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Reptile sex determination is attracting much attention because the great diversity of sex-determination and dosage compensation mechanisms permits us to approach fundamental questions about mechanisms of sex chromosome turnover. Recent studies have made significant progress in better understanding diversity and conservation of reptile sex chromosomes, with however no reptile master sex determination genes identified. Here we describe an integrated genomics and cytogenetics pipeline, combining probes generated from the microdissected sex chromosomes with transcriptome and genome sequencing to explore the sex chromosome diversity in non-model Australian reptiles. We tested our pipeline on a turtle, two species of geckos, and a monitor lizard. Genes identified on sex chromosomes were compared to the chicken genome to identify homologous regions among the four species. We identified candidate sex determining genes within these regions, including conserved vertebrate sex-determining genes爬行动物的性别决定一直备受关注，其性别决定和剂量补偿机制的多样性是我们理解复杂的性染色体转换机制的绝佳材料。近年来，关于爬行动物性染色体多样性和保护的研究取得了一系列重要进展，但目前爬行动物的主效性别决定基因被报道和研究地极少。该文发展了一套将基因组学和细胞遗传学结合起来的研究性染色体的方法。我们首先通过显微解剖获得性染色体特异的探针，再与转录组和基因组测序相结合，从而探索研究了多个澳大利亚非模式爬行动物的性染色体多样性。我们在一种龟、两种壁虎和一种巨蜥中测试了我们的流程。通过将性染色体上的基因与鸡的基因组相比较，我们推断了这四个物种与鸡的同源的候选性别决定区域。我们在这些区域中发现了候选的性别决定基因，包括在脊椎动物中比较保守的性别决定基因

Significance Reptiles have an extraordinary variety of mechanisms to determine sex. The best candidate sex-determining gene in our model reptile (the Australian central bearded dragon) is the key vertebrate sex gene nr5a1 (coding for the steroidogenic factor 1). There are no sex-specific sequence differences between nr5a1 alleles on the sex chromosomes, but the Z- and W-borne alleles are transcribed into remarkably different alternative transcripts. We propose that altered configuration of the repeat-laden W chromosome affects the conformation of the primary transcript to generate more diverse and potentially inhibitory W-borne isoforms that suppress testis determination. This is a mechanism for vertebrate sex determination, in which epigenetic control regulates the action of a gene present on both sex chromosomes., Pogona vitticeps has female heterogamety (ZZ/ZW), but the master sex-determining gene is unknown, as it is for all reptiles. We show that nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1), a gene that is essential in mammalian sex determination, has alleles on the Z and W chromosomes (Z-nr5a1 and W-nr5a1), which are both expressed and can recombine. Three transcript isoforms of Z-nr5a1 were detected in gonads of adult ZZ males, two of which encode a functional protein. However, ZW females produced 16 isoforms, most of which contained premature stop codons. The array of transcripts produced by the W-borne allele (W-nr5a1) is likely to produce truncated polypeptides that contain a structurally normal DNA-binding domain and could act as a competitive inhibitor to the full-length intact protein. We hypothesize that an altered configuration of the W chromosome affects the conformation of the primary transcript generating inhibitory W-borne isoforms that suppress testis determination. Under this hypothesis, the genetic sex determination (GSD) system of P. vitticeps is a W-borne dominant female-determining gene that may be controlled epigenetically.

Reptile sex determination is attracting much attention because the great diversity of sex-determination and dosage compensation mechanisms permits us to approach fundamental questions about sex chromosome turnover and evolution. However, reptile sex chromosome variation remains largely uncharacterized and no reptile master sex determination genes have yet been identified. Here we describe a powerful and cost-effective “chromosomics” approach, combining probes generated from the microdissected sex chromosomes with transcriptome sequencing to explore this diversity in non-model Australian reptiles with heteromorphic or cryptic sex chromosomes. We tested the pipeline on a turtle, a gecko, and a worm-lizard, and we also identified sequences located on sex chromosomes in a monitor lizard using linked-read sequencing. Genes identified on sex chromosomes were compared to the chicken genome to identify homologous regions among the four species. We identified candidate sex determining genes within these regions, including conserved vertebrate sex-determining genes pdgfa, pdgfra amh and wt1, and demonstrated their testis or ovary-specific expression. All four species showed gene-by-gene rather than chromosome-wide dosage compensation. Our results imply that reptile sex chromosomes originated by independent acquisition of sex-determining genes on different autosomes, as well as translocations between different ancestral macro- and micro-chromosomes. We discuss the evolutionary drivers of the slow differentiation, but rapid turnover, of reptile sex chromosomes.

