Chromosoma (Berl.)71, 167-181 (1979)
CHROMOSOMA
9 by Springer-Verlag 1979
Behaviour of Sex Chromosome Associated Satellite DNAs
in Somatic and Germ Cells in Snakes
Lalji Singh, Ian F. Purdom and Kenneth W. Jones
Institute of Animal Genetics, University of Edinburgh, West Mains Road, Edinburgh EH9 3JN,
Scotland
Abstract. Sex chromosome associated satellite DNAs isolated from the snakes
Elaphe radiata (sat III) (Singh et al., 1976) and Bungarus fasciatus (Elapidae)
(minor satellite) are evolutionarily conserved throughout the suborder Ophidia. An autosome limited satellite D N A (B. fasciatus major satellite) is
not similarly conserved. Both types of satellites have been studied by in
situ hybridisation in various somatic tissues and germ cells where it has
been observed that the W sex chromosome remains condensed in interphase
nuclei. In growing oocytes however, the W chromosome satellite rich heterochromatin decondenses completely whilst the autosomal satellite rich regions remain condensed. Later, the cycle is reversed and the W chromosome
condenses whilst the autosomal satellite regions decondense. In a primitive
snake (Eryx johni johni) where the sex chromosomes are not differentiated
and where there is no satellite D N A specific to them, these phenomena
are absent. - The differential behaviour of autosomal and sex chromosome
associated satellite DNAs is discussed in the light of gene regulation.
Introduction
The W sex chromosome in female common Indian krait, B. caeruleus which
is late replicating forms a characteristic W chromatin body in interphase nuclei
of various tissues and stains differentially by the C-banding procedure (RayChaudhuri et al., 1970, 1971 ; Singh and Ray-Chaudhuri, 1975). A satellite D N A
(satellite III) which is mainly present in female snakes is concentrated on the
W chromosome and is conserved throughout the suborder Ophidia (Singh et al.,
1976). The nuclotide sequences of snake sex chromosome associated satellite
DNAs are also represented in birds which suggests that functional constraints
may have limited sequence divergence.
These sex chromosome D N A sequences can be studied throughout the cell
cycle in somatic and germ cells of different species particularly in interphase
and prophase stages in which chromosomes are normally unidentifiable. These
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168
L. Singh et al.
stages have previously not been accessible to such scrutiny because of the lack
of a probe specific for a single chromosome. In the present investigation we
report the behavioural differences between sex chromosome associated satellite
DNAs and autosomal satellite D N A in different cell types and at different
stages of the cell cycle.
Experimental Procedures
Preparation of DNA. The following species of snakes used in the present investigation were obtained
from India. They are Bungarus caeruleus, Bungarus faseiatus, Naja naja naja (Family Elapidae);
Natrix piseator, Elaphe radiata (Family Colubridae); Enhydrina schistosa (Family Hydrophiidae)
and Eryx johni johni (Family Boidae). DNA was isolated from liver, kidney, testis and heart
tissues from each individual and processed separately. Total blood of N. piseator from 25 females
and 5 males was pooled according to sex. Ovaries from all 25 individuals were pooled for DNA
extraction. The extraction method of Marmur (1961) was used with the inclusion of repeated
phenol-chloroform, RNAase and pronase treatments.
Isolation and Purification of Satellite DNAs. Satellite III from E. radiata f~ and the major and
minor satellite DNAs from B. faseiatus ~_ total DNAs were isolated by the method of Jensen
and Davidson (1966) at Ag + DNA ratios of 0.20 and 0.25 respectively and purified by repeated
CszSO4 and CsC1 gradients in the MSE 8 • 40 Ti rotor for 80 h at 32K rpm at 25 ~ C. Full details
of this procedure are given in Singh et al. (1976).
The buoyant densities were determined in neutral CsC1 in a Spinco model E analytical centrifuge
at 25 ~ C, 44,000 rpm for 20 h using Micrococcus lysodeikticus DNA (p= 1.731 g.cm-3) as density
marker. The buoyant densities of major and minor satellite DNAs were 1.700 and 1.709 g.cm-3
respectively. The buoyant density of E. radiata satellite III was 1.700 g.cm-3 as earlier reported
by Singh et al. (1976).
