Download Linkage arrangement in the vitellogenin gene family of Xenopus

Volume 14 Number 22 1986
Nucleic Acids Research
Linkage arrangement in the vitellogenin gene family of Xenopus laevis as revealed by gene
segregation analysis
Jean-Luc Schubiger* and Walter Wahli
Institut de Biologie Animale, University de Lausanne, Batiment de Biologie, CH-1015 Lausanne,
Switzerland
Received 25 September 1986; Accepted 21 October 1986
ABSTRACT
Using restriction fragment length polymorphism (RFLP) we have analyzed
the segregation of alleles of the different vitellogenin genes of Xenopus
laevis. The results demonstrate that the four genes whose expression 1s
controlled by oestrogen, form two linkage groups. The genes Al, A2 and Bl
are linked genetically whereas the fourth gene, the gene B2, segregates
Independently. The possible origin of this unexpected arrangement 1s
discussed.
INTRODUCTION
Vitellogenin, the precursor of the yolk proteins, 1s encoded 1n a small
family of four related genes 1n Xenopus laevis (1). These genes are
strictly regulated by oestrogen 1n the liver of mature females where they
are coordinately expressed (2,3). The four genes can be divided Into two
main groups (A and B) showing a sequence divergence of about 20% within the
coding regions. Each group can then be subdivided into two closely-related
subgroups (Al and A2, Bl and B2) that show approximately 5% sequence
divergence 1n the exon sequences. The 201 divergence between the A and B
genes suggest that an A and a B gene arose by a first duplication of an
ancestral gene about 150 million years ago (4). Later, about 30 million
years ago, a whole genome duplication seems to have occurred 1n Xenopus
laevis (5,6). If this were the case, it would have resulted in an A-B gene
linkage on one chromosome and a second A-B gene linkage on a second
chromosome that arose during the genome duplication event (7). The latter
event would have generally produced pairs of genes 1n Xenopus laevis, with
each member of a given pair on a separate chromosome. In agreement with
this proposal, pairs of related genes have been observed that code for
© IRL Press Limited, Oxford, England.
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albumins (8), giobins (9), ribosomal proteins (10,11) and actins (12).
Structural studies of the four vitellogenin genes have revealed some
features which are consistent with the proposed model of their evolution,
while some others contradict 1t. The strongest support comes from the
linkage between the genes Al and Bl (7), and from the similar degree of
divergence within the homologous exons of the A1/A2 and B1/B2 gene pairs
(1). In contrast, the corresponding introns and flanking regions of the A
genes are much more different than the corresponding introns and flanking
regions of the B genes (13,14). This is inconsistent with a simultaneous
appearance of the two A genes and the two B genes as a result of a genome
duplication. Clearly, the origin of this gene family could be better
understood once Its genomic organization has been elucidated. Furthermore,
knowledge of the organization of the vitellogenin genes might also be
beneficial
for analyses of their oestrogen
dependent
transcriptional
induction, as well as their acquisition of inducibility during development
(15,16). Using RFLPs as allelic markers of different members of the gene
family, we have determined the linkage groups of these genes as a first
step towards this goal.
MATERIAL AND METHODS
Animals
The adult animals that were analyzed were obtained from the African
Xenopus Facility (Clareinch, South Africa 7740).
Analysis of genomic DNA
DNA was prepared from either erythrocyte or hepatocyte nuclei, which
were isolated from adult Xenopus, tadpoles at stage 60-64 (17) or nine
month-old froglets. The DNA was digested with a 5 to 10-fold excess of
restriction endonuclease, electrophoresed on a 0.7S or 0.8S agarose gel and
transferred to a nitrocellulose filter (18).
Preparation of radioactive probes
H13 clones containing the fragments Indicated 1n the text were used to
prepare probes. Single strand "+" DNA was Isolated, annealed with a primer
complementary to the 5' region of the M13 multiple cloning sites and the
primer was elongated with the Klenow fragment of £. coli DNA polymerase in
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the presence of a - [ 32 P] - dATP (3,000 C1/mmol), unlabelled dCTP, dGTP and
dTTP for 60 m1n at 15°C as described (19). The labelled probes had a
Q
specific activity of 1-2 x 10 cpm per gg of input DNA. Preparation of nick
Q
translated DNA probes (specific activity 1-3 x 10 cpm/ug) was performed as
described (20).