The suborder Serpentes is divided into 2 infraorders, Scolecophidia and Alethinophidia, which diverged at an early stage of snake diversification. In this study, we examined karyotypes of 4 scolecophidian species (Letheobia simonii, Xerotyphlops vermicularis, Indotyphlops braminus, and Myriopholis macrorhyncha) and performed FISH with 18S-28S rDNA as well as microchromosomal and Z chromosome-linked genes of Elaphe quadrivirgata (Alethinophidia) to investigate the karyotype evolution in the scolecophidian lineage. Diploid chromosome numbers of X. vermicularis and L. simonii were 30 (16 macrochromosomes and 14 microchromosomes) and 32 (16 macrochromosomes and 16 microchromosomes), respectively. The karyotype of a female M. macrorhyncha consisted of 15 macrochromosomes and 19 microchromosomes, including a heterochromatic microchromosome, indicating the presence of a heteromorphic chromosome pair. E. quadrivirgata Z-linked genes mapped to chromosome 4 of M. macrorhyncha, not to the heteromorphic pair. Therefore, M. macrorhyncha may have differentiated ZW sex chromosomes which are not homologous to those of E. quadrivirgata. One of the E. quadrivirgata microchromosomal genes mapped to the terminal region of chromosome 4q in X. vermicularis, suggesting that fusions between microchromosomes and macrochromosomes occurred in this species. rDNA was localized in different macrochromosomal pairs in the 2 diploid scolecophidian snakes examined here, whereas the gene location in a microchromosomal pair was conserved in 5 alethinophidian species examined. These results might imply the occurrence of chromosome fusions in the scolecophidian lineages. In I. braminus, a unique parthenogenetic snake with a triploid karyotype (21 macrochromosomes and 21 microchromosomes), morphological heteromorphisms were identified in chromosomes 1 and 7. Such heteromorphisms in 2 chromosomes were also observed in individuals from distant locations in the broad distribution range of this species, suggesting that the heteromorphisms were fixed in the genome at an early stage of its speciation.

Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female‐specific 250‐bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.

The pluripotency-associated transcriptional network is regulated by a core circuitry of transcription factors. The PR domain-containing protein, PRDM14, maintains pluripotency by activating and repressing transcription in a target gene-dependent manner. However, the mechanisms underlying dichotomic switching of PRDM14-mediated transcriptional control remain elusive. Here, we identified C-terminal binding protein 1/2 (CtBP1/2) as a component of the PRDM14-mediated repressive complex. CtBP1/2 binding to PRDM14 depends on CBFA2T2, a core component of the PRDM14 complex. The loss of Ctbp1/2 impaired the PRDM14-mediated transcriptional repression required for pluripotency maintenance and transition from primed to naïve pluripotency. Furthermore, CtBP1/2 interacted with the PRC2 complexes, and the loss of Ctbp1/2 impaired PRC2 and H3K27me3 enrichment at target genes after Prdm14 induction. These results provide evidence that the target gene-dependent transcriptional activity of PRDM14 is regulated by partner switching to ensure the transition from primed to naïve pluripotency.

Sex chromosomes in some reptiles share synteny with distantly related amniotes in regions orthologous to squamate chromosome 2. The latter finding suggests that chromosome 2 was formerly part of a larger ancestral (amniote) super-sex chromosome and raises questions about how sex chromosomes are formed and modified in reptiles. Australian dragon lizards (Agamidae) are emerging as an excellent model for studying these processes. In particular, they exhibit both genotypic (GSD) and temperature-dependent (TSD) sex determination, show evidence of transitions between the two modes and have evolved non-homologous ZW sex microchromosomes even within the same evolutionary lineage. They therefore represent an excellent group to probe further the idea of a shared ancestral super-sex chromosome and to investigate mechanisms for transition between different sex chromosome forms. Here, we compare sex chromosome homology among eight dragon lizard species from five genera to identify key cytological differences and the mechanisms that may be driving sex chromosome evolution in this group. We performed fluorescence in situ hybridisation to physically map bacterial artificial chromosome (BAC) clones from the bearded dragon, Pogona vitticeps&rsquo, ZW sex chromosomes and a nucleolar organising region (NOR) probe in males and females of eight Agamid species exhibiting either GSD or TSD. We show that the sex chromosome derived BAC clone hybridises near the telomere of chromosome 2q in all eight species examined. This clone also hybridises to the sex microchromosomes of three species (P vitticeps, P. barbata and Diporiphora nobbi) and a pair of microchromosomes in three others (Ctenophorus pictus, Amphibolurus norrisi and Amphibolurus muricatus). No other chromosomes are marked by the probe in two species from the closely related genus Physignathus. A probe bearing nucleolar organising region (NOR) sequences maps close to the telomere of chromosome 2q in all eight species, and to the ZW pair in P. vitticeps and P. barbata, the W microchromosome in D. nobbi, and several microchromosomes in P. cocincinus. Our findings provide evidence of sequence homology between chromosome 2 and the sex chromosomes of multiple agamids. These data support the hypothesis that there was an ancestral sex chromosome in amniotes that gave rise to squamate chromosome 2 and raises the prospect that some particular property of this chromosome has favoured its role as a sex chromosome in amniotes. It is likely that the amplification of repetitive sequences associated with this region has driven the high level of heterochromatinisation of the sex-specific chromosomes in three species of agamid. Our data suggest a possible mechanism for chromosome rearrangement, including inversion and duplication near the telomeric regions of the ancestral chromosome 2 and subsequent translocation to the ZW sex microchromosomes in three agamid species. It is plausible that these chromosome rearrangements involving sex chromosomes also drove speciation in this group.