Transcription of Satellite DNA. Satellite DNAs were transcribed by using equimolar amounts of
3H-ATP (spec. act. 29 Ci/m mol), 3H-UTP (spec. act. 21 Ci/m mol), 3H-GTP (spec. act. 15 Ci/
m mol), and 3H-CTP (spec. act. 18 Ci/m mol), 2 Ixg of satellite DNA and 2 units of E. coIi DNA
dependent RNA polymerase type I as described by Moat et al. (1975).
Labelling of Satellite DNA. Satellite DNAs were 3H labelled by means of the nick translation
procedure described by Rigby et al. (1977). Equimolar (15-20 gM) of 3H-dATP (spec. act. 30 Ci/
m mol), 3H-dTTP (spec. act. 30 Ci/m mol), 3H-dGTP (spec. act. 16.4 Ci/m mol) and 3H-dCTP
(spec. act. 20 Ci/m mol) and 1-2 gg of satellite DNA was used for each reaction.
Filter Hybridization and (Topt) Determination. DNA was denatured and loaded onto filters
(HAWP 0.45 ~t, 13 mm) (Gillespie and Spiegelman, 1965). Each filter contained a known amount
of D N A + 2 ~tg heterologous DNA as carrier where appropriate. The Denhardt procedure (1966)
was adopted for DNA-DNA hybridization on filters. Optimal rate curves for cRNA and nick
translated DNA (Fig. 1) followed the protocol of Birnstiel et al. (t972) and Moat et al. (t975).
In the case of DNA-DNA hybridization 2 washes of 15 min each were performed after hybridization.
Slide Preparations of Cells from Ovary and Other Tissues. Ovary, liver, kidney, spleen and brain
ceils from B. caeruleus, B. faseiatus, N.n. naja, N. piscator, E.j. johni and E. schistosa were dissected
out in tissue culture MEM medium. Cells were squeezed out using toothed forceps, centrifuged
at 1,200 rpm for 5 min and directly fixed in 3:1 methanol acetic acid 3 times, 10 min each. One
drop of concentrated cell suspension was air-dried on each slide previously acid cleaned. No hypotonic pretreatment was used. Slides were stored dry at 5~ C and were used when required.
Chromosome Preparation. Chromosomes were prepared from short term leukocyte cultures by the
usual air-drying procedure. Cultures were treated with colchicine (0.015 ~tg/ml) for 4 h, 0.075 M KC1
for 8 rain and fixed in 1 : 3 acetic acid methanol. Slides were stored at 5~ C until used.
Behaviour of Satellite D N A s in Snakes
1. Temperature dependence on the initial rate of cRNA-sateIlite D N A (zx--zx)
and nick translated satellite DNA-satellite
D N A ( o - - o ) hybrid formation. B. fasciatus ~- minor satellite c R N A and nick
translated D N A were hybridized to filter
discs containing 0.05 lag of B. fasciatus
D N A + 3 gg of M. lysodeikticus D N A as
carrier at each of the temperatures indicated in a pre-warmed solution of c R N A
or nick translated D N A in 3 • SSC ( c R N A
and nick translated D N A concentration
~0.02 gg/ml) for 30 and 40 rain respectively. Filters hybridized with c R N A were
washed, RNAased, dried and counted. For
hybridizing with nick translated D N A , the
Denhardt (1966) procedure was used
Fig.
169
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In situ Hybridization. The procedure described by Jones (i973) was followed with minor modifications. Slides were heat denatured according to the procedure described by Singh et al. (1977).
cRNA. In the case of B. fasciatus major satellite D N A localization on chromosomes its complementary R N A was used for hybridization. Slides were hybridized with 5 lal cRNA/slide at 58 ~ C (Top0
in 3 x SSC at a c R N A concentration of 0.6 gg/ml for 3 h (spec. act. 1.7 • l0 T counts/min/~tg).
Hybridized slides were treated with R N A a s e (20 gg/ml) for 30 min at 37 ~ C in 2 x SSC, washed
overnight in 2 litre of 2 x SSC at 4 ~ C and dried through an alcohol series.
