BIOLOGY OF REPRODUCTION 83, 893–900 (2010)
Published online before print 11 August 2010.
DOI 10.1095/biolreprod.110.085019
Minireview
What Makes an Egg Unique? Clues from Evolutionary Scenarios
of Egg-Specific Genes1
Xin Tian,3,4,5,6 Joel Gautron,7 Philippe Monget,3,4,5,6 and Géraldine Pascal2,3,4,5,6
UMR85,3 Physiologie de la Reproduction et des Comportements, INRA, Nouzilly, France
UMR6175,4 CNRS, Nouzilly, France
Université François Rabelais de Tours,5 Tours, France
Haras Nationaux,6 Nouzilly, France
UR83 Recherches Avicoles,7 INRA, Nouzilly, France
The avian egg, which contains the egg yolk, the egg white, and
the eggshell, represents the mostly advanced amniotic egg in
oviparous vertebrates. In mammals, this reproductive strategy of
laying egg has gradually evolved toward placentation. In order
to better understand the unique status of the avian egg in the
evolution of the vertebrate reproduction, we investigated the
evolution of some Gallus gallus egg-specific protein-coding
genes. Based on our finding and other recent research, we have
summarized here that gene formation (such as ovalbumin genes,
ovocalyxin-36 and apovitellenin-1 encoding genes in the G.
gallus), gene divergence between G. gallus and mammals (such
as the ovocalyxin-32 gene with its ortholog, the mammalian
RARRES1, and the ovocleidin-116 with its ortholog, the
mammalian MEPE), and gene loss (egg-expressed genes lost
during the evolution of the mammals, such as vitellogenin and
RBP encoding genes) play significant roles in the evolution of
egg-specific genes.
chicken, developmental biology, egg, evolution, female
reproductive tract, G. gallus, gamete biology, gene regulation,
ovalbumin, ovocalyxin-32, ovocalyxin-36, ovocleidin-116, ovum,
phylogeny
INTRODUCTION
Vertebrate embryo development is typically characterized
by two main strategies: viviparity (the embryo develops inside
mother’s body) and oviparity (egg laying). In general, bony
fishes, amphibians and reptiles present various degrees of
oviparity and viviparity [1–3], whereas all the birds and
prototherian mammals (monotremes) are oviparous, and
therian mammals are viviparous.
In spite of different reproductive models, the egg and
especially the amniotic egg (that emerged from ancestral
1
X.T. is supported by an INRA postdoctoral fellowship.
Correspondence: Géraldine Pascal, Physiologie de la Reproduction et
des Comportements, UMR 6175, centre INRA 37380 Nouzilly, France.
FAX: 33 02 47 42 77 43; e-mail: [email protected]
2
Received: 25 March 2010.
First decision: 24 May 2010.
Accepted: 28 July 2010.
Ó 2010 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
893
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reptiles), has played an important role during the evolution of
vertebrate reproduction [4, 5]. In most insects, fishes, and
amphibians, the egg is covered by one or two envelopes [6]. In
birds and some reptiles, the egg has developed with several
extraembryonic membranes (chorion, allantois, amnion, and
yolk sac), a shell been involved in exchanges of gas and water
between the embryo and the atmosphere, and the shell is a
physical barrier against mechanical forces. Although most
mammals (Eutheria) have developed a placenta, formed by the
apposition of extraembryonic membranes (chorion, allantois,
and yolk sac), the evolutionary traces from the amniotic egg
could lead to the conclusion that mammals are as amniotic
animals [7, 8].
The avian egg contains three main compartments (the yolk,
the egg white, and the eggshell) whose components play
important roles in protecting and nourishing the embryo. The
biological role of the Gallus gallus (Table 1) egg is that of a
natural container of nutrients and bioactive molecules for the
extrauterine development of the embryo [9, 10]. It is
noteworthy that the egg contains all components essential for
the development of a reproductive cell into a mature chick. The
egg contains the proteins (egg white and yolk), the lipids
(yolk), and all vitamins and minerals necessary for embryonic
development. Because of its importance in avian reproduction
as well as its application in pharmaceutical, cosmetic, and food
industries, the egg has attracted research interests for decades.
