Heterochromatin as a factor affecting X-inactivation in interspecific female vole hybrids (Microtidae, Rodentid)
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Genet. Res., Camb. (1991), 58, pp. 105-110 With 1 text-figures Printed in Great Britain 105
Heterochromatin as a factor affecting X-inactivation in
interspecific female vole hybrids (Microtidae, Rodentid)
SUREN M. ZAKIAN,* TATYANA B. NESTEROVA, OLGA V. CHERYAUKENE
AND MICHAIL N. BOCHKAREV
Institute of Cytology and Genetics, Academy of Sciences of the USSR, Siberian Department, Novosibirsk 630090, USSR
(Received 16 August 1990 and in revised form 18 March 1991)
Summary
Female interspecific vole hybrids were examined for the expression of the G6PD and GALA genes
on the X chromosomes. When one of the parents was a species with a heterochromatin block on
the X, and the other parent was M. arvalis, without a heterochromatin block on the X,
preferential expression of the genes of the M. arvalis X was consistently observed. When both
parental species had heterochromatin on the X, the parental forms of G6PD and GALA were in
about equal proportions in the hybrid females. The results of the cytological identification of the
active and inactive X on the metaphase spreads in the hybrid females are in agreement with the
biochemical results. It is suggested that the observed phenomenon may be due to a nonrandom
inactivation of the X chromosome containing a heterochromatin block in crosses involving M.
arvalis and by a random inactivation in those with both parents having heterochromatin blocks on
the X chromosomes. These results support our previous suggestion that heterochromatin has an
effect on X inactivation in female interspecific vole hybrids.
1. Introduction 2. Material and methods
X-chromosome inactivation in female mammals is a The four vole (Microtus) species used in the experi-
unique phenomenon of the regulation of the activity ments were M. arvalis, M. subarvalis, M. kirgisorum
of the genetic apparatus (Lyon, 1961,1974). There are and M. transcaspicus. The wild-caught voles were
many models offering to explain the mechanisms of maintained and bred in the vivarium of this Institute.
the X inactivation (Gartler & Riggs, 1983; Grant & Hybrids between the four species were produced. The
Chapman, 1988). While not fully accounting for the number of females from each cross and the direction
phenomenon, the models do not appear to be mutually of the crosses are given in Table 1.
exclusive. Facts relevant to the X inactivation derived Hybrids derived from matings of M. subarvalis with
from traditional and, more importantly, new ex- the other vole species were analysed electrophoretic-
perimental models, would improve our understanding ally for the expression patterns of glucose-6-phosphate
of the phenomenon and help us to gain insight into dehydrogenase (G6PD) in the erythrocytes, lung,
the mechanisms regulating the expression of the X liver, heart, kidney, brain, spleen and muscle; those
chromosome genes in mammals during development. derived from mating of M. arvalis with the other
We have previously suggested a connection between species, as well as the M. transcaspicus x M. kirgisorum
the presence of large heterochromatin regions in the X interspecific hybrids, were examined for the electro-
chromosome and preferential inactivation of these phoretic expression of a-galactosidase (GALA) in the
chromosomes in female hybrids from certain Microtus brain. Electrophoresis was carried out in 13% starch
species crosses (Zakian et al. 1987). This new paper gels. Electrophoretic conditions and preparation of
presents further evidence on this problem, based on the samples for assaying G6PD and GALA, as well as
additional crosses and biochemical and cytological the staining techniques for their visualization, were all
tests which enable us to distinguish between active as described elsewhere (Zakijan et al. 1984). De-
and inactive X chromosomes and to elucidate the termination of the specific activity of G6PD in the
effect of heterochromatin on the process of X erythrocytes of all the studied vole species was based
inactivation in hybrid voles. on a previously described method (Serov & Zakijan,
* Corresponding author 1977). Extraembryonic tissues and embryonic organs
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. https://doi.org/10.1017/S0016672300029748S. M. Zakian and others 106
Table 1. Number of hybrid vole females obtained in different crosses
M. arvalis M. subarvalis M. kirgisorum M. transcaspicus
cJX-inactivation in interspecific vole hybrids 107
•f*ttt
It •Hb
1 2 3 4 5 6 7
Fig. 3. Electrophoretic patterns of G6PD in the
erythrocytes of M. subarvalis (1), M. arvalis (2, 8) and in
the Fj hybrids from M. subarvalis x M. arvalis (3-7). Hb,
haemoglobin.
