytes (MOGi-cre) or
whole brain (Nestin-cre).
In addition, we are interested in the role of antigen presenting cells (APC) in the induction
of EAE or in tolerance to the autoantigen. For that, we generated a mouse strain in
which a myelin-derived peptide can be expressed in either one of a given APC population,
such as dendritic cells, macrophages or B-cells, to investigate the role of these different
populations in disease induction or prevention. Finally, our group is interested in
lymphocyte development and signaling. We explore the signaling events initiated from the
B-cell receptor (BCR). Specifically, we are interetsed in signals transmitted from IgG1type
BCR, and compare it to the signals produced from IgM BCR.
68
Ari Waisman
Apoptotic signals in brain inflammation
Multiple sclerosis (MS) is an autoimmune disease
of the human central nervous system (CNS)
characterized by an accumulation of lymphocytes
and macrophages in the CNS. These inflammatory
cells are thought to play a pivotal role in the CNS
damage, namely demyelination and subsequent
destruction of axons. Experimental autoimmune
encephalomyelitis (EAE) is a common model in
rodents and monkeys for MS. EAE is induced by
immunization with myelin or myelin antigens.
Pathogenesis is caused mainly by antigen specific
CD4+ T cells. The result of this inflammatory process
is oligodendrocyte (ODC) death and axonal injury.
Two major interactions were shown to play a role
in T cell and macrophage mediated tissue damage
in the CNS: TNF-α secreted by T-cells and
macrophages and Fas ligand (FasL) expressed by
the invading cells. TNF-α and FasL act via TNF
receptor 1 (TNFR1) and the Fas receptor (FAS),
respectively. The Fas receptor belongs to the Ig
superfamily, which transmit apoptosis signals after
interaction with FasL. Two natural mutant mouse
strains, the lpr and gld mice that contain mutations
in the fas and fasL genes respectively, were used
to show the importance of Fas-FasL interaction in
EAE. The lpr strain is less susceptible to EAE
induced by immunization with myelinoligodendrocyte-glycoprotein (MOG) peptide 3555 in C57BL/6J mice.
Although a role for Fas-FasL has been
demonstrated in the development of EAE, it
remains unclear which Fas expressing cell types in
the CNS are killed by FasL positive T cells. To
answer whether Fas expression by ODC is
necessary for the death of ODC during the course
of EAE, we generated a mouse strain in which the
fas gene was deleted specifically in ODC, utilizing
the Cre-loxP system.
To delete the fas gene specifically in ODC we
generated a mouse strain in, which expression of
Cre recombinase (Cre) is regulated by physiological
control regions of the MOG gene. MOG
expression was previously shown to be restricted
to ODC. To test the role of fas expression by ODC
in development of EAE we immunized MOGi-cre/
fasfl/fl mice with the encephalitogenic MOG peptide
Fig. 1. Less demyelination in mice lacking Fas in ODC
compared to control mice. Central nervous system
sections were stained with Luxol fast blue myelin stain.
As seen, the myelin is almost intact in the experimental
mice in comparison to the control mice.
p35-55. We observed an amelioration of disease
severity but no decrease in disease incidence in mice
lacking fas expression in ODC in comparison to
control mice. These clinical findings could be
assessed by pathological findings, showing less
demyelination of mice without Fas expression in
ODC comparing to control (Fig. 1).
We are now in the process of crossing the Fas
conditional mice to other mice that express brainspecific Cre-recombinase, to assess the role of
other cell types, such as neurons and astrocytes in
brain inflammation. In addition, we are in the
process of generating new mutant mice that will
also be used to investigate the role of apoptosis in
EAE. The first two mutant mice we are generating
are both involved in the inflammatory process of
NFkB. The first of these is NFkB inducing kinase,
NIK, which is involved in transmitting the signal
from surface receptors to the NFkB pathway, thus
leading to the inflammatory response. In addition,
we are in the process of generating a mouse strain
where we can conditionally delete CYLD-1, a
newly discovered proto-oncogene. CYLD-1 was
recently shown to be involved in signaling from the
TNF receptor, via TRAF-2 to the NFkB pathway.