Nick Translated DNA. Nick translated D N A in 0.1 x SSC was denatured by heating to 100~
for 5 min, chilled in ice, frozen down rapidly, lyophilized to complete dryness and diluted just
before use to the desired volume and salt concentrations. For E. radiata satellite III (4 lag/ml,
spec. act. 0.7 x 107 counts/min/gg) and B. fasciatus minor (1.7 gg/ml, spec. act. 2.4 x 107 counts/min/
gg) satellite D N A s , 5lal of nick translated satellite D N A was used on each slide. Hybridization
was carried out at 60~ (Top0 for 3h. Hybridized slides were dried through an ethanol series,
treated with nuclease S~ type III 5 units/slide in 5 gl of nuclease S~ buffer
( l m M Z n S O 4 + 0 . 1 m M N a C l + 3 0 m M N a acetate pH4.5) for 1 h at 39~
washed in 2 x S S C
at 55 ~ C for 40 rain, washed overnight in 2 x SSC at 4 ~ C, passed through an ethanol series and
air-dried. Slides which were used for the detection of R N A transcripts in the cell were not denatured.
Autoradiography. Hybridized slides were dipped in Ilford K 2 nuclear emulsion diluted 50:50 with
distilled water at 40 ~ C. Slides were exposed for 20-60 d at 4 ~ C, developed in K o d a k D l 9 b developer
at 5~5~ for 12 rain and fixed in Ilford H y p a m for 6 rain diluted with distilled water (1:4) at
the same temperature, rinsed with cold distilled water, with ethanol, with phosphate buffer p H 6.8
and stained in giemsa (2 ml G i e m s a + 5 0 ml of buffer p H 6.8) for 20~,0 min, rinsed in buffer
pH 6.8 and air dried. Rinsing with ethanol increases considerably the stainability of the material.
If required, slides can be destained in 3:1 methanol:acetic acid, rinsed in ethanol, air dried and
restained for appropriate time without any deterioration of the preparation. Photographs were
taken on Agfa Gevaert Copex Pan 35 m m film using a Zeiss research microscope with camera
attachment.
170
L. Singh et al.
Results
W Satellite Location in Highly Evolved Snakes
In situ hybridization of nick translated E. radiata f~ satellite III DNA with
the cells of kidney, liver, brain, spleen, blood, bone marrow and ovary of
B. caeruleus, B. fasciatus, E. schistosa, N. piscator, N.n. naja and E.j. johni
prepared by direct fixation and heat denatured revealed a high concentration
of grains exclusively in a single region of interphase nuclei corresponding to
the W chromatin body (Fig. 2) in all the tissues and in all the species examined
excepting E.j. johni. In the case of ovary two main types of nuclei were observed:
Smaller, abundant, nuclei of follicular epithelial cells which surround the oocyte
closely until maturation, and the spherical oocyte nuclei themselves. The latter
are enormously variable in size reflecting the growth cycle of the oocyte. The
distribution of grains in the follicular nuclei (Fig. 3 a) was essentially similar
to that seen in the case of the somatic tissues (Fig. 2) but in the oocyte nuclei
there was a marked difference which was developmental stage dependent. In
smaller oocytes grains were concentrated in a single point exactly in the same
way as observed in other tissues (Fig. 3a, the large nucleus). A remarkable
dispersion of grains was observed along with the enlargement in the size of
the growing oocytes (Fig. 3b f). In larger oocytes the concentration of grains
was no longer restricted to a single spot, instead grains were homogeneously
distributed occupying larger and mostly peripheral areas surrounding a prominent nucleolus (Fig. 3f). E. radiata satellite III sequences are predominantly
localised on the W chromosome (Singh et al., 1976). The grain distribution
pattern in the nuclei shows the physical state of the W sex chromosome and
its behaviour. The W sex chromosome which was so highly condensed in interphase nuclei of other tissues and in early oocytes appears to decondense as
the growth of the oocyte progresses.
