186
Gene transfer
Isolation of trans-acting regulatory mutations in Drosophila using
alcohol dehydrogenase gene fusions for chemical selection
/. JoseBonner*, Carol Parks, Janice Parker-Thornburg, Mark A. Mortin, and Hugh R. B. Pelham,
Department of Biology, Indiana University, Bloomington, Indiana, USA and Laboratory of Molecular
Biology, Medical Research Council Centre, Cambridge
Alcohol Dehydrogenease (Adh) is an ideal enzyme for chemical selection of mutations affecting its
expression, as Adh activity is required for survival of exposure to ethanol. Conversely, Adh activity renders
exposure to 3-pentyne-l-ol lethal, thus allowing the selection of Adh-deficient flies. We have sought to
adapt this system to the isolation of mutations affecting the expression of developmentally or metabolically
regulated genes for which chemical selections do not already exist. We have used the heat shock system for
our initial studies, as the heat shock genes are readily inducible in the laboratory, and the DNA sequences
involved in their regulation are known. We have fused the 5' flanking sequences of hsp70, and thefirst200
nucleotides of the hsp70 transcription unit to the structural gene for Adh. The fusion is m the 5' untranslated
leader sequence of both genes, and does not affect translational reading frames. We have used this fusion
gene for germline transformation of flies carrying a deletion in their Adh gene. Flies transformed with this
gene fusion exhibit Adh activity only after heat shock, demonstrating that the sequences involved in the
control of Adh transcription have been functionally replaced by the hsp70 sequences.
The activity of Adh, once induced by heat shock, is stable. Males continue to show Adh activity for at
least a week after a one-hour heat shock. Thus Adh can be used in gene fusions of this kind to mark cells
which have previously expressed transcription from the promoter used in the fusion. For example, primary
spermatocytes, which fail to induce Adh activity in response to heat shock, exhibit Adh activity several days
after the shock, as a result of the differentiation of stem cells in which activity was induced.
Because the induction of Adh activity by heat shock allows theseflies(which are otherwise Adh-deficient)
to survive ethanol, we have been able to select dominant, transacting mutations which activate transcription
from the heat shock genes aberrantly (e.g. at 28 °C). We propose that similar trans-acting regulatory
mutations can be isolated which affect the expression of developmentally regulated genes simply by utilizing
a developmentally regulated promoter rather than the promoter of hsp70.
E. De Robertis* (Basel)
No abstract for publication
Gene transfer
DNA sequence and regulation of a Dictyostelium gene isolated by
complementation of a yeast ural mutant
187
M. Jacquet, E. Boy-Marcotte, M. Faure, M. Kalekine and H. Garreau, University Paris-Sud 91405
Orsay-Cedex, France
A strain of Saccharomyces cerevisiae carrying a mutation in the ural gene was transformed with fragments
of Dictyostelium discoideum DNA inserted into a plasmid capable of replication in E. coli or yeast. Rare
prototrophic colonies were recovered that all contained the parent plasmid and an insert of Dictyostelium
DNA. Plasmids recovered from the protrophic yeasts could be used to transform E. coli to antibiotic
resistance. The E. coli transformants harbored plasmids capable of transforming yeast ural mutants to
prototrophy. These recombinant plasmids contained a fragment 1-9 kbp long, containing the coding and
flanking sequences (/. Mol. Appl. Genet. 1982, 1, 513-525). The base sequence was determined by the
Maxam and Gilbert technique. There is one open reading frame which does not contain any intron. The
upstream sequence is very (A+T) rich and contains long stretches of A and T, as observed in other
Dictyostelium genes. In transformed yeasts, two sites for transcription initiation were located close to the
ATG initiation codon by SI mapping. In some recombinant plasmids, isolated after amplification of E. coli,
a 400 bp deletion was observed in the 5' flanking region. This deletion reduced the level of transcription in
transformed yeasts. This 1-9 kbp fragment is unique in Dictyostelium DNA and corresponds to a single
mRNA 1300 bases long. This mRNA is expressed during vegetative growth and its level decreases during
development.
