130
Specific genes in development
Effect of temperature on the expression of genes for DNA ligase in
Axolotl ldevelopment
Y. Andeol 1,J. C. David 2 andJ. Signoretl. 1Laboratoire de biologie du Diveloppement, University de
Caen, 14032 Caen, France. 2Laboratoire de Biochimie du Diveloppement, University de Rennes I, Campus
Beaulieu, 35042 Rennes, France
A heavy form of DNA ligase (8S) replaces the light form (6S) of enzyme when Axolotl egg is fertilized or
artificially activated at 18 °C. When eggs either fertilized or activated are maintained at 10 °C the heavy
form of enzyme does not appear and the light DNA ligase remains undiminished.
Fertilized eggs develop normally, although slowly, at 10 °C with but the light molecular form of DNA
ligase, and no trace of the heavy form reported as specific of developmental stages at 18 °C. Thus,
considering that the two molecular forms of DNA ligase are involved normally in different processes as it is
generally assumed, we must admit that their peculiar roles are not strictly exclusive, because the light form
of enzyme is able to vicariate the heavy form at low temperature.
Nuclear transplantations have been performed inter- or intra-species (AxolotVPleurodeles), with blastula
or gastrula donor embryos. When nuclei taken from embryos raised at 10 °C are implanted in egg cytoplasm
maintained at 18 °C they express the light form of DNA ligase. Nuclei taken from donors raised at 18 °C and
implanted in egg cytoplasm maintained at 10 °C express the heavy form of DNA ligase.
It can be concluded that the switching on of the gene for the heavy DNA ligase that occurs when an
Axolotl egg enters cleavage is temperature dependant. The regulated state of the gene, either activated or
inactivated, appears then stabilized and independent of temperature following nuclear transplantation.
Actin and myosin genes, and their expression during myogenesis
in the mouse
Margaret Buckingham*, Serge Alonso, Paul Barton, Gabriele Bugaisky, Arlette Cohen, Philippe Daubas,
Ian Garner, Adrian Minty, Benoit Robert and Andre" Weydert, Department of Molecular Biology, Pasteur
Institute, 25, rue du Dr. Roux, 75015 Paris, France
During the initial formation of skeletal muscle fibres and their subsequent maturation, different isoforms
of the contractile proteins accumulate. Using recombinant probes, the transcripts coding for actins, myosin
heavy chains and myosin alkali light chains have been characterized both at different stages of foetal muscle
development in the mouse in vivo, and at the onset of muscle fibre formation in a differentiating mouse
muscle cell line. In the case of the actin and myosin, light chain multigene families transcripts of a gene
expressed as a major species in adult cardiac tissue accumulate during skeletal muscle development,
although not with the same kinetics. In contrast, the myosin heavy chain family has developmental isoforms
specific to skeletal muscle; at birth in the mouse, a foetal mRNA is rapidly replaced by one encoding adult
myosin heavy chains. The strategy of gene expression during development is thus different for each of
these multi-gene families.
The organization of the genes concerned has been investigated using a genetic approach in the mouse.
Genes expressed in the same muscle phenotype are not linked. Genes within the actin or myosin light chain
families are also not linked. The two alkali light chains of adult skeletal muscle are a special case, since they
are encoded by a common 3' genomic region with two different contiguous 5' termini. Cardiac and skeletal
muscle myosin heavy chain genes map on different chromosomes, but there is evidence that the genes
expressed within these tissues during development may be linked. These results indicate that regulation is
trans-acting, with possible sequential activation of myosin heavy chain genes in cis, within a developing
muscle. The 5' upstream sequences of the actin and myosin genes and their differential response to
developmental signals are under investigation.
BUCKINGHAM, M. & MINTY, A. (1983). Contractile protein genes. In Eukaryotic Protein Genes (eds N.
MacLean, S. Gregory & R. Flavell), Ch. 21, pp. 365. London: Butterworths.
MINTY, A., ALONSO, S., GURNET, J.-L. & BUCKINGHAM, M. (1983). / . Moi Biol. 167, 77.
WEYDERT, A., DAUBAS, P., CARAVATTI, M., MINTY, A., BUGAISKY, G., COHEN, A., ROBERT, B. &
BUCKINGHAM, M. (1983). /. Biol. Chem. 258, 13867.
Specific genes in development
Gene hierarchies in developmental processes
131
Wilfred G. Chen, Vernon Scoon and Robert A. Reid, Carlton Medical Centre and Victoria Laboratory, San
Fernando, Trinidad, West Indies
Most theories of differentiation are based on the principle that the cytoplasms of early cleavage cells differ
in respect of endogenous chemical factors or chemical disparities created by positional effects, and that
these influence genes leading to amplification and diversification of differences in the resulting clones - a
process compounded and eventually stabilised and maintained by various short- and long-range interactions
between cells and tissues. On the basis of this model, we have attempted to review developmental processes
in terms of hierarchies of genes - the concept of key organisational genes, the products of which activate or
inhibit subsets of genes which may themselves influence, through their products, the expression of other
genes. The classic examples of primary and secondary responses of organisms to gene activation are the
effects of mammalian steroid hormones and the steroid insect moulting hormone ecdysone on the
transcription of primary target genes and the subsequent responses of other genes to the translation
products. A survey of a range of organisms suggests that these principles operate in developmental
processes from the earliest stages of embryogenesis through to the mature adult. The evidence for putative
hierarchial systems will be expressed in tabular form, some speculative hypotheses proposed and
experimental strategies for testing these discussed.
Histone gene expression during the cell cycle in avian erythroblasts
J. R. Coleman*1, A. J. Robins 2 andJ. R. E. Wells 2.1Division of Biology and Medicine, Brown University,
Providence, RI, U.S.A. 2 Department of Biochemistry, University of Adelaide, Adelaide, Australia
H2A.F is an extremely variant H2A core histone sequence discovered by screening a cDNA clone bank
constructed from embryonic chicken RNA (Harvey etal. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 2819.).
H2A.F is an unusual histone gene in that it contains introns (4), has a poorly conserved TATA box, lacks
the normal histone gene 3' terminator, and codes for a protein that diverges in 43 % of its amino-acid
residues from the most abundant avian H2A protein. Like histone H5 transcripts, H2A.F mRNA is
polyadenylated and may exhibit some tissue specificity in distribution. Using aphidicolin, a reversible
inhibitor of DNA polymerase alpha, we have investigated the cell cycle dependence of H2A.1, H5 and
H2A.F expression in an avian erythroblast cell line (Beug et al. (1982). /. Cell. Physiol. Suppl. 1, 195.).
Exposure to 5 /u-g/ml aphidicolin led to a 97 % inhibition of 3H-Tdr incorporation within minutes.