The role of antigen presenting cells (APC) in
the induction of EAE
T-cells recognize antigenic peptides in the context
of MHC molecules. There are major types of
MHC molecules, class I and class II. CD4+
lymphocytes are activated by peptides when
presented by MHC class II. During development
69
Ari Waisman
(A)
(B)
Fig. 2. (A) Short schematic explanation for the ROSA26
locus targeted with a STOP cassette and the mini-gene
coding for the MOG peptide. Only after Cre-mediated
deletion of the STOP cassette, the MOG peptide is
expressed.
(B) T cells specific to the MOG peptide were labeled
with the fluorescent dye CFSE. CFSE is distributed to
the daughter cells, and therefore one can follow the
number of divisions by looking at the histogram in using
FACS. As seen, the T cells divided in 3 days at least 5
times, but only when the DC were taken from the mice
where the MOG peptide is genetically loaded on MHC
class II.
in the thymus, thymocytes undergo a process of
negative selection in which self-reactive T-cells are
deleted when they encounter self-peptides
presented by MHC molecules. This process is
termed negative selection and results in tolerance.
In some cases where the self-protein is not
expressed in the thymus, self-reactive T-cells may
escape deletion and be present in the lymphoid
70
organs. Such an example is T-cells that recognize
the myelin protein MOG, and specifically the MOG
peptide p35-55. As MOG is expressed only in the
brain, MOG-specific T-cells are not deleted, and
upon encounter with MHC class II occupied with
this peptide will proliferate and possibly induce
EAE
We have developed in the lab a mouse model to
investigate the process of peripheral tolerance. We
generated a mouse strain in which the MOG
peptide p35-55 is presented on MHC class II
molecules of selected antigen presenting cells
(APC), such as B-cells, macrophages or dendritic
cells (DC). From the mice where the MOG
peptide was expressed on all APC we isolated the
different APC. These APC where then cultured with
MOG-reactive T-cells. Seen in Fig. 2 is the proliferative response of the T-cells when incubated
with the dendritic cells coming from the mice where
the MOG peptide is expressed – while the same
type of T-cells did not proliferate when incubated
with dendritic cells from the control mice..
To be able to express the MOG peptide in DC,
we are in the process of generating mice in which
the Cre recombinase is expressed under DCspecific promoters. To this end, we have
constructed vectors that allow us to insert the
coding region of Cre under the control of CD11c
and the first promoter of CIITA (CpI), both shown
before to be expressed almost exclusively in DC.
Following B-cell receptor development and
signaling by gene targeting
B-cells recognize antigens via the B-cell receptor
(BCR). Upon engagement of the BCR with
antigen, the co-signaling molecules Igα and Igβ
are phosphorylated and the signal transduction
pathway is induced. Mature B-cell express IgM
and IgD as their BCR, but later can switch also to
other classes such as IgG1.
In a normal wild type mouse, IgG1+ B-cells
comprise only 0.1-0.2% of the total B-cells. This
makes it very difficult to study these cells
biochemically, as they are very hard to purify, and
harvest in large numbers. To better understand the
role of IgG1 in signaling, we generated a mutant
mouse strain in which all the B-cell express IgG1
Ari Waisman
Selected Publications
Kirshner, S.L., Waisman, A., Zisman, E., Ben-Nun,
A., Mozes, E. (1997) T-cell receptor expression
and differential proliferative responses by Tcells specific to a myasthenogenic peptide. Cell
Immunol. 180, 20-28.
Ruiz, P.J., Wolkowicz, R., Waisman, A., Hirschberg,
D.L., Carmi, P., Erez, N., Garren, H., Herkel, J.,
Karpuj, M., Steinman, L., Rotter, V., Cohen, I.R..
(1998) Idiotypic immunization induces
immunity to mutated p53 and tumor rejection.
Nature Medicine. 4, 710-713.