Fig. 2. Interphase nuclei of spleen from B. caeruleus 2 hybridized in situ with E. radiata ~_ satellite
III nick translated DNA. Grains are exclusively localized in the W chromatin body
Behaviour of Satellite DNAs in Snakes
171
Fig. 3a-g. Cross hybridization of E. radiata $ satellite III nick translated DNA with the developing
oocytes of B. caeruleus. Oocyte preparations were heat denatured for 5 sec, hybridized as described
in experimental procedures and exposed for 1 month. Bar- 10 gm. a Grains are concentrated in
the W chromatin body. The large nucleus is an early oocyte and two smaller nuclei are follicular
epithelial cells, b-f Note remarkable dispersion of grains along with the enlargement in the size
of nuclei of the oocytes mostly occupying peripheral areas representing the decondensation of
the W chromosome. Also note a prominent nucleolus surrounded by grains (t). g An oocyte
in late pachytene stage (which has not been hybridized), showing a prominent nucleolus and
conspicuously condensed W chromosome. At this stage some autosomes not the W are associated
with the nucleolus
B. fasciatus $ m i n o r satellite D N A ( p = 1.709 g - c m - 3 ) is also p r e d o m i n a n t l y
d i s t r i b u t e d a l o n g the entire length of the W c h r o m o s o m e a n d is conserved
t h r o u g h o u t the snake groups in the same way as is the satellite III of E. radiata
b u t does n o t hybridize with it (our m a n u s c r i p t in preparation). N o t surprisingly
B. fasciatus ~_ m i n o r satellite D N A showed precisely the same d i s t r i b u t i o n in
somatic a n d in oocyte nuclei as described for E. radiata satellite III when
hybridized its nick t r a n s l a t e d D N A at its t e m p e r a t u r e o p t i m u m (60 ~ C) u n d e r
similar conditions.
W Satellite Location in Primitive Snakes
E. Radiata satellite III a n d B. fasciatus m i n o r satellite D N A s are present in
very m u c h reduced q u a n t i t y in the g e n o m e of primitive snakes b e l o n g i n g to
the family Boidae (our u n p u b l i s h e d data) a n d c o n t r a r y to the W c h r o m o s o m e
of highly evolved species are n o t c o n c e n t r a t e d o n a particular c h r o m o s o m e .
I n growing oocytes of a primitive snake E.j. johni hybridized with E. radiata
satellite III nick t r a n s l a t e d D N A the very few grains that were observed were
172
L. Singh et al.
scattered over the nucleus. C o m p a r a t i v e l y m o r e grains in a similar r a n d o m
d i s t r i b u t i o n were o b s e r v e d after h y b r i d i z a t i o n with B. fasciatus m i n o r satellite
n i c k t r a n s l a t e d D N A . H o w e v e r , a differential c o n c e n t r a t i o n was o b s e r v e d within
the nucleolus.
W Satellite Location in Snakes with Two W Chromosomes
In cases where the sex c h r o m o s o m e c o n s t i t u t i o n includes two W c h r o m o s o m e s
(Singh, 1972a; Singh, 1972b) as for e x a m p l e in the sea snake E. schistosa,
h y b r i d i z a t i o n o f E. radiata satellite I I I a n d B. fasciatus m i n o r satellite D N A
p r o b e s shows t h a t related sequences are d i s t r i b u t e d on b o t h ( W I a n d W2).
A t p a c h y t e n e the two W c h r o m o s o m e s are c o n d e n s e d a n d s e p a r a t e in 75%
instances o b s e r v e d (Fig. 4 b ) b u t a s s o c i a t e d in the r e m a i n d e r (Fig. 4a) a n d all
are distinctly s e p a r a t e d f r o m the p r o m i n e n t nucleolus as f o u n d in o t h e r species
(Fig. 3 g). Later, however, in the g r o w i n g oocyte, b o t h W c h r o m o s o m e s decondense s y n c h r o n o u s l y a n d b e c o m e closely a s s o c i a t e d to give a p a t t e r n o f grains,
after in situ h y b r i d i z a t i o n o f the relevant D N A p r o b e s , similar to t h a t seen
Fig. 4a-e. Cross hybridization of nick translated B. fasciatus ~_ minor satellite DNA with the
oocytes of sea snake, E. schistosa having multiple W chromosomes (W1, W/). Bar= 10 gm. a
Note the concentration of grains at one place indicating that W1 and W2 are associated together.