Gene regulatory networks underlying cellular pluripotency are controlled by a core circuitry of transcription factors in mammals, including POU5F1. However, the evolutionary origin and transformation of pluripotency-related transcriptional networks have not been elucidated in deuterostomes. PR domain-containing protein 14 (PRDM14) is specifically expressed in pluripotent cells and germ cells, and is required for establishing embryonic stem cells (ESCs) and primordial germ cells in mice. Here, we compared the functions and expression patterns of PRDM14 orthologues within deuterostomes. Amphioxus PRDM14 and zebrafish PRDM14, but not sea urchin PRDM14, compensated for mouse PRDM14 function in maintaining mouse ESC pluripotency. Interestingly, sea urchin PRDM14 together with sea urchin CBFA2T, an essential partner of PRDM14 in mouse ESCs, complemented the self-renewal defect in mouse Prdm14 KO ESCs. Contrary to the Prdm14 expression pattern in mouse embryos, Prdm14 was expressed in motor neurons of amphioxus embryos, as observed in zebrafish embryos. Thus, Prdm14 expression in motor neurons was conserved in non-tetrapod deuterostomes and the co-option of the PRDM14-CBFA2T complex from motor neurons into pluripotent cells may have maintained the transcriptional network for pluripotency during vertebrate evolution. This article has an associated ‘The people behind the papers’ interview.

The molecular mechanism underlying the hemolytic and cytolytic processes of cobra cytotoxins (CTXs) is not yet fully elucidated. To examine this, we analyzed the amino acid sequences, hemolytic and cytotoxic activities, and affinities to phospholipids of the five major CTXs purified from the venom of Indian cobra, Naja naja. CTX2, CTX7, and CTX8 belonged to S-type, and CTX9 and CTX10 to P-type. Comparisons of CTX7 with CTX8 and CTX9 with CTX10 revealed similar primary structures and hemolytic and cytolytic activities. CTX2, whose primary structure was rather different from the others, showed several times weaker hemolytic and cytolytic biological activities than the others. The comparison of CTX2 with CTX7 suggested the importance of Lys30 in loop II for the strong hemolytic and cytolytic activities of S-type CTXs. Cloning of 12 CTX cDNAs from the Naja naja venom cDNA library revealed that 18 out of 23 substitutions found in CTX cDNAs were nonsynonymous. This clearly indicated the accelerated evolution of CTX genes. Multiple sequence alignment of 51 kinds of CTX cDNAs and calculations of nonsynonymous and synonymous substitutions indicated that the codons coding the three loops' regions, which may interact with the hydrophobic tails of phospholipids, have undergone an accelerated evolution. In contrast, the codons coding for amino acid residues considered to participate in the recognition and binding of the hydrophilic head groups of phospholipids, eight Cys residues, and those likely stabilizing β core structure, were all conserved.

Highly repetitive DNA sequences of the centromeric heterochromatin provide valuable molecular cytogenetic markers for the investigation of genomic compartmentalization in the macrochromosomes and microchromosomes of sauropsids. Here, the relationship between centromeric heterochromatin and karyotype evolution was examined using cloned repetitive DNA sequences from two snake species, the habu snake (Protobothrops flavoviridis, Crotalinae, Viperidae) and Burmese python (Python bivittatus, Pythonidae). Three satellite DNA (stDNA) families were isolated from the heterochromatin of these snakes: 168-bp PFL-MspI from P. flavoviridis and 196-bp PBI-DdeI and 174-bp PBI-MspI from P. bivittatus. The PFL-MspI and PBI-DdeI sequences were localized to the centromeric regions of most chromosomes in the respective species, suggesting that the two sequences were the major components of the centromeric heterochromatin in these organisms. The PBI-MspI sequence was localized to the pericentromeric region of four chromosome pairs. The PFL-MspI and the PBI-DdeI sequences were conserved only in the genome of closely related species, Gloydius blomhoffii (Crotalinae) and Python molurus, respectively, although their locations on the chromosomes were slightly different. In contrast, the PBI-MspI sequence was also in the genomes of P. molurus and Boa constrictor (Boidae), and additionally localized to the centromeric regions of eight chromosome pairs in B. constrictor, suggesting that this sequence originated in the genome of a common ancestor of Pythonidae and Boidae, approximately 86 million years ago. The three stDNA sequences showed no genomic compartmentalization between the macrochromosomes and microchromosomes, suggesting that homogenization of the centromeric and/or pericentromeric stDNA sequences occurred in the macrochromosomes and microchromosomes of these snakes.