The recent development of high-throughput methods used in
combination with the newly available G. gallus genomic
sequence [11] has dramatically increased data on egg proteins.
By using proteomic and transcriptomic techniques, more than
several hundred protein components have been identified from
G. gallus egg white [12–15], egg yolk [16, 17], and egg
vitelline membrane [18] as well as more than 500 proteins from
the eggshell matrix [19, 20]. These high-throughput studies
constitute by far the most comprehensive data set of G. gallus
egg proteins and offer basic information for further evolutionary studies and functional research on avian eggs. For instance,
many of the numerous protein components in the egg are
ubiquitously expressed proteins (such as actin, ubiquitin, and
histones) [12, 13, 17–19]. In general, these nonspecific egg
proteins are widely distributed across vertebrates, including
bony fishes and mammals, suggesting their conserved
evolution and common functions shared among these animals.
However, there are also many proteins that are expressed only
in the hen reproductive organs and play important roles during
ABSTRACT
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TIAN ET AL.
TABLE 1. Correspondences between Latin species name and common
species name.
Latin species name
Gallus gallus
Meleagris gallopavo
Taeniopygia guttata
Homo sapiens
Xenopus tropicalis
Danio rerio
Oryzias latipes
Ornithorhynchus anatinus
Monodelphis domestica
Anolis carolinensis
Common species name
Chicken
Wild turkey
Zebra finch
Human
Frog
Zebrafish
Medaka
Platypus
Opossum
Anole lizard
EGG-SPECIFIC PROTEINS ORIGINATED BY SPECIFIC
GENE FORMATION IN G. GALLUS
As the amniotic egg is a specific organ of avian reproductive
system, it is presumed that new genes arise to reinforce the
protection and nourishment of the oocyte/embryo during its
development into the egg. Through genomic comparison, we can
illustrate this hypothesis of the G. gallus-specific origin of egg
genes when no orthologs are found in other nonavian lineages.
Ovocalyxin-36
Ovocalyxin-36 (OCX-36) has been recently identified as a
new G. gallus eggshell-specific protein [23]. It is detected only
in the regions of the oviduct where eggshell formation takes
place; it becomes incorporated into the shell membranes and
the calcified eggshell predominantly in the inner part of the
shell [23]. The sequence comparison reveals that OCX-36 is
related to the antibacterial protein superfamily BPI/LBP/
PLUNC with 20%–27% identities between OCX-36 and three
BPI/LBP/PLUNC members (BPIL3, LPLUNC3, and
LPLUNC4). Thanks to the phylogenetic tree (Fig. 1A), we
identified the orthologous relationships among the G. gallus
OCX-36 gene and other mammals/G. gallus common BPI/LBP/
PLUNC genes. In the G. gallus genome, the OCX-36 gene is
nested in a tandem arranged BPI/LBP/PLUNC gene cluster on
the chromosome 20, and it is in particular next to BPIL3,
LPLUNC3, and LPLUNC4. The synteny analysis (Fig. 1B)
reveals, first, that at least one PLUNC gene was present in a
ancestral genome of vertebrates and, second, that the BPI/LBP/
PLUNC gene cluster is conserved in amniotes (including
birds—G. gallus, Meleagris gallopavo [Table 1]; no data on
Taeniopygia guttata because of a lack of information on this
part of genome—and mammals), except that OCX-36 is
specifically present in the Galliformes genomes but not in
any mammalian genomes. By further tblastn [25] searches, we
did not find any OCX-36 counterpart in either mammals or
bony fishes, suggesting again the unique presence of OCX-36
in the G. gallus (birds). Thus, we hypothesize that the eggshellspecific OCX-36 gene arises from a tandem duplication of an
Ovalbumin and Its Two Related X and Y Proteins
Another example concerns the specific duplication of
ovalbumin genes in the G. gallus. The major protein of egg
white, ovalbumin, synthesized in the hen oviduct [28], is
consumed as a nutrient supplemental to the yolk in developing
avian embryos. Its two related proteins named ovalbumin X
and ovalbumin Y are also concentrated in the egg white [13].