Cytoplasmic RNA blot analyses using nick-translated probes from cDNA clones demonstrated that H2A.1
transcripts had virtually disappeared after 2 h of aphidicolin treatment, whereas H2A.F and H5 transcripts
remained approximately
unchanged in abundance. Removal of aphidicolin after 21-24 h was followed by a
dramatic increase in 3H-Tdr incorporation and the re-appearance of a strong H2A.1 mRNA band. No
comparable increase in H2A.F or H5 mRNA abundance was seen. Thus H2A.F and H5 are unusual histone
genes in that their expression is not tightly coupled with DNA synthesis. While H5 is a linker histone that
replaces HI during erythroid cell differentiation and is postulated to play a role in the cessation of gene
activity, H2A.F is presumed to be a core histone that may contribute to an unusual nucleosome structure.
Antibody localization, in situ hybridization and gene transfer studies are being pursued to explore further
the functional significance of H2A.F expression.
132
Specific genes in development
Transcriptional expression of histone gene variants during development
of Xenop us
1
2
Olivier H. J. Destrie , Mary M. Bendig , Rosalia T. M. De Laafx and Johanna G. Koster x. lAnatomisch
Embryologisch Laboratorium, University of Amsterdam, Academisch Medisch Centrum MeibergdreeflS,
1105 AZ Amsterdam, The Netherlands. 2Celltech Limited, 250 Bath Road, Slough, Berkshire SL1 4DY
Using previously cloned Xenopus nucleosomal core histone genes as hybridization probes, a genomic
DNA library of Xenopus laevis was screened for histone gene clusters. From over 200 histone-gene
containing clones identified, 36 were selected as possibly containing HI histone genes by hybridization to a
probe derived from a sea-urchin HI histone gene. These 36 clones were further analyzed by hybrid-selected
translation for the definitive presence of HI histone genes. The genes for three different HI histone variants
were found: HI A, H1B and H1C. Mapping of the histone genes within each clone showed that at least three
different gene arrangements can occur within a cluster and that the type of HI histone variant present in a
cluster may be related to the cluster type. Sl-mapping experiments indicated that histone genes found in
different cluster-types can be expressed in oocytes. Also, the HI gene found in one cluster-type was
expressed in at least three different cell-types: oocytes, gastrula stage embryos, and erythroblasts.
The expression of several individual histone genes (from different clusters; nucleosomal and very
lysine-rich) was quantitated by hybridization of RNA blots at different formamide concentrations. On a
cellular basis, the overall expression of histone genes strongly increases during gastrula stage and slows
down from early neurula stage. An H1C gene is only expressed in later stages (taUbud), at a low level. In
testis only a few genes are expressed (of the Xl-hi-1 type). In erythroblasts the concentrations of H1B
mRNA is relatively high compared with other tissues/stages.
DNA strand break formation and the involvement of ADPRT activity in
cytodifferentiation
F. Farzaneh \ R. Zalin \ D. Brill \ S. Fton 2, J. C. David2 and S. Shall1.1Cell and Molecular Biology
Laboratory, University of Sussex, Brighton BN1 9QG. 2Laboratoire de Biochimie du Development,
University de Rennes, France
We have previously demonstrated that during the spontaneous differentiation of primary avian skeletal
myoblasts in culture, DNA strand breaks are formed. DNA strand breaks are also formed during the
induced differentiation of the human promyelocytic leukaemia cells, HL-60. The transient accumulation of
these DNA strand breaks is not due to a general deficiency in the DNA repair capacity of these cells. Both
dimethylsulphate and ^radiation induced DNA damage is repaired as efficiently in the post-mitotic
terminally differentiating cells as in their proliferating precursors. The chromatin enzyme, adenosine
diphosphoribosyl transferase (ADPRT) is involved in the religation of these strand breaks and the
expression of the differentiated phenotype in these cells.
ADPRT is present in all nucleated cells in animals, plants and lower organisms, with the exception of a
number of terminally differentiated cells in which the nuclei have ceased transcription. This enzyme, which
catalyses the formation of mono-, oligo- and poly (ADP-ribose) - modified chromatin proteins from NAD,
is entirely dependent on DNA containing strand breaks for its activity. Therefore, ADPRT activity is
stimulated by induction of DNA strand breaks; this activity is required for efficient DNA excision repair,
probably because it regulates DNA ligase and possibly topoisomerase activity. During the cytodifferentiation of primary avian myoblasts, in correlation with the formation of DNA strand breaks, there is an
increase in ADPRT activity. The inhibition of this activity by either enzyme inhibitors or substrate
deprivation (by nicotinamide starvation) inhibits the cytodifferentiation, but not the proliferation, of these
cells. ADPRT inhibitors also block the induced differentiation but not the proliferation of the HL-60 cells.
ADPRT activity is also required for the differentiation of Trypanosoma cruzi, mitogen stimulation of
human peripheral blood lymphocytes and the expression of foetal functions in cultured adult rat
hepatocytes.
Changes in DNA molecular weight during the cytodifferentiation of myoblasts, mitogen stimulation of
lymphocytes and the induced differentiation of promyelocytes suggests that DNA strand break formation
and rejoining, modulated by ADPRT activity, may be required in a general process involved in cellular
differentiation. Such a process may be gene amplification, DNA transpositions, and/or regional chromatin
relaxations mediated by topoisomerase activity.
Specific genes in development
Transcription of RNA sequences coding for ribosomal proteins in
lampbrush chromosomes
133
Tiziano Fulceri, Giovanna Giovinazzo and Giuseppina Barsacchi-Pilone, Istituto di Istologia ed
Embriologia, via A. Volta4, 56100 Pisa, Italy
We are studying the nature of transcripts of lampbrush chromosomes of Triturus by hybridizing in situ
H-labeled probes to the RNA fibrils attached on the loops. 3H cRNA was synthesized in vitro from cloned
cDNA sequences coding for ribosomal proteins. The clones are pXom 62, pXom 92 and pXom 102 from
Xenopus laevis, which code for ribosomal proteins S8, L14 and LI, respectively (Amaldi et al. 1982). The
Triturus species tested are vulgaris meridionalis and cristatus carnifex.
Autoradiographically detectable hybrids were found at specific loops in each case, with the number of
labeled loops ranging around ten. In either species, the labeled loops include both normal and landmark
loops, and may appear in a heterozygous condition. In some instances the labeling covers part of the loop
axis only, suggesting that the probed sequence is not transcribed within all the transcription units of the
same loop.
Experiments are under way to define the characteristics of the transcription of ribosomal protein genes,
during the lampbrush stage of oogenesis.
AMALDI, F., BECCARI, E., BOZZONI, I., Luo, Z. X. & PTERANDREI-AMALDI, P. (1982). Nucleotide sequences
of cloned cDNA fragments specific for six Xenopus laevisribosomalproteins. Gene 17, 311-316.