The sex chromosomes in Sauropsida (reptiles and birds) have evolved independently many times. They show astonishing diversity in morphology ranging from cryptic to highly differentiated sex chromosomes with male (XX/XY) and female heterogamety (ZZ/ZW). Comparing such diverse sex chromosome systems thus provides unparalleled opportunities to capture evolution of morphologically differentiated sex chromosomes in action. Here, we describe chromosomal mapping of 18 microsatellite repeat motifs in eight species of Sauropsida. More than two microsatellite repeat motifs were amplified on the sex-specific chromosome, W or Y, in five species (Bassiana duperreyi, Aprasia parapulchella, Notechis scutatus, Chelodina longicollis, and Gallus gallus) of which the sex-specific chromosomes were heteromorphic and heterochromatic. Motifs (AAGG)n and (ATCC)n were amplified on the W chromosome of Pogona vitticeps and the Y chromosome of Emydura macquarii, respectively. By contrast, no motifs were amplified on the W chromosome of Christinus marmoratus, which is not much differentiated from the Z chromosome. Taken together with previously published studies, our results suggest that the amplification of microsatellite repeats is tightly associated with the differentiation and heterochromatinization of sex-specific chromosomes in sauropsids as well as in other taxa. Although some motifs were common between the sex-specific chromosomes of multiple species, no correlation was observed between this commonality and the species phylogeny. Furthermore, comparative analysis of sex chromosome homology and chromosomal distribution of microsatellite repeats between two closely related chelid turtles, C. longicollis and E. macquarii, identified different ancestry and differentiation history. These suggest multiple evolutions of sex chromosomes in the Sauropsida.

The first report of reptile sex reversal in the wild and rapid transition between genetic and environmental sex determination in the Australian bearded dragon (Pogona vitticeps) There have been repeated evolutionary transitions in reptiles between genetic and temperature-dependent sex determination, the regulatory process that initiates differentiation of the gonads in the early embryo to form either testes or ovaries. Various mechanisms have been proposed to explain the transition, including a role for sex reversal. Clare Holleley et al. present the first report of reptile sex reversal in the wild, associated with rapid transition between genetic and environmental sex determination. In a study of the Australian bearded dragon (Pogona vitticeps), they observe sex reversal at the warmer end of the animals geographic range. When sex-reversed females mate with normal males, the chromosomal sex determination system is lost and temperature-dependent sex determination is established. It is not known whether climate-induced changes in sex determination are advantageous or detrimental to the process of evolutionary adaptation. Sex determination in animals is amazingly plastic. Vertebrates display contrasting strategies ranging from complete genetic control of sex (genotypic sex determination) to environmentally determined sex (for example, temperature-dependent sex determination)1. Phylogenetic analyses suggest frequent evolutionary transitions between genotypic and temperature-dependent sex determination in environmentally sensitive lineages, including reptiles2. These transitions are thought to involve a genotypic system becoming sensitive to temperature, with sex determined by gene–environment interactions3. Most mechanistic models of transitions invoke a role for sex reversal3,4,5. Sex reversal has not yet been demonstrated in nature for any amniote, although it occurs in fish6 and rarely in amphibians7,8. Here we make the first report of reptile sex reversal in the wild, in the Australian bearded dragon (Pogona vitticeps), and use sex-reversed animals to experimentally induce a rapid transition from genotypic to temperature-dependent sex determination. Controlled mating of normal males to sex-reversed females produces viable and fertile offspring whose phenotypic sex is determined solely by temperature (temperature-dependent sex determination). The W sex chromosome is eliminated from this lineage in the first generation. The instantaneous creation of a lineage of ZZ temperature-sensitive animals reveals a novel, climate-induced pathway for the rapid transition between genetic and temperature-dependent sex determination, and adds to concern about adaptation to rapid global climate change.

Telomeres are repeat (TTAGGG) n sequences that form terminal ends of chromosomes and have several functions, such as protecting the coding DNA from erosion at mitosis. Due to chromosomal rearrangements through evolutionary history (e.g., inversions and fusions), telomeric sequences are also found between the centromere and the terminal ends (i.e., at interstitial telomeric sites, ITSs). ITS telomere sequences have been implicated in heritable disease caused by genomic instability of ITS polymorphic variants, both with respect to copy number and sequence. In the sand lizard (Lacerta agilis), we have shown that telomere length is predictive of lifetime fitness in females but not males. To assess whether this sex specific fitness effect could be traced to ITSs differences, we mapped (TTAGGG) n sequences using fluorescence in situ hybridization in fibroblast cells cultured from 4 specimens of known sex. No ITSs could be found on autosomes in either sex. However, females have heterogametic sex chromosomes in sand lizards (ZW, 2n = 38) and the female W chromosome showed degeneration and remarkable (TTAGGG) n amplification, which was absent in the Z chromosomes. This work warrants further research on sex chromosome content, in particular of the degenerate W chromosome, and links to female fitness in sand lizards.