Also note the absence of association between a prominent nucleolus and W sex chromosomes
(W1, W2). b Grains are localized on two small univalent chromosomes (W1, W2). c-e Early oocytes
showing exclusive localization of grains in the nucleolus
Behaviour of Satellite DNAs in Snakes
173
in species with a single W (Fig. 3 c-f). Moreover, the W chromosomes are now
closely associated with a prominent nucleolus. In approximately 5% of E.
schistosa oocytes, which represent a particular developmental state, hybridization of the D N A probes described above revealed intense localization of
W associated sequences actually within the nucleolus (Fig. 4c-e). This hybridization was not significantly affected by pretreatment of the preparations with
RNAase and so presumably reflects the presence of W D N A at this site. The
fact that this was the only site of hybridization on these particular nuclei indicates
that most of the W chromosome was embedded in the nucleolus. These particular
oocytes showed relatively very faint staining of their chromatin which was
also rather granular in appearance. They have not been seen in other species,
but since the number of snakes examined is small and the oogenesis is relatively
synchronous in different regions of the ovary, this may reflect sampling differences rather than a significant departure in the details of the process in different
species.
Are W Satellites Amplified in Oocytes ?
The difference in the number of grains observed between the interphase nuclei
of various somatic tissues where W is condensed in a W chromatin body,
and the developing oocytes where W is in a decondensed state, is so great
that one is easily pursuaded that sex chromosome associated satellite D N A s
are amplified during oogenesis. Since pretreatment of slides with RNAase did
not reduce the level of hybridization, R N A is not involved. To test the possibility
of D N A amplification in oocytes 0.05 gg of total ovary and blood D N A from
N. piscator ~_ along with 2 ~tg of M. Iysodeikticus D N A as carrier were loaded
on millipore filter separately and hybridized with nick translated B. fasciatus
minor satellite D N A at 6 0 ~ (Topt) as described in experimental procedure.
Filters containing 2 gg of M. lysodeikticus D N A served as controls. No significant difference was observed between blood and ovary (Table 1). However,
because of the absence of any information in the literature regarding the utiliza-
Table 1. Hybridization of B. fasciatus c~ minor nick translated satellite
DNA with the total DNA of N. piscator ~ from blood and from ovary
bound to millipore filters. Total DNA was denatured and loaded onto
filters. Each filter contained 0.05 lagof total DNA with 2 lag of M. lysodeikticus DNA as a carrier. Filters containing M. lysodeikticus DNA (2 lag/filter) served as controls. Hybridization was carried out for 2.5 h at 60~ C
(ToPT) at labelled DNA concentration 0.015 lag/ml in 3 x SSC following
the Denhardt (1966) procedure. The counts hybridized are the average
of 3 filters
Species
Tissues
Counts per minute hybridized
Natrix piscator $
Ovary
Blood
Control
5,496
4,683
151
174
L. Singh et al.
J
x
E
g
8
E
0.0001
0.0003
0.0005
Complementary DNA (tug)
Fig. 5. Relationship between a m o u n t of D N A on filters and counts of nick translated D N A hybridized using excess nick translated D N A in solution. Varying a m o u n t s of denatured total mouse
D N A (0.005, 0.001, 0.0005 and 0.0001 gg) with 2gg per filter M. lysodeikticus D N A were loaded
on to millipore filters and hybridized with mouse satellite nick translated D N A (0.03 gg/ml) in
3 x SSC at 60 ~ C for 3 h, the Denhardt (1966) treatment was used throughout, M. lysodeikticus
(2 gg per filter) served as controls. The quantity of D N A represented in the graph assumes mouse
satellite to be 10% of the mouse genome
tion of nick translated D N A for quantitative analysis of this kind an excess
of nick translated mouse satellite D N A was hybridized to varying amounts
of total filter bound mouse D N A utilising the Denhardt procedure (1966).