3
Comparative analysis of the 5'-end regions of the vitellogenin genes
from Xenopus and chicken
Jacques-Edouard Germond, Philippe Walker, Marianne Brown-Luedi and Walter Wahli, University de
Lausanne, Faculte" des Sciences, Institut de Biologie animale, CH-1015 Lausanne, Switzerland
In Xenopus laevis and other oviparous vertebrates, vitellogenin, the precursor of the major yolk proteins
is synthesized in the liver of mature females. Vitellogenin synthesis in Xenopus isfirstinducible by oestrogen
at the metamorphic stage 62. Vitellogenesis is thus a suitable system in which to study developmentally and
hormonally regulated gene expression. In Xenopus laevis, four vitellogenin genes have been identified and
characterized. The DNA sequence of their 5'-end regions have been determined, including putative
regulatory sequences upstream of the initiation site of transcription and extending through the first three
exons. These sequences have been compared to the corresponding region of a chicken vitellogenin gene.
The lengths of the three exons analyzed are identical in allfivegenes, namely 53, 21 and 152 bp for Exon I,
II and III, respectively. Divergences in the coding regions show that nucleotide substitutions resulting in
amino-acid replacements accumulate linearly with time. Based on this observation, it is postulated that the
first vitellogenin gene duplication event in Xenopus occurred about 150 million years ago. The accumulation
rate of amino-acid replacement changes suggests that the selective constraints acting upon the analyzed part
of the genes are similar to those acting on globin genes. Blocks of homology, including a palindromic
structure, have been localized in the upstream flanking regions. They may reveal sequences which play an
important role in the control mechanisms of hormonally and developmentally regulated gene expression.
134
Specific genes in development
Molecular analysis of erythropoiesis
P. R. Harrison*, N. Affara, P. S. Goldfarb, K. Kasturi, E. Black, J. Fleming and J. O'Prey, The Beatson
Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G611BD
Erythropoiesis involves the sequential expression of a series of characteristic red cell proteins during
differentiation of committed erythroid progenitor cells, proteins such as spectrin and glycophorin, followed
by the globins, haem and other characteristic enzymes such as carbonic anhydrase, catalase and a specific
lipoxygenase. It had been generally believed that spectrin and glycophorin, like the globins, were specific to
red cells. But recent evidenccby our group and others has shown that spectrin and possibly glycophorin-like
molecules exist in certain other cells (1). This implies that red cell differentiation involves the tissue-specific
regulation of gene families coding for spectrin and possibly glycophorin and other red cell proteins.
A major aspect of our recent work has involved cloning genes or mRNAs coding for characteristic red cell
proteins (2, 3) with a view to elucidating the chromosomal and transcriptional changes involved in the
expression of these genes during red cell differentiation. In particular, we have obtained recombinant
probes for a gene coding for a 19 K polypeptide (epl9) expressed preferentially in erythroblasts as well as in
liver and kidney cells whereas its level in most other tissues is much lower. We had also obtained
recombinant probes for a red cell lipoxygenase mRNA and probably also for a glycophorin gene.
We have used such probes to study the molecular/genetic mechanisms of co-ordinate control of globin and
non-globin genes by measuring their nuclease sensitivity, DNA methylation and transcription patterns in
cell hybrids between erythroblasts and other cell types. Our recent results indicate that the globin and epl9
mRNAs are expressed or repressed in a co-ordinate manner in cell hybrids between erythroblasts (Friend
cells) and haemopoietic (T-lymphoma) or neuroblastoma cell lines respectively. Moreover, the trans-acting
factors involved in suppression of the globin and epl9 genes in the Friend cell X neuroblastoma hybrids act
at the level of the nuclease-sensitive chromatin changes (hypersensitive sites) associated with the activation
of these genes. We are currently testing for regulatory elements surrounding these co-ordinately regulated
genes.
(1) KASTURI et al. (1983). Exp. Cell Res. 144, 241.
(2) AFFARA et al. (1983). Nucl. Acids Res. 11, 931.
(3) GOLDFARB et al. (1983). Nucl. Acids Res. 11, 3517.
Regulation and products of the bithorax complex in Drosophila
melanogaster
DavidS. Hogness*, Philip A. Beachy, Elizabeth Gavis, Peter Harte, Howard Lipshitz, Renato Paro, Debra
Peattie and Shigeru Sakonju, Department of Biochemistry, Stanford University, School of Medicine,
Stanford, CA 94305, U.S.A.
The bithorax complex (BX-C) is defined by a set of homeotic mutations that cause intersegmental
transformations of body parts in the thoracic and abdominal segments of D. melanogaster (reviewed in
Laurence, P. A. and Morata, G. (1983). Cell 35, 595-601.). Isolation of the BX-C DNA and molecular
mapping of its mutations (Bender, W., Spierer, P. and Hogness, D. S. (1983). 7. Mol. Biol. 168, 17-33;
Bender, W., Akam, M., Karch, F., Beachy, P. A., Peifer, M., Spierer, P., Lewis, E. B. and Hogness, D. S.
(1983). Science 221,23-29.), indicates its encompasses -300 kb of DNA. This DNA can be divided into two
parts: a 'Ubx domain' whose functions are required for normal development of the 2nd and 3rd thoracic
segments (T2, T3) and the 1st abdominal segment (Al), and an 'abdominal domain' whose functions are
required for the development of the remaining abdominal segments (A2-A8). The Ubx domain is divided
into the 75 kb Ubx and 25 kb bxd units which are transcription units that encompass the Ubx and bxd
mutations, respectively. The bxd unit is within the Ubx domain because
Ubx mutations inactivate in cis the
same functions as are inactivated by bxd mutations (i.e. the pbx+ and bxd+ functions of the posterior
compartment of T3 and anterior compartment of Al, respectively). Indeed, Ubx mutations inactivate
in cis
all known functions of the Ubx domain, which in+ addition+ to those of the bxd unit, include the ppx+ function
of the posterior compartment of T2 and the abx and bx functions of the anterior compartment of T3. We
report here on the nature and distribution during development of the multiple RNA and protein products
that derive from each of the transcription units in the Ubx domain. We also report on a preliminary
definition of the transcription units in the larger but mutationally less well defined abdominal domain.
Finally, we have begun a molecular analysis of the Poly comb gene, which was initially isolated by J. Lauer
and whose product(s) appears to act as transregulator(s) of BX-C expression.
Specific genes in development
135
The evolution of yolk-protein genes in sibling species of Drosophila
melanogaster
R. KozmaandM. Bownes, Department of Molecular Biology, King's Buildings, Edinburgh University,
MayfieldRoad, Edinburgh EH9 3JR
The yolk-protein genes and yolk-proteins of seven sibling species of Drosophila melanogaster have been
compared as a basis for an evolutionary study. These are D. yakuba, D. erecta, D. teissieri, D. mauritiana,
D. simulans, D. orena and D. melanogaster. Comparisons were made with D. melanogaster for which we
have yolk-protein antibodies, and cloned DNA sequences coding for the three yolk-proteins (YP1, YP2 and
YP3) which can be used as probes to compare DNA and RNA sequences.
1. The cross-reactivity of haemolymph YPs for the different species was compared, using antibodies raised
against D. melanogaster yolk-proteins, by SDS polyacrylamide gel electrophoresis.
2. Genomic DNA restriction patterns for each of the three YP-genes were analysed for different species
using Southern blotting.