Hybridization with radioactive probes
Prehybr1dizat1on, hybridization and washing were performed as described
(21). Alternatively, the filters hybridized with dextran sulfate were
prehybridized at 40°C for 4-16 hours 1n Stark's buffer (5x SSC, [1 x SCC =
150 mM NaCl, 15 mM sodium citrate], 25 mM sodium phosphate pH 6,5, 1 x
Denhardt's solution [ref. 22], 250 ug/ml denaturated salmon sperm DNA, 50S
formamide and 2% glycine; ref. 23). Hybridization was performed at 40°C in
4 volumes of Stark's buffer and 1 volume of 50* dextran sulfate for 20-60
hours in the presence of 1-3 x 10 cpm of probe per ml of hybridization
hours in the presence of 1-3 x 10 cpm of probe p<
solution. Washing was carried out as described (21).
RESULTS
Restriction fragment length polymorphisms (RFLP) as ailelic markers for the
genes Al, A2 and B2
Based on our analysis of genomic clones, three RFLPs were chosen as
genetic markers for the vitellogenin genes Al, A2 and B2. A polymorphism 1n
Intron 12 of the gene Al gives rise to either a 1.1 kb or a 1.5 kb EcoRI
fragment (Figure la; ref. 24). Similarly, a RFLP 1n the 5' flanking region
of the gene A2 gives either a 6.2 kb or a 6.6 kb Hindlll fragment (Figure
lb; ref. 24). Finally, the gene B2 EcoRI RFLP 1s also 1n the 5' end region
of the gene and 1t shows more variation, yielding fragments of 2.25, 2.5,
and 2.7 kb (Figure lc, ref. 21). We Interpret the fact that none of the
several animals tested shows more than two bands to mean that these
fragments represent allelic forms of their respective genes.
Linkage groups within the viteilogenin gene family
A breeding pair of frogs that were Imported from Africa was chosen to
illustrate the segregation of the viteliogenin alleles (Figure 2 ) . As shown
below analysis of their offspring allows determination of the linkage groups
within the vitellogenin gene family. The male was heterozygous and had the
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a
UO»Ckn»i>
10*. H». 110
10S.1W
5
A1 O«n«
y
*
si a*«•
Animal
T
1
z/ -J
H-f-
\
i.i m
01 5
2
3
4
10 U>
— 1.5
m
^^^
—
— 1.1
—
5- u Oaiu T
•
.
. 1 •• , 1 . . .
01
12S.127
e*ttr
124.125
62 hb
5
5
6
10 U>
_6.6
— 6.2
•V
I \
/
H-4-.
01 5
— 2.7
— 2.5
— 2.25
iai.za.za.m z.7u>
Figure 1 Restriction fragment length polymorphisms (RFLP) within the Al,
A2 and B2 gene loci. Each scheme gives a map of the cloned region of the
different genes with the EcoRI sites (panel a and c) or Hindi 11 sites
(panel b ) . The polymorphic region is enlarged. The M l v genomic clones
containing an allele corresponding to one of the polymorphic restriction
fragment are given on the left (24,14). The EcoRI restriction sites are
indicated by an open circle, the Hindi 11 sites by a square. The hatched
boxes show the regions used as probes in the hybridizations.
On the right, autoradiograms are shown presenting the different alleles
observed. In each lane, 10 ug (5 ug for panel c) of Xenopus laevis genomic
DNA prepared from erythrocytes of different animals was digested either by
EcoRI (panel a and c) or Hindi 11 (panel b ) , electrophoresed on a agarose
gel and transferred to a nitrocellulose membrane. For a and b the fragments
used as probe were inserted in a M13 cloning vector and the radioactive
probe was synthesized by elongation of a primer hybridized to the "+"
strand. For c the fragment was nick-translated. Prehybridization, hybridization and washing were performed in the absence of dextran sulfate.
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Parantal animals
•* — ^^^^B
G»n« B2 ^
*^ " —*2
*3 --
%
* 271
27
Offspring
.221
—
26225—
— 27
H
« M I ^T
^
«MW
-«^
_2«
—22S
Figure 2 Genotype, with respect to the gene Al, A2 and B2 polymorphisms,
of the male and female parental animals, as well as of three of their
offspring. 10 ug of genomic DNA prepared from erythrocytes (parental
animals) or from whole tadpoles at stage 60-64 (offspring) were digested by
EcoRI (genes Al and B2) or HindiII (gene A2).