Phylogeny shows that these three proteins belong to the
ovalbumin-related serpin protein family (SERPINB) [29]. The
SERPINB proteins inhibit serine and/or papain-like cysteine
proteinases and protect cells from exogenous and endogenous
proteinase-mediated injury [30]. Ovalbumin is one of few
members of this family that is not a protease inhibitor. It is due
to deviations in the sequence compared with the consensus
sequence for SERPINB proteins [31]. No experimental data are
available on the protease inhibitory activity of ovalbumin X
and Y. The gene tree from online TreeFam database (http://
www.treefam.org/cgi-bin/TFinfo.pl?ac¼TF352619; simplified
in Supplemental Fig. S1A, all Supplemental Data are available
online at http://www.biolreprod.org) suggests that ovalbumin
and the ovalbumin-like genes X and Y are specifically
duplicated in the G. gallus and form a monophyletic group.
In the G. gallus and M. gallopavo genome, the X, Y, and
ovalbumin genes are tandemly localized within an SERPINB
gene cluster (containing 10 SERPINB genes) on chromosome
2. In the H. sapiens genome, this SERPINB gene cluster is split
into two loci on chromosomes 6 and 18 (Supplemental Fig.
S1B). Recently, evolutionary studies [31, 32] have suggested
that first duplication events of SERPINB genes have occurred
450 million years ago (before the split of bony fishes and
tetrapods), further duplication events resulted in at least six
genes before the divergence of birds and mammals, and, more
recently, lineage-independent duplication events may have
happened in bony fishes, birds (giving formation to the three
egg-specific ovalbumin genes), and mammals (leading to three
squamous-cell-carcinoma-related genes), respectively. These
data show that different species lineages diversified their own
serine proteinase inhibitors to protect the body. In particular,
after the duplication in G. gallus, ovalbumin lost protease
inhibitory activity [33], whereas the paralogs gene Y and gene
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the development of the embryo, such as ovalbumin [21],
vitellogenins [22], ovocalyxin-36 [23], and ovocleidin-116
[24].
The evolution of these egg-specific proteins is associated
with the unique status of avian eggs in the evolution of
vertebrate reproduction. In this article, we present an overview
on the evolution of egg-specific proteins by evaluating the
significance of gene formation, gene divergence, and gene loss
to maintain the specificity of the avian egg. The aim is to offer
new information on the evolution of the reproductive system in
vertebrates.
ancestral BPI/LBP/PLUNC gene after the divergence of birds
and mammals.
Additionally, another egg-specific gene TENP (its protein is
concentrated in the egg white and in the eggshell) is also
localized in the same synteny in the G. gallus genome (also for
the M. gallopavo genome but data not shown; Fig. 1B). Both
phylogeny and genomic comparison (Fig. 1) suggest that the
Galliformes TENP gene is orthologous to the mammalian
BPIL1, which is highly expressed in tonsils, especially in
hypertrophic tonsils in Homo sapiens (Table 1) [26]. We have
likely observed a case of functional divergence after a
duplication event between TENP and BPIL1 genes, TENP
gene expression being different in mammals compared with the
Galliformes BPIL1 gene.
In mammals, the BPI/LBP/Plunc proteins, being key
components of the innate immune system, are well known
for their involvement in defense against bacteria [23, 27]. Thus,
the potential role of OCX-36 and TENP is proposed to be
associated with the innate defense of the egg against pathogens.
The specific expression of both genes in avian eggs, especially
the unique duplication of OCX-36 in the Galliformes, shows
how egg-laying birds diversified their antimicrobial property in
protecting the eggs through a duplication-driven adaptive
process.
EVOLUTION OF G. GALLUS EGG GENES
895
X have hinge regions that deviate less from the consensus
SERPINB sequence, and these genes, particularly gene Y, may
encode inhibitory serpins [31]. As genes described below, these
results imply a functional divergence among the ovalbumin
duplicates, the significance of which remains unclear.
EGG-SPECIFIC PROTEINS ASSOCIATED WITH
DIVERGENCE EVOLUTION
After speciation, orthologous genes subjected to different
environmental pressures may undertake independent evolution
in different species. The different pressures of selection may
allow these commonly originated genes to diverge in function.
During the evolution of eggs, for instance, the eggshell is a
specific structure evolved in some reptiles, birds, and
monotremes but disappeared in therian mammals. However,
the orthologs of some eggshell-specific genes can still be
expressed with function in non-egg-laying mammals, suggesting a distinct evolutionary process of these genes in mammals
compared with birds.