3. In situ hybridization of the three D. melanogaster cloned YP-genes to polytene chromosomes of all
species were carried out to determine the locations of each of the genes.
YP1, YP2 and YP3-like genes have been identified in all the sibling species. All these genes appear to be
single-copy and to have an X-chromosome location. Also YP1 and YP2-like genes appear to be closely
linked in these sibling species. The results suggest that phylogenetic trees will vary according to the criteria
used for their construction.
Changes in the pattern of rDNA spacer lengths in ontogenesis of
Triturus vulgar is
Karin Kraus and Klaus Lohmann, Institutfur Biologie I, Universitdt Freiburg, Albertstrasse 21a, D-7800
Freiburg, West Germany
In most eukaryotes the coding regions of the repeated rRNA cistrons are separated from their neighbours
by non-transcribed spacers (NTS). In the present study we investigated the Eco RI restriction pattern of
rDNA of Triturus vulgaris and of some other urodeles by Southern transfer and hybridization with
nick-translated Xenopus rDNA (cloned by P. Wellauer) isolated from the plasmids pXlrll and pXlrl2.
In order to study the inter-individual spacer heterogeneity, the offspring of single females were analysed.
The results show that there is a large inter-individual variability in the tested population of T. vulgaris with
about 12 spacer length classes varying between 8 and 13 kilobases. The intra-indiyidual heterogeneity,
however, is rather limited (2-5 bands only). In some cases remarkable differences in the pattern of size
classes as well as in the relative abundance of different fragments between the progeny of the same female
were observed. To test for the possibility that these differences might be caused by somatic changes, the
rDNA patterns from various organs of single animals were estimated. Thereby it became evident (in four of
five cases studied) that the pattern of ribosomal genes is not constant but varies within the same animal,
probably due to differential replication in early development.
After hybridization with r l l , the 28S coding fragments become visible in two bands, a prominent one of
5-3 kb and a weak band of 5-9 kb. As shown by restriction mapping, the 5-9 kb fragments are interrupted by
an intron, 0-6 kb in length, located in a 1-6 kb Bgl II fragment at the 3' end of the Eco RI fragment. This is,
so far as is known, the first evidence for the existence of introns in the ribosomal DNA of vertebrates.
Furthermore, it has been shown by the comparison of some other urodeles that the Xenopus model of
rapid divergency of spacer sequences in evolution cannot be generalized. Then, in T. helveticus and in
Ambystoma mexicanum the rDNA spacers are homogeneous in size (11-5 kb and 4-0 kb, respectively). This
is an unexpected result since homogeneous spacers were found in some insects only. Supported by the DFG
(Lo 194/4-2).
136
Specific genes in development
Expression of DNA ligase genes by Ram spermatid nuclei implanted in
amphibian eggs
M. Loir x, J. Lefresne 2, /. Signoret 2 andJ. C. David 3 . xLaboratoire de Physiologie des Poissons INRA
Campus de Beaulieu, Rennes, France. 2Laboratoire de Biologie du Diveloppement University de Caen 14032
Caen, France. 3Laboratoire de Biochimie du Diveloppement LA CNRS256 University de Rennes I, Campus
de Beaulieu, 35042 Rennes, France
Two consecutive forms of DNA ligase (6,3S and 7,5S) can be found during Ram germ cell maturation.
The 6,3s form is observed in spermatocytes and round spermatids. Nuclei of spermatocytes and spermatids
have been injected into activated eggs of Axolotl or Pleurodeles, either enucleated or not. Besides the
specific DNA ligase of the recipient system, an additional peak of enzymatic activity is evident on sucrose
gradient analysis.
This peak corresponds to a sedimentation coefficient characteristic of Ram donor cells and is different
from recipient eggs. Moreover, the activity is insensitive to antisera directed against Axolotl DNA ligases.
This result is interpreted as a consequence of gene expression of Ram nuclei in Amphibian cytoplasm.
The alternative form produced after nuclear transplantation - either 6,3 or 7,5S - is in agreement with the
situation observed in donor germ cells.
The methylation pattern of tRNA genes in Xenopus laevis
N. Maclean* andS. Talwar, Department of Biology, Southampton University, Southampton, Hants SO9
3TU
The methylation sensitive restriction enzymes Hha I and Hpa II were used to analyse the methylation
pattern of a tRNA gene cluster in germ line and somatic DNA of Xenopus laevis. A single tDNA repeat
containing 8 tRNA genes was studied and all copies were found to be fully modified in sperm DNA. In the
DNA from erythroid cells, hepatocytes, kidney and brain, most tDNA repeats were found to be fully
modified. However, in a fraction of the repeats, specific demethylated sites can be detected, giving rise to a
pattern which does not vary significantly from one tissue to another. Although our results do not allow a
straight forward correlation between hypomethylation and tRNA gene transcription, they are in agreement
with the observation that hypomethylation accompanies differentiation and development.
Other evidence, suggesting that DNA methylation plays a causative role in gene regulation during
development, will be briefly reviewed.
TALWAR, S., POCKLINGTON, M. J. & MACLEAN, N. (1984). Nuc. Acids Res. 12, 2509-2517.
Specific genes in development
137
Haemoglobin transition in Xenopus laevis is controlled at the cellular
level and occurs precociously after induction of anemia
R. P Muller, H. J. Widmer and R. Weber, Zoologisches lnstitut, Universitat Bern, CH-3012 Bern,
Switzerland
In Xenopus laevis, haemoglobin transition occurs at metamorphosis and involves complete replacement
of larval by adult haemoglobins, indicating a co-ordinate switch in the activity of the genes coding for larval
and adult globin subunits. In situ hybridization of cloned cDNAs to erythroblasts of anemic larvae at
metamorphosis revealed the existence of two types of erythroid-like cells containing either larval or adult
globin mRNAs. This suggests that haemoglobin transition is controlled at the cellular level in that cells
which express the larval genes are replaced by new ones committed to express the adult genes.
To determine whether the change in globin synthesis is due to gene switching in a single cell population,
larval or the
anemia,
a transient
increase in labelling of erythroid-like cells was observed in the liver and the peripheral blood, the peak at
three days after induction of anemia coinciding with enhanced mitotic activity of these cells. This indicates
that the liver and the peripheral blood are the main sources of larval erythroid cells. In situ hybridization
with cloned cDNA-probes revealed that these cells are already committed to express the larval globin genes.
Anemia does not change the qualitative pattern of transcribed mRNA species, but causes precocious
transition from larval to adult globin gene expression without any sign of morphological metamorphosis.
The distribution of lactate dehydrogenase isozymes in Xenopus laevis
tadpole tail
Louise Muntz, Janet E. Hornby, Patricia A. J. Gough, Department of Pure and Applied Zoology, The
University, Whiteknights, Reading, Berkshire RG6 2AJ
Observation of living Xenopus laevis tadpoles shows that, just as the heart beats rhythmically, so the tip of
the tadpole tailflickerscontinuously, to maintain the tadpole's feeding position in mid-water. The rest of the
tail is only used for sudden swimming movements. Histological and ultrastructural studies have shown that
this behaviour correlates with the distribution of red and white muscle in the tail (Dalooi, 1977).