Fig. 3. Shown above is a schematic representation of the
IgH locus in the IgG1i mice. After VDJ rearrangement,
the only constant region that can be transcribed is the
one coding for IgG1. Spleen cells were gated for B-cells
using CD19 specific antibody. Shown are gated B-cells
further stained for IgM and IgG1. In the left panel is
shown the analysis of B-cells from a mouse with wild
type Iga molecule, while on the right a mouse with
truncated Iga.
as their BCR. This mutant mouse was crossed also
to mice that carry mutations in the Igα gene. We
found that B-cells can develop also when their BCR
is IgG1 instead of IgM, although their development
is partially impaired in the early stages of
development in the bone marrow. This impairment
was further studied in mice having the IgG1 mutation
on one locus while having the wild type locus on
the chromosome. These mice had only 10-20% of
their B-cells expressing IgG1 in contrast to the
expected 50%. Surprisingly, we found that when
crossed to a mouse strain that carries a truncated
Igα molecule which cannot signal, the mice now
had 80% of the B-cells expressing IgG1. These
results indicate that IgG1 BCR can most likely signal
independently from Igα, at least partially. Evidence
for such an assumption comes also from the finding
that IgG1 BCR has a long intra-cellular signaling
tail that could potentially signal, while IgM and IgD
do not.
Ruiz, P.J., Waisman, A., Mozes, E. (1998) Anti-T-cell
receptor therapy in murine experimental
systemic lupus erythematosus. Immunol. Lett.
62, 1-8.
Erez-Alon, N., Herkel, J., Wolkowicz, R., Ruiz, P.J.,
Waisman, A., Rotter, V., Cohen, I.R. (1998)
Immunity to p53 induced by an idiotypic
network of anti-p53 antibodies: generation of
sequence-specific anti-DNA antibodies and
protection from tumor metastasis. Cancer Res.
58, 5447-5452.
Blank, M., Waisman, A., Mozes, E., Koike, T.,
Shoenfeld, Y. (1999) Characteristics and
pathogenic role of anti-beta2-glycoprotein I
single-chain Fv domains: induction of
experimental antiphospholipid syndrome. Int.
Immunol. 11, 1917-1926.
Dayan, M., Segal, R., Sthoeger, Z., Waisman, A.,
Brosh, N., Elkayam, O., Eilat, E., Fridkin, M.,
Mozes, E. (2000) Immune response of SLE
patients to peptides based on the
complementarity determining regions of a
pathogenic anti-DNA monoclonal antibody. J.
Clin. Immunol. 20, 187-194.
Babbe, H., Roers, A., Waisman, A., Lassmann, H.,
Goebels, N., Hohlfeld, R., Friese, M., Schroder,
R., Deckert, M., Schmidt, S., Ravid, R.,
Rajewsky, K. (2000) Clonal expansions of
CD8(+) T-cells dominate the T-cell infiltrate in
active multiple sclerosis lesions as shown by
micromanipulation and single cell polymerase
chain reaction. J. Exp. Med. 192, 393-404.
Buch, T., Uthoff-Hachenberg, C., Waisman, A.
(2003) Protection from autoimmune brain
inflammation in mice lacking interferon
regulatory factor-1 is associated with Th2 type
cytokines. Int. Immunol. 15, 855-859.
71
Thomas Wiehe
Thomas Wiehe
Professor
Thomas Wiehe studied Mathematics and
Philosophy at the University of Erlangen and
received his PhD in Biology from the
University of Jena in 1995. He spent two postdoctoral years with Montgomery Slatkin at the
University of California, Berkeley, studying
mathematical models in population genetics. In
1997 he joined the group of André Rosenthal,
IMB-Jena, working in comparative genomics
and in 1999 he became group leader at the
Max Planck Institute for Chemical Ecology.
In 2001 he took on a position as an independent
group leader in bioinformatics at the Free
University, Berlin. He joined the Department
of Genetics of the University of Cologne in
January 2003.