The sand lizard (Lacerta agilis, Lacertidae) has a chromosome number of 2n = 38, with 17 pairs of acrocentric chromosomes, one pair of microchromosomes, a large acrocentric Z chromosome, and a micro-W chromosome. To investigate the process of karyotype evolution in L. agilis, we performed chromosome banding and fluorescent in situ hybridization for gene mapping and constructed a cytogenetic map with 86 functional genes. Chromosome banding revealed that the Z chromosome is the fifth largest chromosome. The cytogenetic map revealed homology of the L. agilis Z chromosome with chicken chromosomes 6 and 9. Comparison of the L. agilis cytogenetic map with those of four Toxicofera species with many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) showed highly conserved linkage homology of L. agilis chromosomes (LAG) 1, 2, 3, 4, 5(Z), 7, 8, 9, and 10 with macrochromosomes and/or macrochromosome segments of the four Toxicofera species. Most of the genes located on the microchromosomes of Toxicofera were localized to LAG6, small acrocentric chromosomes (LAG11-18), and a microchromosome (LAG19) in L. agilis. These results suggest that the L. agilis karyotype resulted from frequent fusions of microchromosomes, which occurred in the ancestral karyotype of Toxicofera and led to the disappearance of microchromosomes and the appearance of many small macrochromosomes.

Evaluating homology between the sex chromosomes of different species is an important first step in deducing the origins and evolution of sex-determining mechanisms in a clade. Here, we describe the preparation of Z and W chromosome paints via chromosome microdissection from the Australian marbled gecko (Christinus marmoratus) and their subsequent use in evaluating sex chromosome homology with the ZW chromosomes of the Kwangsi gecko (Gekko hokouensis) from eastern Asia. We show that the ZW sex chromosomes of C. marmoratus and G. hokouensis are not homologous and represent independent origins of female heterogamety within the Gekkonidae. We also show that the C. marmoratus Z and W chromosomes are genetically similar to each other as revealed by C-banding, comparative genomic hybridization, and the reciprocal painting of Z and W chromosome probes. This implies that sex chromosomes in C. marmoratus are at an early stage of differentiation, suggesting a recent origin.

Background Satellite DNAs (stDNAs) are highly repeated sequences that constitute large portions of any genome. The evolutionary dynamics of stDNA (e.g. copy number, nucleotide sequence, location) can, therefore, provide an insight into genome organization and evolution. We investigated the evolutionary origin of VSAREP stDNA in 17 monitor lizards (seven Asian, five Australian, and five African) at molecular and cytogenetic level. Results Results revealed that VSAREP is conserved in the genome of Asian and Australian varanids, but not in African varanids, suggesting that these sequences are either differentiated or lost in the African varanids. Phylogenetic and arrangement network analyses revealed the existence of at least four VSAREP subfamilies. The similarity of each sequence unit within the same VSAREP subfamily from different species was higher than those of other VSAREP subfamilies belonging to the same species. Additionally, all VSAREP subfamilies isolated from the three Australian species (Varanus rosenbergi, V. gouldii, and V. acanthurus) were co-localized near the centromeric or pericentromeric regions of the macrochromosomes, except for chromosomes 3 and 4 in each Australian varanid. However, their chromosomal arrangements were different among species. Conclusions The VSAREP stDNA family lack homogenized species-specific nucleotide positions in varanid lineage. Most VSAREP sequences were shared among varanids within the four VSAREP subfamilies. This suggests that nucleotide substitutions in each varanid species accumulated more slowly than homogenization rates in each VSAREP subfamily, resulting in non-species-specific evolution of stDNA profiles. Moreover, changes in location of VSAREP stDNA in each Australian varanid suggests a correlation with chromosomal rearrangements, leading to karyotypic differences among these species. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-1044-6) contains supplementary material, which is available to authorized users.

Chromosomal mapping of the butterfly lizards Leiolepis belliana belliana and L. boehmei was done using the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences. The karyotype of L. b. belliana was 2n = 36, whereas that of L. boehmei was 2n = 34. The 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, while the 5S rRNA genes were found in the pericentromeric region of chromosome 6 in both species. Hybridization signals for the (TTAGGG)n sequence were observed at the telomeric ends of all chromosomes, as well as interstitially at the same position as the 18S-28S rRNA genes in L. boehmei. This finding suggests that in L. boehmei telomere-to-telomere fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located or, alternatively, at the secondary constriction of chromosome 1. The absence of telomeric sequence signals in chromosome 1 of L. b. belliana suggested that its chromosomes may have only a few copies of the (TTAGGG)n sequence or that there may have been a gradual loss of the repeat sequences during chromosomal evolution.