An electrophoretic comparison of tissue homogenates from Xenopus tradpole hearts, tail tip and tail base
has been made using Titan III cellulose acetate Zip Zone system (Helena). The base of the tadpole tail,
containing mainly white striated muscle, has five main lactate dehydrogenase (LDH) isozymes; in older
tadpoles, the major isozyme in the tail base is the slowest moving isozyme, as found in mammalian skeletal
muscle. By contrast, the heart, as in mammals, contains only the faster isozymes, with the fastest isozyme
being the major component. The major isozyme in the tip of the tail, containing mainly red striated muscle,
is the fastest isozyme, as in the heart, but the other LDH isozymes are present in small quantities. A
developmental study shows that in the base of the younger tails the faster isozymes are at slightly higher
concentrations than in older tails.
Comparisons are being made between Xenopus tadpole tails and those of other amphibian species with
different swimming behaviour; and adult amphibian heart and skeletal muscle.
The LDH isozymes of mouse kidney have been used as a standard for electrophoresis of the amphibian
LDH isozymes. The amphibian isozymes show a similar pattern of five main bands but seem to be slower
and have a smaller range of mobility than the mouse isozymes.
DALOOI, M. R. K. (1977). A study of the notochord, musculature and nervous system of the tail of Xenopus
laevis (Daudin, 1802) with particular reference to flickering movement. Ph.D. Thesis, Department of
Zoology, University of Reading.
138
Specific genes in development
Tissue-specific expression of 6-crystallin gene as demonstrated by the
intranuclear injection of the cloned genes
T. S. Okada*, Hisato Kondoh, Shigeo Hayashi and Kunio Yasuda, Institute for Biophysics, Faculty of
Science, University of Kyoto, Kyoto, Japan
Crystallins are a group of proteins known to be synthesized actively only in lens. Among them, 6crystallin is a highly abundant protein in chicken lens and has been extensively utilized as a molecular
marker for studying lens differentiation. We have injected the cloned genes of chicken 6-crystallin, which
contain a continuous stretch of this particular gene, into nuclei of various mouse cells in primary cultures.
Since 6-crystallin is totally absent in mice cells, such xenogenic injection system should permit us to detect
the transient expression of injected genes, if any, in a highly specific manner. The following results have
been obtained.
(1) The cloned 6-crystallin gene of chicken is expressed, though transiently, in the mouse lens epithelial
cells as efficiently as in the homologous chicken lens cells with a correct splicing of the gene transcripts. (2)
The expression is relatively 'tissue-specific', i.e. it is most inefficient in the non-lens tissues examined. (3)
Experiments with the promotor-replaced 6-crystallin gene indicate that regions located upstream from the
5' ends are responsible for the 'tissue-specific' expression. (4) Deletion mapping analysis revealed that the
region responsible for the expression resides between -50 and -100 bp upstream to the first exone.
The present system provides the first demonstration of the preferential expression of introduced cloned
genes in the homologous cell types of different species and could be used to study the 'tissue-specific'
expression of specific genes in cell differentiation.
Methylation of specific sequences in male mouse germ-line versus
somatic cell DNAs
Carola Ponzetto-Zimmerman and Debra J. Wolgemuth, Department of Human Genetics and Development
and The Center for Reproductive Sciences, Columbia University College of Physicians and Surgeons, 630
West 168th Street, New York, NY 10032, U.S.A.
During spermatogenesis, there exists the unique situation of the regulation of genes involved in meiosis
concomitant with the processing of the germ cell DNA to serve as a totipotent genome for the early embryo.
A property of the genome that has been the focus of some of our investigations in this regard is that of
methylation of cytosine residues. Recently, we showed that subsets of the mouse major satellite sequences
are undermethylated in germ cell versus somatic cell DNA (Nucl. Acids Res. 12, 2807-2822). This
under-methylation was not characteristic of all repetitive elements, since 'R'-type sequences, believed to be
part of the LIMd family of interspersed repeat sequences, were similarly methylated in somatic and
spermatogenic DNAs. We have extended this study to include unique (or low-copy) and moderately
repetitive sequences, with an emphasis on sequences which are expressed in the testis. To this end, we
analyzed DNAs from cells in various stages of spermatogenesis, as well as from mature spermatozoa, and
compared the methylation patterns with DNA from somatic tissues. Cells were purified by sedimentation at
unit gravity in a Celsep apparatus into pachytene spermatocyte, early spermatid, and late spermatid
populations. DNAs were isolated from these separated cells, from mature sperm from the cauda epididymis
and vas deferens, and from liver and brain, digested with the methylation-sensitive isoschizomers Msp I and
Hpa II, and subjected to Southern blot analysis using cloned DNA probes. Albumin, a gene not transcribed
in the male germ-line, was more heavily methylated in the germ line DNAs than in liver DNA. B-tubulin
was also more heavily methylated in the spermatogenic DNAs, as compared to liver DNA, even though the
gene is expressed in both cell types. In contrast, B-actin is relatively hypomethylated in spermatogenic
versus liver DNA. Two conclusions may be drawn to date. First, there does not appear to be a clear
correlation between the level of methylation and transcriptional activity in the germ-line of the genes
examined. This must be qualified, however, in that only those sequences which contain Msp I-Hpa II sites
would have been detected. Second, at least with respect to the housekeeping genes studied thus far,
methylation patterns do not appear to vary within the stages of spermatogenesis, even though there are
differences in the transcriptional activities.
Specific genes in development
139
Isolation and characterisation of cell-type specific cDNA clones from
Dictyostelium discoideum
A. J. Richards, T. Phillpots, A. J. Corney andB. D. Homes, Department of Biology, University of Leeds,
Leeds LS2 9JT
A major attraction of Dictyostelium discoideum as a system for the analysis of development is that
essentially only two cell types are produced: spore and stalk cells. Isolation and characterisation of genes
expressed in only one cell type from this organism, therefore, should allow elucidation of the regulatory
mechanisms which lead to cell-type specific gene expression.
After starvation, D. discoideum amoebae aggregate to form multicellular pseudoplasmodia which
differentiate into fruiting bodies consisting of spore and stalk cells. Prespore and prestalk cells (the
precursors of spore and stalk cells) are first observed after the formation of pseudoplasmodia (~ 12 h) and
terminal differentiation of spore and stalk cells begins during culmination (~ 18-20 h).
A cDNA clone bank was thus constructed using 18 h poly (A) + mRNA. We have isolated 49 cDNA
clones encoding mRNAs absent prior to 13 h of development but which then accumulate to high levels in
only spore cells during late development. Using Southern blot analysis these clones have been grouped into
22 families with 1-17 members per family. The size of the polypeptide encoded by each family of clones has
been determined using hybrid selection.