Group members
Nora Pierstorff, PhD Student
Anton Malina, Programmer
Bioinformatics and Population Genetics
The group works on theoretical aspects of population genetics as well as on bioinformatical
methods in evolutionary and comparative genomics. Co-workers are currently being hired.
Population Genetics
We are concerned with the question of how to
localize those sites in a genome which play a crucial
role for phenotypic adaptation of a species. It is
well known that beneficial mutations lead to a loss
of genetic variability in its chromosomal vicinity —
a fact that has been termed genetic hitchhiking. We
are intersted to model, to quantify and to statistically
test such genetic footprints as they depend on
evolutionary mechanisms. While ground-breaking
work has been done during the last decade by
various researchers, biologically more realistic and
genome-data oriented models must be deviced and
for example epistatic effects of multiple gene
72
Thomas Wiehe
Fig. 1. Measurements of genetic variability in
microsatellite markers along a stretch of 600 kb in D.
melanogaster chromosome III (data from B. Harr). A
potentially beneficial mutation has caused the reduction
seen at position 500kb.If this is true, a theoretical model
(red curve) could help to map the position of the mutation
as well as to determine other evolutionary parameters.
interactions need to be incorporated. Developing
such models and the respective statistical tests is
therefore an ongoing and challenging task.
Bioinformatics
Comparative genome analysis became one of the
standard routes to „compute“ the gene inventory
of an organism. For example, estimates on the total number of human genes were significantly altered after the complete mouse genome became
available and could be compared to the human
chromosomes. More importantly, the comparison
of genomes, transcriptomes and proteomes of
closely related organisms is expected to lead to a
better unserstanding of gene regulation and function
and of the gene potential of a species. Work in this
group will initially concentrate on the comparative
analysis of eukaryotic promoters and the evolution
of alternative splice variants.
Fig. 2. In contrast to coding exons, regulatory elements
in promoters of homologous genes are usually not
conserved (upper panel). This makes their automatic
detection difficult. However, potential elements may be
detected via their physico-chemical properties of the
DNA which are sometimes much more conserved than
the naked sequence (lower panel).
Selected Publications
Wiehe, T., Schmid, K., Stephan, W. (2003) Selective
sweeps in structured populations. In: Selective
Sweeps (ed. D. Nurminsky). Landes
Biosciences, Georgetown.
Abril, J., Guigo, R., Wiehe, T. (2003) gff2aplot: a
plotting tool for sequence comparisons.
Bioinformatics (in press).
Guigo, R., Wiehe, T. (2003) Gene prediction accuracy
in large DNA sequences. In: Frontiers in
computational genomics (eds. M. Galperin and
E. Koonin), Caister Academic Press, Norfolk.
Haubold, B., Kroymann, J., Ratzka, A., MitchellOlds, T., Wiehe, T. (2002) Recombination and
gene conversion in a 170kb genomic region of
Arabidopsis thaliana. Genetics 161, 1269-1278
73
Cologne Spring Meetings
Cologne Spring Meetings
The annual Cologne Spring Meeting is organised
by the Institute for Genetics, but co-organisers from
other departments or faculties of the University of
Cologne are often included. The meeting has a
strong tradition. It developed out of the Spring
Course on Phage and Bacterial Genetics, and
has been held annually since the early seventies.
The Cologne Spring Meeting has attracted large
audiences of scientists and their students from all
over Europe. One year ahead of time, the Institute, upon proposal, selects a scientific topic for the
upcoming Spring Meeting. For each of our meetings,
outstanding speakers from all over the world are
invited who are among the leaders in their fields.
Traditionally, the schedule of the meeting leaves
ample time for discussion with the speakers. A
unique feature of this meeting is that it is open to
the audience, i.e. no registration is required, and
the admission is free. This is one of the reasons
why the Cologne Spring Meeting has always
attracted a large number of even very young
students providing them with an early contact to
74
international outstanding scientists as well as an
exposure to a wide range of approaches and
research areas.