Injection of cloned genes into rainbow trout eggs
N. Maclean and S. Talwar, Department of Biology, Southampton University, Southampton, Hants SO93TU
Fertilised eggs of rainbow trout, Salmo gairdneri, have been injected with cloned DNA at either the
four-cell stage or the blastomere stage of development. The DNA used is a mouse metallothionein gene
within the E. coli plasmid pBR 322 and presumptively transformed fish fry have been assayed for increased
resistence to the toxic effects of cadmium sulphate in solution. Since the trout egg is retained within a very
thick chorion which is impermeable to even quite small molecules, a special injection technique has been
devised, involving the use of one glass needle within another.
An outline will also be given of the isolation of a trout metallothionein gene sequence from a rainbow
trout gene library, and future experimental work involving the injection of other novel gene sequences
briefly indicated.
188
Gene transfer
Gene transfer in the sea-urchin Stronglycentrotuspurpuratus
A. P. McMahon, C. N. Flytzanis, B. R. Hough-Evans, K. Katula, F. Teng, R. J. Britten, E. H. Davidson,
Division of Biology, California Institute of Technology, Pasadena, California, 91125 USA
We have developed a simple, rapid microinjection system for introducing DNA into the 80 /u,m sea-urchin
egg. The fate of injected DNA has been studied during early embryogenesis (up to 96 h post injection), in
late larvae (5 weeks post injection) and in the juvenile sea-urchin following metamorphosis (4-5 months
post injection).
Unfertilised eggs were injected with ~2 pi of a variety of DNA solutions, fertilised in situ and cultured to
the relevant developmental stage. The results indicate that when DNA is injected as linear molecules, rapid
ligation occurs to form high molecular weight concatenates which undergo ~ a 70-fold replication by the
blastula stage (24 h post fertilisation). In contrast, injected supercoiled molecules persist as either
supercoiled or relaxed circular molecules and do not replicate.
When individual, 5-week larvae (50,000 cells) were examined, 50-85 % were found to contain injected
sequences, and, in over half of these, larvae sequences were present at more than 1 copy per cell. Following
metamorphosis there is a significant reduction in the number of transformants (5-10 %) and from the
analysis of extracted DNA by genomic blots, the complex pattern of restriction fragments suggests that
DNA is integrated into the genome in these individuals. A junction fragment representing the site of
integration in one individual has been cloned and sequenced and is under analysis.
We are currently examining transformed embryos and larvae for the expression of a variety of
microinjected genes. Preliminary results on this work will be presented.
Plasmids in Dictyostelium discoideum
A. Noegel, B. Metz, K. Williams and D. L. Welker, Max Planck Institute for Biochemistry, 8033 Martinsried,
West Germany and MacQuarie University, Sydney, Australia
The presence of a plasmid in the lower eukaryote Dictyostelium discoideum has been reported previously.
In the studies presented here, we show the presence of plasmids in several independent wild isolates of
Dictyostelium discoideum. Using a modified 'lysis in the gel' procedure originally described for bacteria, we
have screened several strains. From a total of 25 strains, five contained plasmid DNA. Three of the
plasmids, Ddpl (from NC4), Ddp2 (WS380B) and Ddp3 (OHIO) are described here. They are isolated as
supercoiled DNA molecules from nuclei and have sizes of 13«8 kb (Ddpl), 5*8 kb (Ddp2) and approximately 27 kb (Ddp3). Restriction enzyme analysis does not show similarity between the three plasmids.
Furthermore, in Southern blot experiments no homology could be detected among these plasmids. There
does also not exist any detectable homology to either mitochondrial or nuclear DNA of D. discoideum. The
copynumber of the plasmids varies between 50 to 100 copies for Ddp3 to about 300 copies for Ddp2 and the
plasmid DNA represents between two to five percent of total nuclear DNA.
The coexistence of various plasmids in one cell has been examined by constructing diploid cells. No
exclusion or incompatibility behavior has been observed so far.
The presence of plasmids in eukaryotic cells is not uncommon. However, they are usually homologous to
mitochondrial or chromosomal DNA and are localized in the mitochondria or nucleus. The features of the
Dictyostelium discoideum plasmids (localization in the nucleus, lack of homology to any other cellular
DNA) brings them close to the 2 fxm circle of yeast. Functions have not been associated with the plasmids so
far.