Northern blot analysis has been used to determine the kinetics of accumulation during development of
mRNAs corresponding to the cDNA clone families. This technique has also been used to determine the
effect of known regulators of developmental gene expression on the accumulation profiles.
Stalk-specific and non-cell type specific clones have also been isolated and should prove valuable for
future analyses of the regulation of spore-specific gene expression.
Inhibition of terminal deoxynucleotidyl transferase, DNA polymerases oc
and /3 but not DNA ligases by nalidixic acid, oxolonic acid and pefloxacin
in chicken thymus
R. Rusquet and J. C. David, Laboratoire de Biochimie du Dtveloppement, University de Rennes I, Campus
de Beaulieu, 35042 Rennes, France
DNA polymerases a and )3, Terminal deoxynucleotydil Transferase (TdT) and DNA ligases have been
purified to homogeneity from chicken thymus. Quinolone inhibitors of replication (nalidixic acid, NA,
oxolinic acid, OA, pefloxacin, PA) are potent inhibitors of DNA polymerases a and 0 as well as TdT.
DNA polymerase a is inhibited by a non-competitive mechanism while DNA polymerase /Hs inhibited by
a competitive mechanism with Ki of respectively 0-8, 1, 1-4 mM for NA, OA and PA. TdT
is inhibited
either through a competitive mechanism if dpAi0 is used as substrate with Kj of l-10~\ 10~5 and 2-10"4 M
respectively for these inhibitors. When d GTP is used as substrate, the inhibition is uncompetitive. DNA
ligases are not inhibited.
Since these quinolone antibiotics are specific inhibitors of DNA replication, the question is raised as to
why they inhibit both DNA polymerase ex and f$ and not DNA ligases in eucaryotic cells. Moreover, the
major question is why these drugs are inhibitors of TdT, an enzyme with very unclear functions. These
results are discussed in terms of the possible involvement of this enzyme in DNA replication and repair.
140
Specific genes in development
Stability of the regulated state of the gene for DNA ligase following
nuclear transplantation in Amphibia
/. Signoret1, J. C. David 2 andJ. Lefresne l. 1Laboratoire de Biologie du Dtveloppement, University de
Caen, 14032 Caen, France. 2Laboratoire de Biochimie du diveloppement, University de Rennes, Campus
Beaulieu, 35042 Rennes, France
The gene for the heavy form of DNA ligase can be found either activated or inactivated in particular cell
types. The enzyme possesses specific molecular variants among species that unable to identify the specific
origin of any peak of DNA ligase activity in a complex system.
Nuclear transplantation in Urodele egg cytoplasm have established that the transferred nuclei always
retain the activated or inactivated state of the gene exhibited before the operation. The enzymatic pattern of
the host cytoplasm and the activity or inactivity of the native gene have no influence on the expression of the
implanted gene.
A stable and autoreproducible change occurs in the nucleus when the gene is activated under the influence
of a critical cytoplasmic event. Nevertheless this trigger is not sufficient by itself and the nucleus needs to be
in a sensitive state such as the female pronucleus. Another nucleus exposed to the same cytoplasmic
influence can remain with its gene for the heavy form of DNA ligase in the inactivated state. The change
from the inactivated to the activated state of the gene has been experimentally induced in 'non-sensitive'
nuclei by mild treatments considered to displace some protein components from native chromatin.
Thus the genetic properties of the nucleus are subject to specific modifications under the action of natural
or experimental factors. This nuclear differentiation, far from irreversible, yet possesses a significant degree
of stability following nuclear transplantation and cell multiplication.
Amelogenin gene expression during mouse molar organogenesis
H. C. Slavkin*, M. L. Snead, M. Zeichner-David, C. Bessem and P. Bringas, Jr., Laboratory for
Developmental Biology, School of Dentistry, University of Southern California, Los Angeles,
Calif. 90089, U.S.A.
Epithelial-mesenchymal interactions have been found to mediate mouse molar tooth organogenesis.
During embryogenesis, cranial neural crest-derived ectomesenchyme cells differentiate into dental
ectomesenchyme which in turn mediates tooth morphogenesis, histogenesis and cytodifferentiation. We are
interested in when#and how dental ectomesenchymal cells induce terminal epithelial differentiation and the
differential gene expression of amelogenin polypeptides. To identify when inner enamel epithelial cells first
express enamel-specific gene products, we have analyzed cytoplasmic RNA from developing mouse molar
tooth organs (e.g. cap stages through crown stages of tooth organogenesis) by hybridization to a cloned
cDNA probe (pm5/5). In addition to Northern blot analyses of molar tooth organs from timed-pregnant
Swiss Webster mice ranging from 16-days' gestation through 2-days' postnatal age, we also used
monospecific polyclonal antibodies with indirect immunofluorescent microscopy, immunoprecipitation of
metabolically labeled epithelial-specific proteins, one- and two-dimensional slab gel electrophoresis and
electrophoretic transfer to nitrocellulose membranes and immunodetection assays. We now report that the
de novo expression of amelogenin-encoding mRNAs as well as immunodetection of amelogenin
polypeptides were first detected at Theiler stage 27 during mouse embryogenesis. Our data suggests that
dental ectomesenchyme-derived induction of adjacent inner enamel epithelia results in both nascent
transcription and translation of amelogenin gene products. The expression or consequence of
ectomesenchyme-mediated induction in the mouse mandibular first molar takes place at birth in vivo.
Comparable studies carried out in vitro using serumless, chemically defined media suggest that amelogenin
gene expression does not require hormonal mediators. We further suggest, therefore, that amelogenin gene
expression in terms of transcription and translation is not under strict hormonal regulation and that the
putative ectomesenchyme-derived inductive signals may indeed modulate differential gene expression in a
responding epithelia.
These studies in part were supported by research grants DE-02848 and DE-07006, NIDR, NIH, USPHS.
Specific genes in development
The metabolism of 20-OH ecdysone in adult male Drosophila
141
T. Smith and M. Bownes, Department of Molecular Biology, University of Edinburgh, MayfieldRoad,
Edinburgh, EH93JR
The adult male Drosophila provides an intriguing model for the study of the induction of yolk protein
synthesis. The female begins to produce yolk proteins soon after emergence, but in the male the yolk
protein genes are not normally expressed. The fat bodies of adult males, however, can be stimulated to
produce yolk proteins by the injection of 20-OH ecdysone (500 ng is routinely used). The rate of yolk
protein synthesis reaches a peak after 8-12 h and then declines to undetectable levels by 24 h. This is in
contrast to the female in which yolk protein synthesis continues for several days after emergence.
Can the rapid decline in the rate of synthesis in the male be explained by loss of 20-OH ecdysone
following injection? In order to investigate this possibility, the rate of metabolism of 20-OH ecdysone,
following its injection into adult flies, was determined. Within ten minutes, most of the 20-OH ecdysone had
been metabolised into compounds of higher polarity. By 4 h, none of the original 20-OH ecdysone remained
and by 24 h, the total levels of ecdysteroid were down to pre-injection values.