The 2003 Cologne Spring Meeting organised
by T. Langer, J. Dohmen, B.Kisters-Woike (Institute for Genetics) and M. Scheffner (Medical
Faculty) was on „Cellular Quality Control“. This
meeting brought together scientists studying a
variety of important cellular quality control systems,
which are studied in organisms ranging from bacteria
to humans. The presentations covered the quality
control of DNA and RNA, as well as the quality
control of proteins, mediated by chaperones and
the ubiquitin/proteasome system in eukaryotes, and
by proteases cooperating with substrate selecting
adaptor proteins in bacteria. In other sessions, the
role of quality control in organellar biogenesis, in
cell cycle as well as in immunity and disease was
discussed.
Cologne Spring Meetings
Cologne Spring Meetings of the recent past
2000
„Protein Machines and
Subcellular Organisation“
Organizers: M.Leptin, C.Kocks,
K.Reiners
(Institut for Genetics)
2001
„Evolutionary Genetics and
Bioinformatics“
Organizers: D.Tautz,
J.Howard (Institut for
Genetics), D.Schomburg (Institute for Biochemistry)
2002
„Immunity“
Organizers:
J.Howard, C.Kocks,
M.Cramer (Institut for
Genetics) M.Krönke
(Medical Faculty)
The upcoming 2004 Cologne Spring Meeting which is being organised by K. Schnetz, M. Leptin,
K. Reiners (Institut for Genetics) and A. Noegel (Medical Faculty) will be on „Cell Dynamics“. This
meeting will cover various aspects of subcellular and cellular movements.
75
The Center for Mouse Genetics
The Center for Mouse Genetics
Mouse Genetics at the Institute for
Genetics
Since Klaus Rajewsky introduced mouse genetics
into the Institute at the beginnng of the 1970s this
field has acquired enormous importance in biological
and especially biomedical research. The mouse is
an excellent, and sometimes the only possible,
model system with which to investigate mechanisms
and test hypotheses with high human and medical
relevance. The genome sequence is now in that state
which is frequently described as “complete”,
meaning that it is unlikely that significant areas of
protein coding sequence remain to be discovered.
Furthermore, as creatively exploited by Rajewsky,
targeted germ-line modification is now easier in this
species than in any other standard research
organism except Saccharomyces cerevisiae.
Mouse reverse genetics is an indispensable part of
any high level genetics laboratory which operates
in the near-human domain. Until the retirement of
Rajewsky the mouse reverse genetics capability of
the Institute was developed and operated through
the Rajewsky Abteilung. In planning for the future
of the Institute, and with the intention to dedicate
not only the Rajewsky chair but also the Doerfler
chair to mouse studies, and including Howard’s
mouse-oriented research, it seemed appropriate to
separate the mouse resources from a single Abteilung and establish a Center for Mouse Genetics
(CMG) available to all and answerable directly to
the Vorstand. Since the retirement of Rajewsky in
March 2002, the CMG has come into existence
with the scientific, adminstrative, technical and
animal caretaker staff as listed above. The CMG
reports to the Vorstand through a management
group which presently includes Drs Plück and
Lichtenberg and Professors Brüning and Howard.
The resources controlled by the CMG and its
staff include the mouse accommodation itself, as
well as the cell culture and microinjection equipment
necessary for mouse germ-line modification
procedures. The technology of mouse reverse
genetics by homologous recombination in ES cells
76
Personnel
Scientific Management
Dr Anne Plück
Adminstrative Management
Dr Ursula Lichtenberg (pt)
Secretary
Renate Pingen (pt)
Technical Support
Sonja Becker
Brigitte Hülser (pt)
Pia Scholl (pt)
Frank Steiger
Animal Caretakers
Gisela Küster
Kerstin Marohl
Margit Molsberger
Agathe Stark
Sabine Paschlies
Gina Piper
Tanja Tropartz (pt)
Christina Viktorius (pt)
Claudia Wolf (pt)
pt: Part-time
A two-week old baby CB20 (albino) mouse derived from
an embryo injected with C57BL/6 ES cells carrying a
targeted mutation in the IIGP1 resistance GTPase gene
on chromosome 18. The pigmented fur patches of C57BL/
6 origin demonstrate chimerism.