Gene transfer
Transcriptional enhancers of immunoglobulin genes
189
D. Picard \ T. Gerster \ T. Leanderson 2 , W. Schaffner \ andE. Serfling*1.]institute ofMolecular Biology
II, University of Zurich, Honggerberg, CH-8093 Zurich. 2Basel Institute of Immunology, CH-4005 Basel,
Switzerland
Recently, a lymphocyte-specific enhancer has been identified downstream of the joining region in
immunoglobulin heavy chain genes. We have also shown direct evidence for a functionally similar enhancer
within the large intron of the kappa light chain gene of the mouse. The kappa enhancer is, however, less
active than the heavy chain gene enhancer. No enhancer was found within a cloned lambda I light chain
gene. We note that enhancer activity correlates with the time of immunoglobulin gene activation during B
lymphocyte differentiation. Surprisingly, in transfection experiments, the heavy chain gene enhancer is
highly active in B cells and even in pre-B cells, in contrast to the low expression level of the endogenous
heavy chain gene in these cells. It therefore appears that the activation of immunoglobulin genes during
lymphocyte differentiation is a multi-step process directed by enhancers as well as other components.
W. Richardson (London)
No abstract for publication
190
Gene transfer
Persistence and expression of genes injected into fertilized Xenopus
eggs
G. U. Ryffel, U. Wagner, D. B. Muelner 1 andA.-C. Andres 1, Kernforschungszentrum Karlsruhe, Institut
fur Genetik, Postfach 3640, D-7500 Karlsruhe 1, West Germany. (x Present address: Ludwig Institute for
Cancer Research, Inselspital, CH-3012 Bern, Switzerland)
To define regulatory DNA sequences involved in the expression of the albumin and vitellogenin genes, we
inject gene variants into fertilized eggs of Xenopus and follow the fate of these DNA molecules throughout
development and differentiation. In general, a mosaic distribution of the injected DNA is observed between
and within the various tissues of the developing frog, indicating that no integration occurred before the first
cleavage stage. The persisting DNA may be partially integrated but can also be found in an episome-like
form. The unintegrated form is not supercoiled and usually rearranged.
Using several genes, including a vitellogenin minigene, a chimaeric vitellogenin-gpf gene (constructed by
P. Walter and W. Wahli) as well as the chicken conalbumin gene, we detect transcriptional activity in the
tailbud embryo resulting in the appearance of the corresponding polyadenylated RNA. At this early
developmental stage 10 to 50 gene copies per cell are present and it seems that this relative high copy
number of injected genes cannot be inactivated, as the endogenous genes are, at this developmental stage.
At later stages, i.e. in hepatocytes of frogs containing the injected genes in the liver, the persisting genes are
quiescent and cannot so far be activated by estrogen.
The T-DNA of Ti-plasmids as a model system to study the developmental
biology of plants
J. Schell*1*2, M. Van Montagu 2, L. Willmitzer \ J. Schroder \ H. Joos 2, D. Inzi 2, L. Otten 1, /. P.
Hernalsteens 2, K. Spanief 1 and P. Zambryski 2. lMax-Planck-Institutfur Ziichtungsforschung, D-5000
Koln 30, West Germany. 2Laboratorium voor Genetika, Rijksuniversiteit Gent, B-9000 Gent, Belgium
Large plasmids in Agrobacterium tumefaciens (Ti) and A. rhizogenes (Ri) are known to endow these
bacteria with the capacity to transfer a defined DNA fragment (T-DNA) into the plant cell nucleus and to
covalently integrate this T-DNA segment in chromosomal DNA, thus creating a new locus at a number of
possible sites. Under normal circumstances only the T-region of the 77-plasmid is inserted in the
chromosomal DNA and thus §tably maintained. No functions located within the T-region are required for
either transfer or integration. The plasmid derived T-DNA was shown to consist of a number of well-defined
transcriptional units transcribed by the-host polymerase II and coding for a number of different functions
involved in the regulation of plant differentiation. Thus separate shoot-suppressing and root-suppressing
functions have been identified. By a combination of in vivo and in vitro recombinant DNA techniques it was
possible to eliminate, by deletion mutations, each of the differentiation-controlling one genes, either singly
or in various combinations.