SummaryHeparan sulfate (HS) proteoglycans modulate the activity of multiple growth factors on the cell surface and extracellular matrix. However, it remains unclear how the HS chains control the movement and reception of growth factors into targeted receiving cells during mammalian morphogenetic processes. Here, we found that HS-deficient Ext2 null mutant mouse embryos fail to respond to fibroblast growth factor (FGF) signaling. Marker expression analyses revealed that cell surface-tethered HS chains are crucial for local retention of FGF4 and FGF8 ligands in the extraembryonic ectoderm. Fine chimeric studies with single-cell resolution and expression studies with specific inhibitors for HS movement demonstrated that proteolytic cleavage of HS chains can spread FGF signaling to adjacent cells within a short distance. Together, the results show that spatiotemporal expression of cell surface-tethered HS chains regulate the local reception of FGF-signaling activity during mammalian embryogenesis.

Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition.

Protobothrops flavoviridis (Crotalinae) venom gland phospholipase A 2 (PLA 2 ) isozyme genes have evolved in an accelerated manner to acquire diverse physiological activities in their products. For elucidation of the multiplication mechanism of PLA 2 genes, a 25,026 bp genome segment harboring five PLA 2 isozyme genes was obtained from Amami-Oshima P. flavoviridis liver and sequenced. The gene PfPLA 2 encoded [Lys 49 ]PLA 2 called BPII, the gene PfPLA 4 neurotoxic [Asp 49 ]PLA 2 called PLA-N, the gene PfPLA 5 basic [Asp 49 ]PLA 2 called PLA-B, and PfPLA 1(ψ) and PfPLA 3(ψ) were the inactivated genes. The 5′ truncated reverse transcriptase (RT) elements, whose intact forms constitute long interspersed nuclear elements (LINEs), were found in close proximity to the 3′ end of PLA 2 genes and named PLA 2 gene-coupled RT fragments (PcRTFs). The facts that PcRTFs have the stem–loop and repetitive sequence in the 3′ untranslated region (UTR) which is characteristic of CR1 LINEs suggest that PcRTFs are the debris of P. flavoviridis ancestral CR1 LINEs, denoted as Pf CR1s. Since the associated pairs of PLA 2 genes and PcRTFs are arranged in tandem in the 25,026 bp segment, it is thought that an ancestral PLA 2 gene- Pf CR1 unit ( PfPLA - Pf CR1) which was produced by retrotransposition of Pf CR1 by itself to the 3′ end of PLA 2 gene duplicated several times to form a multimer of PfPLA - Pf CR1, a cluster of PLA 2 genes, in the period after Crotalinae and Viperinae snakes branched off. Recombinational hot spot of a 37 bp segment, named Scomb , was found in the region 548 bp upstream from the TATA box of PLA 2 genes. Thus, it could be assumed that multiplication of PfPLA - Pf CR1 occurred by unequal crossing over of the segment, - Scomb - PfPLA - Pf CR1- Scomb- . The Pf CR1 moieties were afterward disrupted in the 5′ portion to PcRTFs. The detection of two types of PcRTFs different in length which were produced by elimination of two definitive sequences in Pf CR1 moiety possibly by gene conversion clearly supports such process but not multiplication of the PLA 2 gene-PcRTF unit.

The butterfly lizard (Leiolepis reevesii rubritaeniata) has the diploid chromosome number of 2n = 36, comprising two distinctive components, macrochromosomes and microchromosomes. To clarify the conserved linkage homology between lizard and snake chromosomes and to delineate the process of karyotypic evolution in Squamata, we constructed a cytogenetic map of L. reevesii rubritaeniata with 54 functional genes and compared it with that of the Japanese four-striped rat snake (E. quadrivirgata, 2n = 36). Six pairs of the lizard macrochromosomes were homologous to eight pairs of the snake macrochromosomes. The lizard chromosomes 1, 2, 4, and 6 corresponded to the snake chromosomes 1, 2, 3, and Z, respectively. LRE3p and LRE3q showed the homology with EQU5 and EQU4, respectively, and LRE5p and LRE5q corresponded to EQU7 and EQU6, respectively. These results suggest that the genetic linkages have been highly conserved between the two species and that their karyotypic difference might be caused by the telomere-to-telomere fusion events followed by inactivation of one of two centromeres on the derived dicentric chromosomes in the lineage of L. reevesii rubritaeniata or the centric fission events of the bi-armed macrochromosomes and subsequent centromere repositioning in the lineage of E. quadrivirgata. The homology with L. reevesii rubritaeniata microchromosomes were also identified in the distal regions of EQU1p and 1q, indicating the occurrence of telomere-to-telomere fusions of microchromosomes to the p and q arms of EQU1.

Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata) was performed by conventional Giemsa staining, Ag-NOR banding, FISH with the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences, and CGH. The karyotype was composed of 2 distinct components, macrochromosomes and microchromosomes, and the chromosomal constitution was 2n = 2x = 36 (L4m + L2sm + M2m + S4m + 24 microchromosomes). NORs and the 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, and the 5S rRNA genes were localized to the pericentromeric region of chromosome 6. Hybridization signals of (TTAGGG)n sequences were observed at the telomeric ends of all chromosomes and interstitially at the same position as the 18S-28S rRNA genes, suggesting that in the Leiolepinae tandem fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located. CGH analysis, however, failed to identify sex chromosomes, suggesting that this species may have a TSD system or exhibit GSD with morphologically undetectable cryptic sex chromosomes. Homologues of 6 chicken Z-linked genes (ACO1/IREBP, ATP5A1, CHD1, DMRT1, GHR, RPS6) were all mapped to chromosome 2p in the same order as on the snake chromosome 2p.

Three novel families of repetitive DNA sequences were molecularly cloned from the Korean field mouse (Apodemus peninsulae) and characterized by chromosome in-situ hybridization and filter hybridization. They were all localized to the centromeric regions of all autosomes and categorized into major satellite DNA, type I minor, and type II minor repetitive sequences. The type II minor repetitive sequence also hybridized interspersedly in the non-centromeric regions. The major satellite DNA sequence, which consisted of 30 bp elements, was organized in tandem arrays and constituted the majority of centromeric heterochromatin. Three families of repetitive sequences hybridized with B chromosomes in different patterns, suggesting that the B chromosomes of A. peninsulae were derived from A chromosomes and that the three repetitive sequences were amplified independently on each B chromosome. The minor repetitive sequences are present in the genomes of the other seven Apodemus species. In contrast, the major satellite DNA sequences that had a low sequence homology are present only in a few species. These results suggest that the major satellite DNA was amplified with base substitution in A. peninsulae after the divergence of the genus Apodemus from the common ancestor and that the B chromosomes of A. peninsulae might have a species-specific origin.

A genetic linkage map for Suncus murinus was previously constructed with 11 marker loci. In this study, we developed 172 new microsatellite and three RFLP markers, and re-constructed a new framework map by combining all markers. The new map comprises 42 markers that are distributed into 12 linkage groups, two of which are assigned to chromosomes, and spans 403.5 cM with an average inter-marker distance of 13.5 cM.

Comparative chromosome painting with mouse (Mus musculus, MMU) chromosome-specific DNA probes was performed for three Muridae species, the Indian soft-furred field rat (Millardia meltada), the spiny mouse (Acomys dimidiatus) and the harvest mouse (Micromys minutus). All probes except for the Y probe were successfully hybridized to the chromosomes of all species, and homologous chromosome segments between mouse and the three species were identified at the molecular level. Comparison of our data with the published data of six other genera (Mus, Rattus, Apodemus, Otomys, Rhabdomys and Cricetulus) of the Muridae suggested that the associations MMU1b/17a, 2b/13a, 5b/11a, 7/19, 10b/17b, 10c/17c, 11b/16a, 12/17d and 13b/15, and the single painted chromosomes and chromosome segments MMU3, 4, 5a, 8a, 8b, 16b, 18 and X were probably contained by the ancestral karyotype of the Muridae, and have been strongly conserved throughout murid evolution.

Ryukyu spiny rats (genus Tokudaia) are indigenous species that are confined to three islands of the Nansei Shoto archipelago, Amami-Oshima, Tokunoshima and Okinawa-jima, Japan. Tokudaia tokunoshimensis from Tokunoshima Island and Tokudaia osimensis from Amami-Oshima Island are closely related taxonomically, although their karyotypes are quite different: the diploid chromosome numbers and sex chromosome constitution are 2n = 45, X0/X0 for T. tokunoshimensis and 2n = 25, X0/X0 for T. osimensis. We conducted comparative chromosome painting with chromosome-specific DNA probes of the laboratory mouse (Mus musculus) to molecularly examine the chromosome homology between T. tokunoshimensis and T. osimensis, and deduced a possible ancestral karyotype of Tokudaia species and the process of evolutionary chromosome rearrangements. The proposed ancestral karyotype with the diploid number of 2n = 48, XX/XY was similar to the karyotype of T. tokunoshimensis, and the karyotype of T. osimensis would then have been established through at least 14 chromosomal changes, mainly centric fusion and tandem fusion, from the ancestral karyotype. The close karyological relationship between the ancestral karyotypes of Tokudaia and Apodemus also suggests that the chromosomal evolution in the Tokudaia-Apodemus lineage has been very slow and has accelerated only recently in the branch leading to T. osimensis.