When counts per min hybridized are plotted against the amount of mouse
D N A on the respective filters a straight line is obtained (Fig. 5) indicating
that the number of counts per'min hybridized is directly proportional to the
quantity of D N A loaded on the filter. Taken together with this control, the
result of the quantitation of satellite D N A in oocyte and soma indicates that
there is no significant amplification of this D N A in the oocyte. The apparent
difference noted from autoradiographic comparison therefore reflects the limitation of this approach to quantitation due to the rapid saturation of the film
that is achieved when sequences are condensed as compared with when they
are decondensed. This conclusion is not materially altered by the fact that
the oocyte is 4n in D N A content at the stages examined. Parallel experiments..
in which blood and ovary D N A of N. piscator $ were hybridized on filters
with a nick translated cloned Xenopus ribosomal D N A sequence also revealed
no evidence of D N A amplification in snake oocytes (Table 2).
W Chromosome-spec~'c Decondensation
The decondensation cycle of W chromosome satellite D N A rich chromatin
is specific for this particular chromosomal region at this particular time in
development. This was shown by hybridizing a radio-labelled D N A probe of
Behaviour of Satellite DNAs in Snakes
175
Table 2. Hybridization of nick translated plasmid DNA containing Xenopus ribosomal genes with the total N. piscator female DNA from blood
and from ovary bound to millipore filters. Each filter contained 5 gg
of totaI DNA. No carrier DNA was used. Filters containing M. lysodeikticus DNA (5 gg/filter) served as controls. Filters were preprocessed by
Denhardt procedure and hybridization was carried out for 18 h at 45~ C
at nick translated DNA concentration of 0.10 gg/ml in 3 x SSC+50%
Formamide. The counts hybridized are the average of 3 filters
Species
Tissues
Counts per minute hybridized
Natrix piscator f~
Ovary
Blood
Control
1,709
1,420
228
a m a j o r , n o n sex-specific satellite D N A f r o m B. f a s c i a t u s to b o t h s o m a t i c cells
a n d to those oocytes w h i c h c o n t a i n d e c o n d e n s e d W a s s o c i a t e d sequences. The
h y b r i d i z a t i o n p a t t e r n revealed t h a t this satellite sequence is m a i n l y localised
in the m i c r o c h r o m o s o m e s (Fig. 6) a n d is a b s e n t f r o m the Z a n d W. It is n o t
c o n s e r v e d e v o l u t i o n a r i l y in r e l a t e d species ( u n p u b l i s h e d data). In s o m a t i c interp h a s e cells a n d early oocytes this sequence is c o n d e n s e d into 1 4 c h r o m o c e n t r e s
(Figs. 6 a n d 7a, d) a n d in later stages o f oocytes o f B. f a s c i a t u s is c o n c e n t r a t e d
in m a i n l y one l o c a t i o n (Fig. 7 b, c, e). This b e h a v i o u r c o n t r a s t s m a r k e d l y with
t h a t o f the W a s s o c i a t e d satellite sequences in the same o o c y t e stages in the
same species. T h e difference is better a p p r e c i a t e d when we also c o m p a r e their
respective a m o u n t in the genome. F r o m the tracings o f a n a l y t i c a l e q u i l i b r i u m
density g r a d i e n t c e n t r i f u g a t i o n (not shown), B. f a s c i a t u s m a j o r satellite D N A
Fig. 6. A female metaphase plate of B. jktsciatus hybridized in situ with the autologous major
satellite cRNA. Chromosomes were heat denatured for 6 sec, hybridized and exposed for 20 d.
Grains are exclusively localized on the microchromosomes. Sex chromosomes Z and W are devoid
of any grains. Note the interphase nucleus showing clustering of grains in chromocentres. Bar = 10 gm
176
L. Singh et al.
Fig. 7a-e. In situ hybridization of B. fasciatus major satellite cRNA with the growing oocytes
of the same species. Bar= t0 lam. a and d Grains are clustered in 2 4 small regions of the nucleus.
b, c and e Grains are concentrated in the centre of the nucleus at one spot indicating the association
of all the microchromosomes in a compact mass. In a comparable size of oocyte of the same
species minor satellite DNA sequences are highly decondensed (see Fig. 3 f)
appears to be 5-6 times more t h a n the m i n o r satellite D N A per haploid genome.