Microarray Facility
is the routine procedure. In addition to this service
work the staff of the CMG explore new technical
developments such as, at present, the use of
tetraploid recipient embryos to guarantee firstgeneration transmission of the desired mutants.
Mouse accommodation
The new mouse rooms on the 4th floor of the
Zülpicher Straße Institute for Genetics are not yet
in service. The rooms are divided between the
4th floor of the older part of the Genetics building
in Zülpicher Straße 47 and the new building. The
older part was first constructed for mouse use in
2000, but not fully equipped or occupied because
of advancing plans for the construction of the new
building. It was clear that the building operations
would make the new rooms unusable.
Nevertheless the existence of the self-contained
mouse unit in the older building has allowed us to
plan this as a high-containment central breeding
complex separate from the main experimental unit,
which will occupy the 4th floor of the new building.
This unit is expected to be in service before the
end of 2003.
The mouse facilities are based exclusively on
individually ventilated cages and racking which,
while more demanding in daily handling, provide a
much higher security against accidentally introduced
microbiological contamination. Routine microbiological screening of sentinel animals and stringent controls on the introduction of new stocks further contribute to microbiological cleanliness. In the
year 2003/2004 we shall be rederiving strains and
stocks from the Weyertal colony into the new
colony in the Zülpicher Straße.
Microarray Facility
The Microarray facility was funded by the Ministry of NRW on the basis of a grant application
of the Institute of Genetics (Prof. Tautz). It was desinged as a central facility open to all research
groups in Cologne. On the long run, it is planned to be integrated it into the ZFG initiative. The
facility is equipped with an „all in one“ Picking, Gridding, Printing robot (GeneTAC G3, Genomic
Solutions), a hybridisation robot (GeneTAC hybridisation station, Genomic Solutions), a four
laser scanner (GeneTAC LS IV, Genomic Solutions) and one 4-channel and one 8-channel
pipetting robot (Multiprobe IIex). Additionally, PCR-machines (DNA Engine Tetrad, MJ Research) an incubator and a 96-well centrifuge are available on request.
Picking: The Picking application allows picking
of colonies and plaques with a 48 pneumatic
pin tool. Inocculation can be performed into 96
standard or deep well plates as well as into 384
well plates. Different input formats are possible:
150mm round petri dishes, 100mm round petri
dishes, bioassay plates (22x22cm), GS culture
plates (100x140mm) and single well plates
(86x128mm).
Gridding: The Gridding application allows arraying
of bacteria, YACs or similar onto membranes from
either 96 type or 384 type well plates by using a
96 pin gridding tool. Membranes fitting to either
bioassay plates or single well plates, which are filled
with agar, can be used as output. A maximum
number of eight output plates can be produced in
one run. On a 22x22 cm membrane, the tool will
print up to 6 unique features, whereas a 12x8 cm
membrane will only have one feature.
Printing: The Micro-arraying application allows
printing of biological samples with a 48 pin tool
onto coated slides. 384 well plates are needed as
input and glass slides with a modified surface for
DNA-attachment are used as output. This process
is also commonly referred to as High Density
Arraying. Concerning the printing pattern, the
system is freely programmable. Custom patterns
can be compiled to specify order of spotting,
77
Training and Support for Graduate Students
distance between the spots and position of positive
as well as negative markers. The generation of
duplicate slides as well as multiple number of
identical spots (up to quintuple) on one slide are
possible.
Hybridisation
The hybridisation station consists of twelve
positions for automated hybridisation and washing
of microarrays. The twelve position unit allows for
six sets of different hybridisations in duplicate.
Protocols can be edited through a touchpad or by
a PC-based software. Up to five different washing
solutions can be used.
Scanning: For Scanning the hybridised arrays a
four laser scanner (GeneTAC LS IV) is available.