The DNA was electrophoresed on a agarose gel along with 3 equivalents
per haploid genome of the clones XXlv 107, 110, 221 or 228 or 6 equivalents
of the clones XXIv 124 or 126 as where indicated. The DNA fragments were
then transferred to a nitrocellulose membrane. The probes used are
indicated in Figure 1, with exception for the gene B2 analysis, where the
probe was the 2.5 kb EcoRI fragment of XXIv 228 (Fig. lc). Hybridization
was performed in the presence of dextran sulfate.
following genotype : gene Al-1.1 and 1.5 alleles, gene A2-6.2 and 6.6
alleles and gene B2-2.25 and 2.7 alleles. The female was similarly heterozygous with the gene Al-1.1 and 1.5 alleles and the gene A2-6.2 and 6.6
alleles, while being homozygous for the gene B2-2.5 allele.
From 29 to 52 progeny of this mating were analyzed, depending on the
gene pair tested. As an example, Figure 2b shows the genotype of 3
offspring of this cross. The qualitative and quantitative analyses are
given in Table I to III. Table I shows the segregation of the gene A2
versus the gene B2 alleles. Given the alleles involved, four different
genotypes are expected if the two genes are linked, while six different
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Table I: Segregation of the A2 and B2 alleles
A2a6;6.2
p
A2:6.6; 6.2
B2: 2.7; 2.25
B2: 2.5; 2.5
Possible gametes
6.6/2.5
6.6/2.7
6.2/2.7
6.6/2.25
6.2/2.25
62125
F1 29 individuals analyzed
Dnked
FREQUENC:IES
observed
expect ed
unlinked
GENOTYPEES
expect;id
linked
unBnked
possible
A2: 6.6; 6.6 / B2: 2.7; 2.5
A2: 6.6; 6.2 / B2: 2.7; 2.5
•
•
7.2
(0)
3,6
4
'
C)
•
7,2
(7.2)
7,2
8
(*)
(*)
•
0
(7.2)
3,6
8
•
0
(7,2)
3,6
2
(*)
•
7,2
(7,2)
7,2
4
•
7,2
(0)
3,6
5
A2: 62; 62 1 B2: 2.7; 2.5
A2:6.6; 6.6 / B2: 2.5; 2^5
A2: 6.6; 6.2 / B2: 2.5; £25
'
A2:6.2; 62 1 B2: 2.5; 2^5
•
TOTAL
29
(29)
29
29
Testing 29 Individuals gives a probabftty of 0.98 that all six genotypes would be found S the genes are not
linked.
In case of linkage, there are two possibilities (the second is given In brakets) for the genotypes expected
depending on which of the aDelea form a linkage group in the male.
Statistical analysis of the expected unlinked frequencies versus the observed ones gives: %2 - 7 ; 0.2 < p < 0.3.
genotypes would result 1f the two genes segregate independently. In the
twenty-nine animals tested, all six genotypes were found with frequencies
1n agreement with the expected values. From this result it appears that the
genes A2 and B2 are not linked, which is Inconsistent with the simple
gene-genome double-duplication model. Thus, we wondered if either the gene
A2 or B2 belongs to the already established Al-Bl linkage group (7).
To answer this question the inheritance of the alleles of the gene Al
relative to those of the gene A2 was analyzed (Table II). In the 36 animals
checked
only
three
out of nine
possible
genotypes
were
observed,
demonstrating that the genes Al and A2 are linked. The gene A2-6.6 allele
was found linked to the gene Al-1.1 allele and the gene A2-6.2 allele is
linked to the gene Al-1.5 allele in both parental animals. Thus, the gene
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Table II: Segregation of the A1 and A2 alleles
A1: 15; 1.1
A1: 15; 1.1
A2: 6.6; 6.2
A2: 6.6; 6.2
P
Possible gametes
1.5/6.6
1.1 /6.6
1.5/6.6
1.1 /6.6
1.5/6.2
1.1 /6.2
1.5/6.2
1.1 /6.2
F1 36 individuals analyzed
FREQUENC IES
GENOTY PES
possible
expected
linked
unlinked
expec•ted
unlinked
linked
observed
•
0
(9)
2,2
0
A1: 1.5; 1.5 / A2: 6.6; 6.2
•
0
(0)
6,6
0
A1:1.5; 1.1/A2: 6.6; 6.6
•
0
(0)
4,4
0
A1: 1.5; 1.1 / A 2 : 6.6; 6.2
' O
•
18
(18)
6,6
17
A1: 1.5; 1.5/A2: 6.2; 6.2
*
•
9
(0)
2,2
10
•
0
(0)
4,4
0
9
(0)
4,4
9
A1: 1.5; 1.5/A2: 6.6; 6.6
(')
A1: 1.5; 1.1/A2: 6.2; 6.2
AV 1 1-1 1 / A 2 6 6-6 6
A l : 1.1; 1.1/A2: 6.6; 6.2
•
0
(0)
4,4
0
A1:1.1; 1.1 / A 2 : 6.2; 6.2
*
0
(9)
2,2
0
36
(36)
C)
TOTAL
36
36
Testing 36 individuals give a probability of 0.95 that all nine genotypes would be found if the genes are
unDnked.