F r o m the above results it is quite clear that these two satellite D N A s , one
being W specific and the other being m i c r o c h r o m o s o m e limited behave differently in growing oocytes, the former being highly decondensed and the latter
being extremely condensed. As oocytes grow in size, however, B. fasciatus m a j o r
satellite D N A sequences which so far remained highly condensed in the centre
of the nucleus gradually decondense forming distinct peripheral loops (Fig. 8 a,
b). In m o r e mature oocytes grains were observed uniformly distributed all over
the nucleus with no sign o f clustering. At this stage the c h r o m a t i n structure
o f the nucleus was no longer prominent. However, the pattern o f grain distribution gave the impression of chromatin loops distributed t h r o u g h o u t the nucleus
(Fig. 8 c, d).
Behaviour of Satellite DNAs in Snakes
177
Fig. 8a-d. In situ hybridization of B. fasciatus major satellite cRNA with the oocytes of the same
species at later stages ofoogenesis. Bar = 10 pro. a and b Note the distribution of grains on chromatJn
forming peripheral loops showing decondensation of satellite rich chromatin of microchromosomes.
c and d Partial photographs of more advanced stages of developing oocytes showing complete
decondensation of satellite rich microchromosomal chromatin as revealed by the grain distribution.
At this stage chromatin structure of the nucleus is no longer prominent and grains are uniformly
distributed all over the nucleus
Discussion
The m a i n findings described in this p a p e r c o n c e r n the b e h a v i o u r o f b o t h sexc h r o m o s o m a l a n d a u t o s o m a l satellite D N A rich h e t e r o c h r o m a t i n during o o g e n esis. T h u s while b o t h types o f h e t e r o c h r o m a t i n are n o r m a l l y c o n d e n s e d
t h r o u g h o u t the soma, b o t h exhibit a c o n s p i c u o u s d e c o n d e n s a t i o n b e h a v i o u r
in the o o c y t e a c c o r d i n g to a t e m p o r a l l y n o n - o v e r l a p p i n g p r o g r a m m e , This sug-
178
L. Singhet al.
gests that each decondensation cycle signifies an address to particular linkage
groups whose functions appear sequentially as part of the programme of oogenesis. From the fact that certain gene clusters, for example the somatic 5S genes
in Xenopus (Brown and Sugimoto, 1974) remain inactive, whilst the requirements
of the oocyte for 5S RNA is met by a specific set of 5S genes, it is clear
that oogenesis involves considerable subtlety in transcriptional control, the basis
of which is unknown. From the present data, we can infer that different satellite
DNA sequences are involved in decondensation at different times, and, in the
case of the W chromosome, there must be sequences interspersed with the
satellite sequences from knowledge of the relative genomal contributions of
the W chromosome and its satellite sequences. The fact that a nucleolus is
associated with the W at this time, does not necessarily imply that transcription
involving ribosomal functions is going on. It is not known whether the W
contains rDNA, but it does have a secondary constriction which in other systems
usually signifies a nucleolus organiser. However by in situ hybridization of
nick translated W satellite DNA to detect possible transcripts complementary
to these sequences, we have so far drawn a blank. Thus, transcription, if it
is occurring, probably involves the interspersed non-satellite DNA. In this case,
the functions of the satellite DNA might be to control the cycle of condensation.
It is easy to imagine that the monotonous nature of satellite nucleotide sequences
could lend itself to the cooperative change of state of an entire chromosome
region in response to a simple signal. It is of interest in this respect that in
cases where the W chromosome is in two separate parts Wl and W2, both
decondense simultaneously. The alternative possibility is that satellite DNA
sequences might be decondensing in response to a specific address to interspersed
genes. However, absence of differential condensations of a presumptive W chromosome and of the sex chromosome associated satellite DNAs from the genome
of adaptively primitive snakes makes the controlling role of the satellite D N A
in the condensation cycle more probable. Conservation of W associated satellite
DNAs and similarity in the decondensation cycle of W chromosome during
oogenesis in various species of snakes as revealed by cross hybridization studies
further testify its controlling function. An analogous system is present in the
flour moth Ephestia kuehniella in which inactivation of the W chromosome
occurs specifically in somatic cells. In the previtellogenic stages of oocyte however, the W chromosome is transcriptionally active as revealed by 3H-uridine
incorporation (Traut and Scholz, 1978). This does not prove that transcriptionally active sequences are in repetitive DNA. It shows, however, that in growing
oocytes decondensation of the W chromosome can be associated with transcriptional activity. By analogy with the Y chromosome in the spermatocytes of
D. hydei (Hess, 1965), the functions most probably being expressed on the
W at this time will be those connected with fertility.