Up to 24 Chips can be loaded and scanned
consecutively. Three internal lasers (532nm Solid
State laser, green, 20mW; 594nm Helium Neon
laser, yellow, 2mW; 633nm Helium Neon laser,
red, 15mW) and one external laser (488nm Argon
Ion laser, blue, 10 to 20mW) allow in
combination with different selectable filter sets the
detection of most of the available fluorochromes,
at least of the Cye3-, Cye5-, Fluorescein- and
Texas Red dyes. The scanning resolution is
selectable down to 1 µm. For Image acquisition
and analysis the GeneTAC Biochip Analyzer and
Integrator software is used.
Data Analysis: The program arraySCOUT
(Lion Bioscience) is implemented on the server
of the RRZK. User accounts are available on
request for researchers within the University
network.
Training and Support for Graduate Students
The Institute offers a Thesis Committee Programme to provide support, supervision and training
for Graduate Students. In addition, two fellowship programmes, jointly run with members of
other Institutes, provide funding for excellent students, and aim to attract highly qualified national and international students.
Thesis Committee Programme
Coordinator: Brigitte von Wilcken-Bergmann
arbitrators mediating between and advising both
sides.
All graduate students at the Institute of Genetics
are encouraged to register for the Thesis
Committee Programme. Each student nominates
two independent scientists in addition to his or her
own supervisor as members of the committee. The
committee meets at regular intervals to discuss the
progress of the students’ work. After the first year,
each student submits a written report on the
current status of the research project. The thesis
committee discusses the report and future
approaches with the student. In addition, the
members of the committee are also available for
any other advice or help that the student might need.
In cases of conflict between student and advisor,
the other two committee members serve as
Graduiertenkolleg ‘Genetik zellulärer
Systeme’
Chairperson: Maria Leptin
78
This programme, funded by the DFG since 1997,
is run in cooperation with research groups from
the Institutes of Developmental Biology, Botany and
the Dept. of Biochemistry of the Medical Faculty.
It was set up with the aim of establishing a joint
selection, supervision and training programme for
internationally recruited graduate students. All
professors participate in the assessment of
applicants. Successful candidates begin their
training with a period of three lab rotations before
they join the research groups for their Ph.D.
Sonderforschungsbereiche
projects. In addition to the meetings with their thesis
committees, the students regularly present their
results to the whole faculty and the other students
during monthly Progress Reports. The students
also organize a series of research talks by invited
speakers.
International Graduate School in Genetics
and Functional Genomics
Chairperson:
Maria Leptin
Coordinator:
Sebastian Granderath
Secretary: Gisa Marxen
The Graduate School was established in 2001 with
funding from the Government of the Land Nordrhein-Westfalen. Seventeen professors from four
Institutes participate, including all of the members
of the Graduiertenkolleg described above. The
School’s aims are the same as those of the
Graduiertenkolleg, but it has a more extended
training period and a more formalized exam
structure. In addition to the 6 months’ rotation
period, the students participate in specialized
courses in modern mouse genetics and genomic
high-throughput technologies.. The Graduate
School also offers courses on scientific writing and
presentations. In addition, foreign students
participate in an intensive German language course
during the first 2 months. As of 2003, these additional activities will also be made available to the
students of the Graduiertenkolleg.
Funding for the Graduate Programs
For the Graduiertenkolleg, the DFG funds a cohort
of 8 to 11 students every three years. In addition
to the fellowships, a small amount of consumables
are provided, as well as resources for administration
and funds for the students to attend meetings and
invite guest scientists.
For the funding period beginning September
2003, we applied for a raise in the level of the
fellowships to match those of the Graduate School
and Graduate Programmes in other institutions.
Likewise, the consumables budget was increased.
The International Graduate School in Genetics
and Functional Genomics was established in 2001.
It is financed by the state of North Rhine
Westphalia for an initial period of four years. The
annual budget comprises ten full fellowships for PhD
students, two postdoctoral fellowships, appropriate
funds for consumables, as well as resources for
administration and coordination, travel funds to
allow students the attendance of conferences and
the invitation of guest scientists.