In case of linkage, there are two possibilities (the second is given In brakets) for the genotypes expected
depending on which of the alleles form a linkage group.
Statistical analysis of the expected finked frequencies versus the observed ones gives: x 2 - 0.834; 0.5 < p < 0.9.
In contrast statistical analysis of the expected unlinked frequencies versus the observed ones gives:
X2 - 60.6; p < 0.001.
A2 belongs
to the Al-Bl
linkage group; however,
Its position and
orientation relative to the Al-Bl complex remain to be elucidated.
Further confirmation that the gene B2 1s at a different genetic locus 1s
confirmed by Its behavior relative to the gene Al (Table III). In the
fifty-two offspring tested all six genotypes anticipated from independent
segregation of the gene Al versus the gene B2 alleles were observed at
frequencies close to the expected values.
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Table III: Segregation of
Possible gametes
the A1 and B2 alleles
A1: 15; 1.1
A1: 15; 1.1
B2: 25; 2.5
B2: 2.7; Z25
1AI23. 1.1/2.5
1.5/2.7
1.1 /2.7
1.5/2.25
1.1 Z2.25
F1 52 individuals analyzed
GENOTYF>ES
possible
A1:15;1.5/B2:Z7;2.5
exp€icted
Inked
unlinked
•
A1:15;15/B2:2.7;25
A1: 1.5; 1.1 /B2:Z7;Z5
A1: 15; 1.1 /B2:2.5;Z25
13 (0)
6.5
9
•
0 (13)
B.5
5
•
13 (13)
13
16
C)
•
13 (13)
13
11
(')
•
0 (13)
6,5
5
*
13 (0)
6,5
6
TOTAL
52 (52)
(')
'
*
A1: 1.1; 1.1 /B2:2.5; 2.25
A1:1.1; 1.1 /B2:2.5; 2^5
•
•
F R E Q U E N DIES
expe<3ed
observed
unlinked
Qrtked
(')
52
52
Tasting 52 individuals gives a probability of 0.99 that the six genotypes would be found if the genes are
unlinked.
In case of linkage, there are two possibilities (the iscond Is given In brakets) for the genotypes expected
depending on which of the alleles form a Dnkage group in the male.
Statistical analysis of the expected unlinked frequencies versus the observed ones gives: x 2 - 4.7; 0.3 < p < 0.5.
DISCUSSION
RFLP within the vitellogenin loci
Previous analysis of numerous vitellogenin genomic clones revealed
several polymorphic regions, some of which we have used here to determine
the linkage groups within the gene family (13,21,24). Frequently, the RFLPs
used are the result of Insertions or deletions 1n Introns or gene flanking
regions. For the gene Al alleles, for example, the longer 1.5 kb EcoRI
fragment from Intron 12 contains a repeated sequence that 1s absent in the
corresponding allelic 1.1 kb fragment (25). Similarly, the polymorphism
observed 1n the 5' end region of the gene B2 1s also due to repeated
sequences (21). The polymorphism 1n the 5' flanking region of the gene A2
1s again the result of an Insertion or deletion event (24) but 1t has yet
to be characterized at the sequence level.
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Definition of the linkage groups within the viteliogenin gene family of
Xenopus laevis
Linkage between the Al and Bl vitellogenin genes has previously been
found by molecular cloning, and the two genes are about 15 kb apart (7).
The general organization and linkage of the whole gene family can formally
be studied
in two ways. The first
is to demonstrate
the linked
or
independent segregation of members of the gene family, and the second 1s to
"walk" along the chromosome (chromosomal walking) from the Isolated genes
towards the next linked member of the family. Here, we have performed an
analysis by exploiting the RFLPs discussed above as allelic markers of the
genes Al, A2 and B2.