In the female, the initial rate of metabolism was not so rapid. No highly polar metabolites were seen
within the first ten minutes; although at this time up to 10 % of the total steroid recovered consisted of a less
polar metabolite migrating in the same position as a-ecdysone.
Isolated fly regions cultured under conditions previously shown to be capable of supporting protein
synthesis failed to metabolise 20-OH ecdysone to the more polar compounds observed in the intact fly,
although again a less polar metabolite was observed. This is tentatively identified as 20-OH ecdysone acetate.
If the continued presence of high levels of 20-OH ecdysone are required for yolk protein synthesis in the
male fly, the rapid metabolism of 20-OH ecdysone to levels below those which need to be injected to
produce the initial stimulation may explain why yolk protein synthesis in the male is not maintained for
longer than 24 h. Mass spectrometry is being employed in an effort to identify the metabolites.
Organization and expression of the globin gene family in Xenopus laevis
J. Stalder*1, W. Meyerhof2, H. Widmer 2, H. Hosbach \ E. Sandmeier \ W. Knochel 2 and R. Weber \
1
Zoologisches Institut, Universitdt Bern, CH-3012 Bern, Switzerland. 2Institutfur Biochemie und
Molekularbiologie FU Berlin, D-1000 Berlin, Germany
In all vertebrates studied so far, hemoglobin synthesis undergoes sequential changes during development
reflecting switches in the expression of the globin genes which encode the polypeptide subunits of
hemoglobins. In amphibians, hemoglobin transition occurs at metamorphosis and is characterized by the
simultaneous replacement of larval by adult globin chains. Therefore, the Xenopus laevis globin system is an
attractive model for studies on the control mechanisms
of developmentally regulated genes.
Analysis of cDNA clones derived from 9 S poly(A)+ RNA from erythroblasts of anemic animals revealed
that in both the larval and the adult stage two main groups of unrelated globin sequences are expressed,
each comprising two closely related a- and 0-sequences.
From two genomic libraries, 9 different larval and adult globin genes have been isolated, and from genetic
analysis, there is evidence for two additional larval fi genes. Together with the adult j3 gene described by
Jeffreys et al. (1980) the X. laevis globin gene family thus comprises 12 genes arranged in two clusters (I and
II), each containing larval (L) and adult (A) globin genes in the same order:
5'_QrL_arL_aA_£A . ^ L ^ l ^ ,
The corresponding globin genes of each cluster are expressed coordinately. To detect putative control
regions of coordinately expressed genes we determined the nucleotide sequence within 1 kb 5' to the a*and
the an genes. We found a relatively conserved region from the cap site to position -290 and 3 boxes of
homology further upstream, each separated by more diverged sequences or regions representing either
deletions or insertions.
Comparison of the 5' flanking region of the crfgene in genomic DNA of tailbud embryos and an adult frog
showed identical sequences up to position -639 except for 9 base changes presumably due to polymorphism. This suggests that transition from the inactive to the active stage of the af gene does not
involve sequence rearrangement in this region. Analysis of chromatin conformation further revealed that
DNase I cuts specifically DNA 5' to the TATA and CCAAT boxes only in chromatin of adult erythrocytes
but not in the chromatin of liver cells or larval erythrocytes, suggesting a correlation between gene activity
and DNase I hypersensitivity.
142
Specific genes in development
Developmental abnormalities of the cochlea in two mouse mutants:
deafness and viable dominant spotting
Karen P. Steel and Gregory R. Bock, MRC Institute of Hearing Research, University Park,
Nottingham NG72RD
Several mouse mutants which originally appeared to show degenerative changes of the inner ear have now
been shown by transmission electron microscopy to have developmental abnormalities in the late stages of
cell differentiation. These mutants fall into two categories: those with neuroepithelial defects, in which the
sensory epithelium is primarily affected and those with cochleo-saccular defects, in which the initial
cochlear abnormality occurs in the stria vascularis.
The deafness mutant (dn/dn) belongs to the neuroepithelial category. Cochlear hair cells do not develop
into their normal mature shape but remain short, and very few synapses with nerve endings are found. Fluid
spaces between hair cells in the organ of Corti are very retarded in opening. Hair cell degeneration follows
these developmental anomalies. The stria vascularis, however, has a normal appearance. These
ultrastructural observations correlate well with physiological data, since the endocochlear d.c. resting
potential (EP), which is produced by the stria vascularis, is of normal magnitude in deafness mice, while
there are no signs of hair cell function or cochlear nerve activity in response to sound stimuli at any stage
during development.
The viable dominant spotting mutant (W/W) belongs to the cochleo-saccular group. The stria vascularis
is thinner than normal and contains very few blood capillaries. This defect may be related to the known
abnormality of neural crest cells in this mutant, since neural crest cells are believed to form the intermediate
cell layer in the normal stria. In 14 out of 16 of these mutants so far examined, no EP could be recorded,
suggesting strial dysfunction. On the other hand, hair cells develop normally, although many outer hair cells
later degenerate. Vf/W mice do show signs of cochlear nerve activity, but respond to sounds at high
intensities only. This mutant may be useful for studying the significance of the endocochlear potential in
normal hearing processes.
Isolation and partial purification of the mRNA coding for Axolotl DNA
ligase 8S and its cell-free translation
P. Thiebaud 1, J. Lefresne 2, J. Signoret2 andJ. C. David 1. lLaboratoire de Biochimie du Diveloppement,
University de Rennes I, Campus de Beaulieu, 35042 Rennes, France. 2Laboratoire de Biologie du
Diveloppement, University de Caen, 14000 Caen, France
Two independent successive forms of DNA ligase are normally present during the Axolotl egg
development. A 8S form is expressed at the first cleavage and it results from a new transcription. This
observation suggested that the 8S DNA ligase messenger RNA might be isolated.
Activated or fertilized eggs can incorporate (3H) undine in a RNA peak which contains a RNA
population (24S) coding for 8S DNA ligase. The poly (A + ) RNA of this population has been purified using
oligo dT cellulose chromatography. The purified mRNA can translate for a 160 K DNA ligase which is
specifically precipitated by the anti-ligase 8S antibody. Surprisingly, the 8S DNA ligase synthesized in vitro
displays a catalytic activity. This protein synthesis results from a new genetic activity. To our knowledge, the
appearance of 8S DNA ligase in Axolotl eggs is the earliest non-maternal genetic event during development.
Specific genes in development
143
Mitochondrial gene expression during oogenesis in Xenopus laevis
Andrew C. Webb, Deborah A. Lucas and Melinda S. Gandy, Department of Biological Sciences, Wellesley
College, Wellesley, Massachusetts 02181, U.S.A.
Mitochondrial DNA (mtDNA) accounts for 99 % of the frog oocyte DNA content. Therefore,
mitochondrial gene transcripts represent a major contribution to the maternally derived mRNA stockpile
bequeathed to the developing embryo.