The Graduate School budget is further
augmented by the PHD/IPP programme of the
DAAD. No fellowships or funds for consumables
are available, the PHD/IPP funds are restricted to
the support of “structural framework” ( i.e.
coordination, language courses, marketing) of
existing international PhD programmes.
Sonderforschungsbereiche
Research groups of the Institute for Genetics
participate in two Sonderforschungsbereiche of the
University of Cologne, the SFB 635 on
“Posttranslational Control of Protein Function” and
the SFB 572 “Festlegung von Zellverbänden und
Musterbildungsprozesse”.
The SFB 635 started in July 2003. It focuses on
mechanisms controlling the cellular activities of
proteins on a posttranslational level and thereby
tackles a problem of central importance in the
postgenomic era. It is now understood that the
maintenance of cellular homeostasis and an
adaptation to changing environmental conditions
depends on a variety of posttranslational control
mechanisms which form complex regulatory
networks in cells. The activity of proteins can be
modulated by covalent modifications, interactions
with proteins or by altering their stability, regulatory
mechanisms which are often interdependent of each
other. Groups within the Sonderforschungsbereich
are in particular interested in protein modifications
by phosphorylation, ubiquitin and ubiquitin-like
79
Sonderforschungsbereiche
molecules and in proteolytic processes in general.
Conserved processes are studied both on a cellular
and molecular level in various organisms from
bacteria to mammals and higher plants to unravel
underlying general principles. From the Institute for
Genetics participating groups include Jens Brüning,
Jürgen Dohmen, Jonathan Howard, Thomas Langer (Coordinator), Maria Leptin and Frank
Sprenger. Other groups joined in from the Institutes for Botany and Biochemistry, the Max-PlanckInstitut für Pflanzenzüchtung and the Institute for
Cell Biology in Bonn. The research is supported
80
by central facilities including the mouse center and
the DNA sequencing facility of the Institute for
Genetics and the service facility for protein analysis
of the Center for Molecular Medicine Cologne
(CMMC).
Five groups of the Institute for Genetics (Thomas
Klein, Maria Leptin, Frank Sprenger, Diethard
Tautz and Wim Damen) participate in Sonderforschungsbereich 572 of the Biological Institutes of
the Unversity for which funding began in 2001.
Research within the SFB 572 (Coordinator W.
Werr, Institut für Entwicklungsbiologie) focuses on
the characterisation of differentiation processes and
there regulation in vertebrates and plants.
How to contact the professors and group leaders?
Jens C. Brüning
Tel.: 0221-470-2467
e.mail: [email protected]
Wim Damen
Tel.: 0221-470-3419
e.mail: [email protected]
Ute Deichmann
Tel.: 0221-470-4588
e.mail: [email protected]
Walter Doerfler
Tel.: 0221-470-2386
e.mail: [email protected]
Jürgen Dohmen
Tel.: 0221-470-4862
e.mail: [email protected]
Jonathan Howard
Tel.: 0221-470-4864
e.mail: [email protected]
Börries Kemper
Tel.: 0221-470-5287
e.mail: [email protected]
Thomas Klein
Tel.: 0221-470-3403
e.mail: [email protected]
Michael Knittler
Tel.: 0221-470-5259
e.mail: [email protected]
Sigrun Korsching
Tel.: 0221-470-4834
e.mail: [email protected]
Thomas Langer
Tel.: 0221-470-4876
e.mail: [email protected]
Maria Leptin
Tel.: 0221-470-3401
e.mail: [email protected]
Benno Müller-Hill
Tel.: 0221-470-2388
e.mail:[email protected]
Karin Schnetz
Tel.: 0221-470-3815
e.mail: [email protected]
Frank Sprenger
Tel.: 0221-470-5259
e.mail: [email protected]
Diethard Tautz
Tel.: 0221-470-2465
e.mail: [email protected]
Ari Waisman
Tel.: 0221-470-3402
e.mail: [email protected]
Thomas Wiehe
Tel.: 0221-470-3843
e.mail: [email protected]
81