The results demonstrate that 1n Xenopus laevis the genes Al, A2 and Bl
are linked, while the gene B2 is located elsewhere, most likely on a
different chromosome. Clearly, the simple gene-genome double-duplication
model (7) only partially explains the present day organization of the gene
family. It has been suggested that Xenopus laevis, with 36 chromosomes, 1s
"tetraploid" after a genome duplication that occurred about 30 million
years
ago
(5,6).
This
could
have
arisen
by
alloploidization
or
alternatively, by autoploidization. The fact that only bivalents, rather
than multivalents, are observed during meiosis could indicate that the
first
of
these
observation
plants,
two
does
genes
possible
not
have
exclude
been
events
took
place
autopolyploidization
described
that
control
(26). However,
since, at
bivalent
this
least
1n
pairing
in
autopolyploids (27,28). Xenopus tropical is with twenty chromosomes would
represent a close relative to the now extinct diploid ancestral
Interestingly,
vitellogenin
1s
encoded
1n
three
genes
in
form.
Xenopus
tropicalis, two closely related type-A genes with about 6% divergence and a
single type-B gene (29). It will be Interesting to see 1f these genes
correspond to the A2, Al-Bl linkage group 1n Xenopus laevis. As shown in
Figure 3, our results suggest that 1n the genus Xenopus an A2-A1-B linkage
group arose before the genome duplication event 1n Xenopus laevis. First
was a duplication to give the A-B gene pair about 150 million years ago
(4), followed much
later by an A2-A1 duplication
shortly
before the
polyploidization event 1n Xenopus laevis. If the genome duplication 1s the
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Nucleic Acids Research
AHopolyplo<dy
AutopolyploMy
A-0
g.n» dupOcaflon
.
|
A1-AI
Bam dupflcation
di^flcadon
*r-AT
Ban* aUninatlan
Friainl day orgentzaflen
Figure 3
Scheme representing the possible origin of the present day
arrangement of the viteTlogenin genes in Xenopus laevis. The star Indicates
that the position and orientation of the gene A2 relative to the Al-Bl
complex are not known.
result of autopolyploidy, then two A genes must have been lost on one
of the duplicated chromosomes. Alternatively, 1f alloploidy took place, one
of the two related species which mated might have only contributed with a B
gene that was still very closely related to the B gene of the other
species. Alternatively both species had an A2-A1-B type linkage group. In
that
case,
two A
genes
must
again
have
been
lost
after the
tetraploidization event. This revised gene-genome duplication hypothesis 1s
compatible with the observation that the two B genes are more closely
related than the two A genes 1n Xenopus laevis.
It has been observed that silent mutations accumulate much more rapidly
in exon sequences than replacement mutations (30). The accumulation of both
has been calculated for the first three exons of the A and the B genes
(4). In both pairs of genes (Al and A2, Bl and B2) replacement mutations
have been Introduced to nearly the same level (3.4 and 3.3%, respectively),
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but more silent mutations are found between the two A genes (28.8%)
compared to the two B genes (22.21). This again suggests that the two A
genes formed shortly before the two B genes. If the latter two genes arose
as result of the genome duplication event 30 million years ago, this
difference suggests that the two A genes might have formed about 10 million
years earlier, that is about 40 million years ago.
Additional variants of the general hypothesis presented here could be
discussed. The chromosomal distribution of the duplicated genes has not
necessarily to follow the polyploidization pattern. Indeed, there is a
variety of possible genomic rearrangement mechanisms by which duplicated
genes might become located on different non-homologous chromosomes. Thus,
only further detailed molecular and comparative analysis of the two
vitellogenin lod in Xenopus laevis, as well as of the corresponding l o d
1n related species, will help to refine our understanding of the phylogeny
of the vitellogenin gene family.
ACKNOWLEDGEMENTS
We thank Drs. Bob H1psk1nd, Anne Seiler, Philippe Walker, and Riccardo
Wittek for comments on the manuscript as well as Hannelore Pagel for
secretarial help. This work was supported by the Etat de Vaud and the Swiss
National Science Foundation.
*
Present address : F. Hoffmann-La Roche and Co AG,
Grenzacherstrasse 124, CH-4002 Basel, Switzerland.
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