These studies indicated that the genes that code for shoot inhibition simultaneously code for a stimulation
of root formation and that, reciprocally, the gene coding for root inhibition simultaneously stimulates shoot
formation. In order to test whether some of the T-DNA genes have an influence on the embryo
development, plant cells containing either individual - or a set of- one genes were obtained and regenerated
into flowering plants by various means. It was thus shown that plants containing the root-suppressing,
shoot-stimulating genes could transmit these genes sexually to seeds. These seeds however would develop
into abnormal seedlings unable to grow roots and developing teratoma-like shoots. By expression of some of
the T-DNA one genes in E. coli it was possible to demonstrate that one of the genes involved in
shoot-suppression and root-stimulation codes for an enzyme converting indole-3-acetamide into
indole-3-acetic acid (auxin). The functions of the other genes are under study. The results indicate that at
least some of the one genes direct the formation of growth factors. Removal of all these tumor-controlling
genes does not affect DNA transfer or integration. Thus it was possible to design modified Ti-plasmids that
can insert foreign genes in plant cells from which normal plants can be regenerated that express the foreign
genes and transmit them sexually with normal Mendelian segregation ratios.
Gene transfer
Viral promoter/enhancer activity in differentiating embryonal
carcinoma cells
191
Merilyn Sleigh and Trevor Lockett, CSIRO Division of Molecular Biology, P. O. Box 184, North Ryde,
NSW, 2113, Australia
The F9 line of mouse embryonal carcinoma cells differentiates in monolayer culture in the presence of
retinoic acid to produce cells with the characteristics of embryonic parietal endoderm. The F9 system thus
provides an in vitro model for studying gene regulatory events of normal embryogenesis.
Retinoic acid-induced differentiation is accompanied by alterations in the expression of both endogenous
and introduced (viral) genes. We have used the chloramphenicol acetyltransferase assay (Gorman et al.
1982) to assess the activity of viral promoter/enhancer combinations after transfection of F9 cells at various
stages of the differentiation process. The ability of the cells to take up DNA increases dramatically as
differentiation proceeds. Even after this is taken into account, transcription from the SV40 early gene
promoter/enhancer is seen to increase during differentiation, approaching that seen in mousefibroblastsand
a parietal yolk sac cell line (PYS). The role of the enhancer sequence in this increased transcription has been
assessed by transfecting with SV40-CAT in the presence of an excess of the enhancer sequence and by
determining the effect of the enhancer on transcription from the herpes simplex virus thymidine kinase
promoter during F9 cell differentiation. Lastly, we have examined transcription from integrated marker
genes under SV40 promoter/enhancer control, to determine whether transcription increases during retinoic
acid-induced differentiation.
GORMAN, C. M., MOFFAT, L. F. & HOWARD, B. H. (1982). Recombinant genomes which express
chloramphenicol acetyltransferase in mammalian cells. Mol. Cell Biol. 2, 1044-1051.
An improved method for cultivation of tobacco protoplasts upon in vitro
genetic transformation
M. R. Smets \J. Vanderleyden \A. P. Van Gool \ F. A. Krens \ G. /. Wullems x andR. A.
Schilperoort 2.1F. A. Janssens Memorial Laboratory of Genetics, Katholieke Universiteit Leuven, Kardinaal
Mercierlaan92, B-3030 Heverlee, Belgium. 2 Department of Plant Molecular Biology, Rijksuniversiteit
Leiden, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
Enhanced growth is observed when in vitro genetically transformed tobacco protoplasts are grown on
filter paper discs put on top of a layer of petunia cells. This method offers two important advantages: (1) the
selection of transformed phytohormone-autotrophic cells starts considerably earlier when compared to
previous methods (Krens et al., 1982). (2) a time consuming transfer of small individual calli to selection
medium can be avoided by transferring the whole top-filter paper disc.
Protoplasts (pps) of Nicotiana tabacum petit Havanna SRI, are isolated and transformed with
Agrobacterium tumefaciens 7Y-plasmid DNA according to Krens et al., (1982). After cell wall synthesis, the
culture medium is stepwise diluted with fresh medium to reduce the sucrose
concentration from 13-6 to 3 %
(w/v). This gradual dilution decreases the cell density to about 5-103 pps/ml. Subsequently these cells are
transferred to Petri dishes. These dishes contain a layer of solidified medium with petunia suspension cells in
late log phase, covered with two Whatman filter paper discs. A small fraction of the tobacco cell suspension
(0-5 ml) is spread on the top filter and subcultured for about two weeks (Horsch & Jones, 1980). When
tobacco cell colonies become visible on the top filter, this filter is transferred to a selection medium lacking
phytohormones. After one month of growth in the selective medium, the surviving colonies are transferred
to fresh culture medium and tested for nopaline dehydrogenase (NpDH) activity. NpDH activity is a clear
indication for the genetically transformed state of a callus. Modification of the in vitro transformation
method by incorporation of a feeder plate culture system allows the screening of a large number of
independent calli. It remains to be assessed what the number is of transformed calli that can be obtained
from a single transformation experiment.