Fig. 4. Electrophoretic GALA patterns from brain tissue
of M. arvalis (1), M. kirgisorum (4), M. transcaspicus (M.
extraembryonic membranes of the mouse is preferen- subarvalis has the same mobility) (6), and their F, female
tially inactivated (Takagi & Sasaki, 1975). From this hybrids from the crosses: $ M. transcaspicus x $ M. arvalis
point of view it appeared of interest to observe how (2), $ M. kirgisorum xS1. M. Zakian and others 108
• w
(a)
(6) (4)
Fig. 5. Metaphase chromosomes from the bone marrow cells of hybrid female voles: M. arvalis x M. subarvalis (a, b), M.
kirgisorum x M. arvalis (c, d), M. transcaspicus x M. arvalis (e,f), M. transcaspicus x M. subarvalis (g, h). Note the dark
staining inactive X (long arrow): M. arvalis (b, d,f), M. subarvalis (a,h), M. kirgisorum (c), M. transcaspicus (e, g), and
the light staining active X (short arrow): M. subarvalis (g), M. transcaspicus (h).
3tt|
t* t t
Fig. 6. Electrophoretic patterns of G6PD in the Fig. 7. Electrophoretic patterns of G6PD in the
erythrocytes of M. arvalis (1), M. subarvalis (6) and in the erythrocytes of M. transcaspicus (1), M. subarvalis (2), in
yolk sacs of hybrid females from M. subarvalis x M. M. transcaspicus x M. subarvalis hybrids in heart (3), in
arvalis crosses (2-5). brain (4), in kidney (5) and in M. kirgisorum x M.
subarvalis hybrids in lung (6).
The salient finding of the present experiments is
that heterochromatin has no effect on X inactivation parental enzymes of the kind we observed for crosses
in the extraembryonic tissues of hybrid voles. This is with M. arvalis (Figs. 4, 7). The proportion of the
in contrast to what we have observed for their somatic G6PD parental forms was almost 1:1 in the hybrids
tissues in this and previous experiments (Zakian et al. from the M. transcaspicus x M. subarvalis cross. The
1987). same was observed for the GALA parental forms in
The involvement of M. transcaspicus in the crosses the brain of hybrids from the $M. transcaspicus x £M.
allowed us to examine the X-inactivation process in a kirgisorum cross. In $ M. kirgisorum xX-inactivation in interspecific vole hybrids 109
Table 3. Summary of relative expression of maternally- and paternally-
derived X chromosomes (X™ and Xp)
Inactive X chromosome
Cross
(female x male) Marker Adult tissue Fetus Yolk sac
ra
arvalis x subarvalis* Kanda X" > X (77 % X")
G6PD X p > Xm X p > Xm
GALA X p > Xm X p > Xm
subarvalis* x arvalis Kanda X m > X" (85% Xm)
G6PD X m > X" X m > X" X" :
GALA X m > X" Xm > Xp
subarvalis* x transcaspicus* Kanda Xm = Xp (54% X m )
G6PD X m = X"
transcaspicus* x subarvalis* Kanda X m = X" (47 % Xm)
G6PD X m = Xn
kirgisorum* x arvalis Kanda X m > X" (89 % X m )
GALA X m > X"
kirgisorum* x subarvalis* G6PD X m Ss X"
transcaspicus* x arvalis Kanda X m > X" (79 % X m )
GALA X m > XP
transcaspicus* x kirgisorum*
* X chromosome has blocks of constitutive heterochromatin
Data from Table 2, Figs. 3, 4, 6, 7, text, and from Zakian et al. 1987
respectively) but was about 1-5-fold lower (103 mU/ lagging replication of an X chromosome carrying
mg of protein) in M. kirgisorum. Consequently, the heterochromatin may possibly be a factor provoking
observed unequal expression of the parental forms of X inactivation. If both Xs carry heterochromatin
G6PD in M. kirgisorum x M. subarvalis appears to be blocks, we should expect inactivation to become a
due to differences in the specific activity of the enzyme matter of chance.
in their parental species rather than to preferential X When seeking to explain our results we are at the
inactivation in one of the parents. crossroads of two different problems, X inactivation
The results of cytological investigation of the active and heterochromatin contribution to the process.
and inactive Xs in the metaphase spreads of hybrid Each problem deserves consideration in its own realm.
females from the M. subarvalis x M. transcaspicus We would rather describe only the phenomenon we
cross are given in Table 2. The inactive X of M. observed. Our following reports will deal with the
transcaspicus and M. subarvalis occurs in almost equal putative mechanisms of the effect of heterochromatin
proportions. Here again, there is a good agreement on X inactivation in female vole hybrids.
between the results of cytological and biochemical
analyses. All results obtained in this and the previous The authors express gratitude to Dr P. M. Borodin for val-
uable comments in the course of the manuscript prepar-
paper (Zakian et al. 1987) are summarized in Table 3. ation. The authors are grateful to Anna Fadeeva for trans-
We attempted to explain the present results in terms lating this paper from Russian into English and to V.
of the various hypothetical mechanisms of X in- Prasolov for the photographs.
activation proposed in the literature. The results seem
to be best accounted for by the replication-expression
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