Ovocalyxin-32
Ovocalyxin-32 (OCX-32) is highly and specifically expressed in the uterine (shell gland) and isthmus regions of the
G. gallus oviduct and concentrated in the eggshell [34].
Sequence comparison by blastp [25] search found that the
OCX-32 protein shares more than 50% similarity (and .30%
identities) with two proteins: latexin (LXN) and retinoic acid
receptor responder protein 1 (RARRES1). Phylogenetic
analysis (Fig. 2A) suggests that the G. gallus OCX-32 is
orthologous to the RARRES1 gene and paralogous to the LXN
gene. As shown in the tree, there is only one latexin-like gene
copy present in the bony fishes, whereas in tetrapods the
ancestral latexin-like gene might have duplicated and given
formation to OCX-32 (in birds)/RARRES1 (in Xenopus
tropicalis [Table 1] and mammals) and their paralog latexin
(LXN). This hypothesis is supported by genomic analyses. In
the G. gallus, M. gallopavo, and T. guttata genomes, OCX-32
is localized next to LXN on chromosomes 9, 11, and 9,
respectively. By comparing with other vertebrate genomes, we
found that the region encompassing ovocalyxin-32 and LXN is
conserved among bony fishes, X. tropicalis, G. gallus, and
mammals (Fig. 2B). In particular, only one LXN-like gene is
found in Danio rerio and Oryzias latipes (Table 1), and
RARRES1 is localized in the orthologous position of OCX-32
in X. tropicalis and mammals. The tandem location of LXN and
OCX-32/RARRES1 implies the tandem duplication before the
divergence of tetrapods. Although originated from the same
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FIG. 1. Gene formation of the eggshell-expressed ovocalyxin-36 in the G. gallus. A) Consensus phylogenetic tree of ovocalyxin-36 and its relatives
reconstructed by the fusion of four separate methods (neighbor joining [NJ], minimum-evolution [ME], maximum parsimony [MP], and maximum
likelihood [ML]). The branches with bootstrap values .80% calculated by at least three methods are underlined in bold. B) The genomic localization of
OCX-36 in the G. gallus and syntenic comparison among four vertebrate species (H. sapiens, M. domestica, G. gallus, and O. latipes). Numbers on the
right of pentagons are gene locations expressed in Mb for different species.
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TIAN ET AL.
ancestral, the three genes show great divergence in function
during evolution. The egg-specific OCX-32 is an eggshell
matrix protein, which reinforces the antimicrobial properties of
the eggshell by providing protection for the developing avian
embryo [35], whereas LXN is the only known endogenous
carboxypeptidase A inhibitor in mammals and plays an
important role in regulating the activity of hematopoietic stem
cells [36], and RARRES1 is expressed in multiple tissues and
suggested to be a tumor suppressor in H. sapiens [37, 38]. Note
that no expression data have been published on LXN in G.
gallus. This functional divergence could be associated with the
adaptation of the different selective pressures. In order to
evaluate whether the divergence between OCX-32 and
RARRES1 is driven by positive selection, we conducted an
evolutionary analysis with PAML [39], but positive selection is
not significant during the evolution of these orthologous genes
(data not shown).
Ovocleidin-116
Another eggshell-specific protein, ovocleidin-116 (OC116), is associated with the calcification of the eggshell in
birds [24]. OC-116 was found also in osteoblast and osteocytes
of cortical bone [40]. The structure of OC-116 clearly shows
that this gene belongs to the secretory calcium-binding
phosphoprotein (SCPP) gene family, which is associated with
the tissue mineralization in vertebrates [41]. In spite of the
sequences of SCPP protein family being highly divergent,
thanks to multiple sequence alignment based on exon
conservation presented in Bardet’s study [42], we were able
to rebuild a phylogenetic tree. It seems to indicate a homology
relationship between OC-116 and other SCPP proteins
(Supplemental Fig. S2A). This hypothesis is widely supported
by several studies [11, 41, 42] and by our synteny analysis
showing that in the G. gallus (also for M. gallopavo and T.
guttata), OC-116 is localized in a mineralization-specific gene
cluster (including bone sialoproteins and dentin matrix acidic
phosphoprotein 1) on chromosome 4 and that this gene cluster
is conserved between birds and mammals (Supplemental Fig.