R. B. & JONES, G. E. (1980). A double filter paper technique for plating cultured plant cells. In
vitro 16, 103-109.
KRENS, F. A., MOLENDUK, L., WULLEMS, G. J. & SCHILPEROORT, R. A. (1982). In vitro transformation of
plant protoplasts with 7Y-plasmid DNA. Nature 296, 72-74.
HORSCH,
192
Gene transfer
Tissue-specific expression of transfected ar-fetoprotein genes in
teratocarcinoma cells and mice
Shirley M. Tilghman*, Richard W. Scott, Thomas P. Vogt, Robb Krumlauf, Robert Hammer and Ralph
Brinster, Institute for Cancer Research, Philadelphia, PA 19111 and the University of Pennsylvania School of
Veterinary Medicine, Philadelphia, PA 19104, USA
Two independent methods have been utilized to examine the sequences within the ar-fetoprotein (AFP)
gene that are required for its tissue-specific activation during development in liver, visceral endoderm and
gut. Firstly, F9 teratocarcinoma cells, which can be induced to differentiate into either parietal or visceral
endoderm in the presence of retinoic acid, have been transformed with modified copies of the mouse AFP
gene. These copies contain a five exon, internally deleted minigene along with 14 kilobase pairs of 5'
flanking DNA. In five of twelve transformants carrying exogenous copies of the AFP gene, the
differentiation to either parietal or visceral endoderm is accompanied by tissue-specific induction of the
exogenous template in a manner qualitatively, but not quantitatively, identical to that of the resident AFP
gene. Identical minigene constructs have also been introduced into the germline of mice via the
microinjection of fertilized eggs and reimplantation into pseudopregnant recipients. We observe that in
approximately 35 % of the mice which are delivered and carry the minigene, the expression of the minigene
template is detected in those tissues which express the AFP gene and absent in all other tissues examined. In
no instance have we observed the expression of the minigene in an inappropriate tissue. The region of DNA
on the minigene which is responsible for its tissue-specific activation has been localized to between 1 and 7
kilobase pairs of DNA upstream of the site of initiation of transcription.
The regulated expression of /3-globin genes
5. Wright \ E. deBoer l,A. Rosenthal 2 , R. Flavell 3 and F. Grosveld*1. 1National Institute for Medical
Research, Mill Hill, London NW71AA. 2 Department of Molecular Biology, The Hebrew University,
Hadassah Med. School, Jerusalem. *Biogen Research Corp., 14 Cambridge Centre, Cambridge,
Massachusetts 02142, USA
The human /Mike globin genes are differentially expressed during the course of development. The five
active genes which are located in a cluster on the short arm of chromosome 11 are expressed at different
times and in different erythroid tissues. The embryonic e-globin gene is expressed in the yolk sac, the foetal
y° and /^-globin genes in the foetal liver and the adult 6- and /5-globin gene in adult bone marrow. To study
this tissue and stage-specific expression, we have used DNA-mediated gene transfer into cultured murine
erythroleukaemia (MEL) cells. Chemically induced differentiation of these cells results in a several hundred
fold increase in transcription of the adult mouse globin genes. These cells thus serve as a model for gene
activation during erythropoiesis and can be used to study the expression of foreign /5-globin genes.
We have introduced into MEL cells a series of human globin gene cosmids and two sets of hybrid genes
constructed from the human jS-globin gene and the human y-globin or murine H-2Kbml genes. Analysis of
the mRNA products from these genes before and after MEL cell differentiation showed that the
human
/3-globin gene is induced specifically in contrast to the human e- or y-globin or murine H-2KbnU genes.
Hybrid genes containing human /5-globin DNA sequences from either the 5' or 3' side of the translation
initiation site were both inducible. Measurement of the relative rate of transcription showed this induction
to be the result of transcriptional activation. We therefore suggest that MEL cell factors regulated j8-globin
gene expression during MEL cell differentiation by interaction in trans with DNA sequences located both 5'
and 3' to the translation initiation site of the /5-globin gene.