A controlling function in decondensation-condensation cycles on the part
of sex-chromosome associated satellite DNA concerned with the expression
of fertility genes could account for the conservation of the sex chromosome
satellite DNA i n t h e snake species examined, as well as the apparent limitation
of these sequences to the sex determining chromosome. Similar sequence conservation has been shown in respect of the Y chromosome in Drosophila species
Behaviour of Satellite DNAs in Snakes
179
(Hennig et al., 1974; Yamasaki, 1977). Similarly, the association of particular
satellite DNAs with the autosomes, and in particular, with the microchromosomes, might signify a role in the specific functions of these chromosomes
during oogenesis. Few genes are known on the microchromosomes in birds,
and none in snakes whose genetics are however essentially uninvestigated. In
birds, one such microchromosomal gene is sarc, which is related to ~he src
sequence of the avian sarcoma virus transforming function (Stehelin et al., 1976),
but which is itself of unknown function (Padgett et al., 1977; Spector et al.,
1978). Decondensation of microchromosomal satellite D N A in oocytes suggests
that microchromosomal genes may function particularly during oogenesis. In
that case sarc gene products may well be more abundant in the oocyte than
at later stages.
Like the W, the microchromosomal heterochromatin remains condensed
in somatic cell nuclei where it is often nucleolus associated. Some of the microchromosomes have ribosomal genes (unpublished data).
In the present study, the finding that the nucleolus apparently contains
most of the W satellite D N A at some stage of oogenesis is unexplained, but
it may be set beside that which shows that D N A extracted from isolated nucleoli
in mammalian cells contain approximately four times as much satellite D N A
as does the remaining chromatin fraction (Schildkraut and Maio, 1968).
In summary, the present observations indicate a close connection between
decondensation behaviour of satellite rich C-band heterochromatin and oogenesis in snakes. They also strongly suggest that the microchromosomes, which
are 50% C-band positive, are involved in some accessory oogenic function.
Because of the general similarities that exist between oogenic processes in a
wide spectrum of animals, we suppose that these phenomena are general and
that a major function of satellite D N A is in connection with oogenesis. The
functions of satellite D N A in this context are not clear, but from the fact
that different satellite rich heterochromatin decondenses at distinctly different
times it seems likely that it is involved in the timing of specific gene action
in oogenesis. The significance of this could be in connection with subsequent
morphogenetic processes which are controlled to an important extent by the
oocyte. It is quite pertinent to focus attention on the facts that mature mammalian oocytes in contrast to somatic cells, have two active X chromosomes (Andina, 1978); female somatic cells in Microtus oregoni have XO constitution
but germ cells are XX (Ohno et al., 1966) and in Crustaceans heterochromatin
diminution takes place from presumptive soma but not from germ cells (Beermann, 1977). Because the oocyte is the most important cell produced by the
organism, it would not be too surprising to discover that there is a concomitant
genomic investment in specifically oogenic mechanisms at the D N A level.
Acknowledgements. Supported by a grant (No. 6-123) from the National Foundation. Expert technical assistance of Ms J. Muir and P. Goldsbrough is gratefully acknowledged.
We are thankful to Prof. S.P. Ray-Chaudhuri, Zoology Department of Calcutta University,
India and Prof. B.K. Bachhawat, Director, Indian Institute of Experimental Medicine, Calcutta,
India for providing their laboratory facilities and to Mr K. Majumdar, Zoology Department,
Calcutta University, India for his generous help in collecting and processing the research material.
180
L. Singh et al.
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Received August 25-September 29, 1978 / Accepted September 29, 1978 by W. Beermann
Ready for press October 4, 1978