S2B). Taken together, these results imply that G. gallus OC116 and mammalian MEPE (matrix extracellular phosphoglycoprotein) likely are orthologs. By a blastp [25] search, we
found, as best alignment, 52% similarity (35% identities)
between the N terminal domains (; 80 amino acids) of G.
gallus OC-116 and H. sapiens MEPE protein. Although it is
also possible that OC-116 and MEPE occur independently in
birds and mammals, the lower similarities between OC-116 and
its paralogs in the G. gallus or between MEPE and its paralogs
in mammals enhance the hypothesis of orthology between OC116 and MEPE. The great divergence of SCPP genes imply the
diversification of function in mineralization well represented
by the functional specificity of G. gallus OC-116 derived by
the divergent and adaptive evolution (i.e., calcification of the
eggshell in birds).
EGG-SPECIFIC PROTEINS LOST IN MAMMALS
The transition from egg-laying and yolk-dependent nourishment in birds and reptiles toward the placentation and
lactation in mammals is characteristic of the evolution of
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FIG. 2. Gene divergence between the G. gallus eggshell-expressed ovocalyxin-32 with its ortholog, the RARRES1 gene in mammals and X. tropicalis. A)
Consensus phylogenetic tree of ovocalyxin-32 and its relatives reconstructed by the fusion of four separate methods (NJ, ME, MP, and ML). The branches
with bootstrap values .80% calculated by at least three methods are underlined in bold. B) The genomic localization of OCX-32 in the G. gallus and
syntenic comparison among five vertebrate species (H. sapiens, M. domestica, G. gallus, X. tropicalis, and O. latipes). Numbers on the right of pentagons
are gene locations expressed in Mb for different species. The sequence with an asterisk in the phylogenetic tree is predicted by Joint Genome Institute
(Walnut Creek, CA), and the protein ID is 292359 (http://genome.jgi-psf.org/cgi-in/dispGeneModel?db¼Xentr4&id¼292359).
EVOLUTION OF G. GALLUS EGG GENES
897
reproduction in amniotes. The egg components, especially
those from the yolk, constitute an essential resource for the
developing oviparous embryo, whereas the placenta establishes
an interchanging platform of nutrients and waste products
between the embryo/fetus and its mother through blood. This
transition is a long and gradual processing, as suggested by the
observation of the intermediate mammals: monotremes (such
as the Ornithorhynchus anatinus [Table 1]), which lay eggs but
possess primitive mammary glands [43], and the marsupials
(such as the Monodelphis domestica [Table 1]), which have
developed a placenta but contain considerably more yolk in
their oocytes than eutherians [44]. Thus, tracing the evolutionary fate of egg-specific genes (especially yolk-specific genes)
in mammals is thought to be helpful in understanding the
transition of the reproductive strategies between birds and
mammals.
Vitellogenins
A spectacular example concerns the progressive loss of
vitellogenin (VTG) proteins in mammals through coevolution
with lactation and placentation [45, 46]. In nonmammalian
oviparous vertebrates, the embryonic development depends
entirely on nutritional reserves derived from VTG proteins,
while mammals have evolved placenta and milk resources to
nourish their embryos and early offspring. In the G. gallus
genome, two VTG (VTG2 and VTG3) genes and two predicted
pseudogenes are localized in tandem on the chromosome 8,
with VTG1 positioning separately in another region on the
same chromosome [45]. By means of sensitive genomic
comparison and evolutionary simulations, Brawand et al. [46]
have recently shown that these three VTG-encoding genes
progressively lost their functions and became pseudogenes
during mammalian evolution (Supplemental Fig. S3). Monotremes, which lactate but lay small parchment-shelled eggs,
retain a functional VTG gene, consistent with their intermediate reproductive state. Meanwhile, the major milk resource
genes, caseins, which have similar biological functions as
VTGs, emerged in the common mammalian ancestor [46]. This
remarkable observation provided evidence that the gradually
reduced egg yolk resource for the mammalian young was
accompanied by the appearance and development of the
alternative resource, lactation and placentation in mammals.