Three cDNAs cloned from Xenopus ovarian pA + RNA (Golden et al. 1980) mapped to adjacent loci on
the 5-6 kb Cfol (HhaT) fragment of mtDNA (0-6-0-85 map units) and were tentatively identified as
complementary to the genes for cytochrome C oxidase (CO) subunits I and II and ATPase (A) subunit 6 by
comparisons with the mammalian mitochondrial genome (e.g. transcript sizes, relative map positions). The
transcription map of the 5-6 kb Cfol fragment also revealed an 800 bp intergenic sequence between the
COII and A6 loci that is absent from mammalian mtDNA.
Evidence of non-co-ordinated expression of these three mitochondrial genes during Xenopus oogenesis
was obtained after hybridization of nick-translated cDNAs to total pA RNA extracted from different
oocyte stages. Relative
quantities of transcripts were assessed by probing (i) Northern blots of CH3HgOH
gels loaded with 3H-poly(U)-titrated samples of oocyte pA + RNA, and (ii) Nal solubilized oocyte
mitochondrial extracts immobilized onto nitrocellulose filters. Although mRNA levels for COI and COII
seem to be co-ordinated throughout oogenesis, the abundance of A6 transcripts appears to be independent
of CO expression. Uncoupling of genes in this fashion implies either differential transcription or unequal
stability of their transcripts. Dideoxynucleotide sequencing of the entire 5-6 kb Cfol fragment and these
three cDNAs has been undertaken to (a) confirm the identity and alignment of the genes, (b) examine the
extent of sequence homology with their counterparts in other phyla, and (c) establish the nature of the 800
bp intergenic sequence.
Our working hypothesis is that Xenopus mtDNA exhibits an intermediate sequence organization in the
evolutionary elimination of introns and spacer regions seen in mitochondrial genomes. As such, the
intergenic region found in Xenopus between the cytochrome oxidase and ATPase subunit loci, if not an
active promoter, may at least represent a remnant of the more uncoupled mitochondrial gene arrangement
that prevails in lower eukaryotes and has been lost in mammals. (Supported by NIH grant HD-14552.)
GOLDEN, L., SCHAFER, U. & ROSBASH, M. (1980). Cell 22, 835-844.
Development biology of human haemoglobins
W. G. Wood*, MRC Molecular Haematology Unit, University of Oxford, John Radcliffe Hospital, Oxford
Haemoglobin remains one of the best defined systems for studying the developmental regulation of
specific gene products. Both the a-(£-ip£-ipac-c&-al) and B-(e-GY-Ay-\lip-6-P) globin gene complexes have
been thoroughly characterised at the DNA sequence level and recent studies have documented changes in
erythroid cell chromatin at different stages of development. Thus, the 5' ends of embryonic e and fetal y
genes are unmethylated in erythroid cells at the time of their expression but methylated when inactive and
DNase 1 hypersensitive sites are present at the 5' end of the Gy , Ay , 6 and /3 genes in fetal erythroid (but
not non-erythroid) cells whereas only those 5' to the adult 6 and ft genes remain in adult erythroblasts.
Evidence from naturally occurring deletion mutants of the /3 complex have invoked cis-active regulatory
sites within the complex, some of which appear to act over considerable distances, while studies of human
cell lines expressing globin genes have demonstrated the existence of trans-acting factors involved in gene
expression, which may be developmental stage specific.
While progress at the molecular level is impressive, the cellular basis of haemoglobin switching - what
initiates these changes at the appropriate time - is much less well-understood. No natural mutants of the
timing process have been described and pathological perturbations of erythropoiesis in fetal life produce
only transient secondary changes. However, using sheep as an animal model, fetal haemopoietic cells of
various ages can be transplanted to an adult animal to determine whether the development of these cells in
an adult 'environment' influences their pattern of haemoglobin synthesis. The fetal cells continue to switch
to adult haemoglobin synthesis but the time of switching is not related to the time post-transplantation but
rather to the post-conceptional age of the cells. Using chromosomal markers, it can be shown that the donor
haemopoietic cells are developing amongst marrow stromal cells of recipient origin. These results,
therefore, are most consistent with the hypothesis that the time of switching is controlled by an intracellular
developmental clock.
144
Specific genes in development
A study of the interaction between the human e globin gene and
chromosomal proteins from an £ globin expressing cell line (K562)
Jing de Zhu and John Paul, The Beatson InstituteforCancer Research, Garscube Estate, Switchback Road,
Bearsden, Glasgow G611BD
A two-dimensional protein-DNA electrophoretic technique has been developed to search for sequence
specific binding proteins. The chromosomal proteins were fractionated from nuclei by K562 cells which
produce the e globin chain. The proteins were separated by SDS-polyacrylamide gel electrophoresis and
Western-blotted on to nitrocellulose paper. The EcoRI/BamHI fragments of the plasmid pATH G which
has a 8 kb insert containing 5-3 kb 5' flanking, the e globin gene and 1*1 kb 3' flanking sequences was
uniformly 32P labelled with the E. coli DNA polymerase. The 32P-DNA fragments were applied to the
immobilized proteins. The DNA associated with individual proteins was extracted with SDS and proteinase
K, and separated on an agarose gel. The dried gels were autoradibgraphed. With this assay, we found that
14 different proteins or sets of proteins (classified by their sizes) in either 0-35 M NaCl extraction or histone
depleted fraction bind the e globin DNA. The relative affinity of proteins to individual segments of the
human e globin gene varies with different proteins.
The chromatin structure of the human e globin gene: studies using both
the indirect end-labelling technique and a modified technique based on
the nick-translation of nuclei assay
Jing de Zhu and John Paul, The Beatson InstituteforCancer Research, Garscube Estate, Switchback Road,
Bearsden, Glasgow G611BD
With an indirect end-labelling technique, we have found a set of nuclease hypersensitive sites (HNS)
around the human e globin gene in several nuclei from cell lines which produce the e globin RNA. They are
a K562 cell line, a Cos7 cell line transiently transformed with a plasmid containing the e globin gene and a
TK-BHK cell line stably transformed by a fusion plasmid containing TK gene and the e globin gene. Some
of the HNS are located on, or close to, the initiation sites of the e globin transcripts, including the canonical
cap site, the -200 bp cap site and sites -850 bp, -1430 bp and -4-5 kb from the canonical cap site
respectively. This location correlation may suggest a common mechanism whereby RNA polymerase can
recognize the peculiar chromatin structure demonstrated by HNS. With a modified technique of the
nick-translation of nuclei
assay, we were able to preferentially label the human e globin gene and 18 s
ribosomal gene with 32P triphosphate nucleotides in K562 nuclei, but not the human ar-lactalbumin gene
which is not expressed in K562 cells whereas in HES nuclei, cells of which produce neither RNAs of the e
globin and the or-lactabumin gene, only the 18 s ribosomal gene is labelled. We also observed that the
labelled sequences still maintain a chromatin structure of higher nuclease sensitivity. This assay provides a
quick and simple assay for surveying the structural alteration of any DNA sequences in nuclei once its
corresponding clone is available.