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FIG. 3. Gene loss of the egg-expressed RBP gene in therian mammals. A) Consensus phylogenetic tree of RBP and its relatives reconstructed by the fusion
of four separate methods (NJ, ME, MP, and ML). The folate receptor 1 (FOLR1) protein sequences are treated as root. The branches with bootstrap values
.80% calculated by at least three methods are underlined in bold. B) The genomic localizations of RBP and RTBDN genes in five vertebrate species (H.
sapiens, O. anatinus, G. gallus, X. tropicalis, and O. latipes). RBP is localized in genomes of the X. tropicalis, the G. gallus, and the O. anatinus but it is not
found in the H. sapiens and O. latipes. RTBDN is present in genomes of O. latipes, the X. tropicalis, the O. anatinus, and the H. sapiens, and even the
syntenic region is missing in the G. gallus genome; some ESTs are found in G. gallus symbolized by a star (in contrast when no EST was found, symbolized
by a crossed circle). Numbers on the right of pentagons are gene locations expressed in Mb for different species. SS turtle, soft-shelled turtle.
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TIAN ET AL.
FIG. 4. Evolutionary scenarios of some
egg-specific genes along the vertebrate
lineages. Gene formation, divergence, and
loss of the main egg-specific genes discussed in this article are presented in the
branches of a life tree. The genes concerning specific formation in the G. gallus (birds)
are shown in pink, the genes concerning
gene divergence are shown in blue, and the
genes concerning gene loss are shown in
green. The presence of a gene is depicted by
a solid symbol, and the loss of a gene is
depicted by a hollow symbol and marked
with ‘‘W.’’ The divergent time of lineages are
referred from Blair and Hedges [50] and
Bininda-Emonds et al. [51].
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Riboflavin-Binding Protein
Another example concerns the loss of the riboflavin-binding
gene in therian mammals. Riboflavin-binding protein (RBP),
synthesized in the oviduct (egg white RBP) and the liver (egg
yolk RBP) of laying hens, plays an important role in the
development of oocyte/embryo. It binds riboflavin (vitamin
B2) and is involved in its transport from the serum into the G.
gallus oocyte/embryo and from the oviduct into the egg white
[47–49]. By a blastp [25] search, we find that the G. gallus
RBP protein is close to the folate receptor family, and it shows
higher identities than its counterparts in bony fishes (such as
44% identities to the D. rerio protein NP_110108566). The
phylogenetic analysis (Fig. 3) suggests that these fish RBP
genes should be orthologous to a mammalian retina-specific
folate receptor gene, retbindin (RTBDN). However, because of
the sequence divergence between the eutherian and the
noneutherian RTBDN, the phylogenetic analysis (Fig. 3) is
not significant in addressing this orthologous relationship,
while the close relationship between RBP and RTBDN is
suggested. Combining phylogenetic analysis and genomic
comparison, we hypothesize that the RBP gene may have been
duplicated from the ancient RTBDN gene in the common
tetrapod ancestor (because no RBP ortholog is identified in
bony fishes) and have been lost in therian mammals, likely
associated with the transmission of the reproductive model. We
failed to identify any RBP pseudogenes from therian mammals,
and it may be explained by the fast and unrestricted mutation
after gene pseudogenization. We note that the RTBDN gene is
present in the genomes of bony fishes, X. tropicalis, Anolis
carolinensis (Table 1), and mammals, but the syntenic region
containing RTBDN is not present in the G. gallus, M.
gallopavo, and T. guttata genomes. Moreover, we failed to
find any ESTs of RTBDN gene in the UniGene data bank (ftp://
ftp.ncbi.nih.gov/repository/UniGene/), even if G. gallus ESTs
of MAST1, GCDH, RNASEH2A, and JUNB genes are found
EVOLUTION OF G. GALLUS EGG GENES
(Fig. 3). These results imply that RTBDN likely was lost during
evolution by genomic rearrangements in bird genomes.
16.
CONCLUSION
ACKNOWLEDGMENTS
We are grateful to John Williams for attention to the English-language
version; Joëlle Dupont, Sophie Réhault-Godbert, and Yves Nys for helpful
discussions; and Jean-Yves Sire for data on OC-116.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
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