All snake species exhibit genetic sex determination with the ZZ/ZW type of sex chromosomes. To investigate the origin and evolution of snake sex chromosomes, we constructed, by FISH, a cytogenetic map of the Japanese four-striped rat snake ( Elaphe quadrivirgata ) with 109 cDNA clones. Eleven of the 109 clones were localized to the Z chromosome. All human and chicken homologues of the snake Z-linked genes were located on autosomes, suggesting that the sex chromosomes of snakes, mammals, and birds were all derived from different autosomal pairs of the common ancestor. We mapped the 11 Z-linked genes of E. quadrivirgata to chromosomes of two other species, the Burmese python ( Python molurus bivittatus ) and the habu ( Trimeresurus flavoviridis ), to investigate the process of W chromosome differentiation. All and 3 of the 11 clones were localized to both the Z and W chromosomes in P. molurus and E. quadrivirgata , respectively, whereas no cDNA clones were mapped to the W chromosome in T. flavoviridis . Comparative mapping revealed that the sex chromosomes are only slightly differentiated in P. molurus , whereas they are fully differentiated in T. flavoviridis , and E. quadrivirgata is at a transitional stage of sex-chromosome differentiation. The differentiation of sex chromosomes was probably initiated from the distal region on the short arm of the protosex chromosome of the common ancestor, and then deletion and heterochromatization progressed on the sex-specific chromosome from the phylogenetically primitive boids to the more advanced viperids.

Comparative chromosome painting was applied to the Indian spiny mouse (Mus platythrix) with mouse (M. musculus) chromosome-specific probes for understanding the process of chromosome rearrangements between the two species. The chromosome locations of the 5S and 18S-28S ribosomal RNA genes and the order of the 119 and Tcp-1 genes in the In(17)2 region of the t-complex were also compared. All the painting probes were successfully hybridized to the Indian spiny mouse chromosomes, and a total of 27 segments homologous to mouse chromosomes were identified. The comparative FISH analysis revealed that tandem fusions were major events in the chromosome evolution of the Indian spiny mouse. In addition, other types of chromosome rearrangements, i.e. reciprocal translocations and insertions, were also included.

Transitions between sex determination systems have occurred in many lineages of squamates and it follows that novel sex chromosomes will also have arisen multiple times. The formation of sex chromosomes may be reinforced by inhibition of recombination and the accumulation of repetitive DNA sequences. The karyotypes of monitor lizards are known to be highly conserved yet the sex chromosomes in this family have not been fully investigated. Here, we compare male and female karyotypes of three Australian monitor lizards, Varanus acanthurus, V. gouldii and V. rosenbergi, from two different clades. V. acanthurus belongs to the acanthurus clade and the other two belong to the gouldii clade. We applied C-banding and comparative genomic hybridization to reveal that these species have ZZ/ZW sex micro-chromosomes in which the W chromosome is highly differentiated from the Z chromosome. In combination with previous reports, all six Varanus species in which sex chromosomes have been identified have ZZ/ZW sex chromosomes, spanning several clades on the varanid phylogeny, making it likely that the ZZ/ZW sex chromosome is ancestral for this family. However, repetitive sequences of these ZW chromosome pairs differed among species. In particular, an (AAT)n microsatellite repeat motif mapped by fluorescence in situ hybridization on part of W chromosome in V. acanthurus only, whereas a (CGG)n motif mapped onto the W chromosomes of V. gouldii and V. rosenbergi. Furthermore, the W chromosome probe for V. acanthurus produced hybridization signals only on the centromeric regions of W chromosomes of the other two species. These results suggest that the W chromosome sequences were not conserved between gouldii and acanthurus clades and that these repetitive sequences have been amplified rapidly and independently on the W chromosome of the two clades after their divergence.

DC-SIGN, a human C-type lectin, is expressed on the surface of dendritic cells (DC), while a closely related human gene, DC-SIGNR or L-SIGN, is found on sinusoidal endothelial cells of liver and lymph node. Both DC-SIGN and DC-SIGNR/L-SIGN can bind ICAM-3 and HIV gp120, and transmit HIV to susceptible cells in trans. Here, we report the cloning of five mouse genes homologous to human DC-SIGN and DC-SIGNR/L-SIGN. Only one gene, named mouse DC-SIGN, is highly expressed in DC, and is not found in a panel of mouse macrophage and lymphocyte cell lines. The other four genes, named mouse SIGNR1 (SIGN-Related gene 1), SIGNR2, SIGNR3 and SIGNR4, are expressed at lower levels in various cells according to RT-PCR and Northern blot analyses on RNA. All the genes of mouse DC-SIGN and SIGNRs map to adjacent regions of chromosome 8 A1.2-1.3. However, like human DC-SIGN, only the mouse DC-SIGN gene is closely juxtaposed to the CD23 gene, while the other four SIGNR genes are located close to each other in a neighboring region. mRNAs of mouse DC-SIGN and three SIGNR genes encode type II transmembrane proteins (DC-SIGN, 238 amino acids; SIGNR1, 325 amino acids; SIGNR3, 237 amino acids; SIGNR4, 208 amino acids), but the SIGNR2 gene only encodes a carbohydrate recognition domain (CRD) without a cytosolic domain and a transmembrane domain (SIGNR2, 178 amino acids). Amino acid sequence similarities between the CRD of human DC-SIGN and the mouse homologues are 67% for DC-SIGN, 69% for SIGNR1, 65% for SIGNR2, 68% for SIGNR3 and 70% for SIGNR4 respectively. However, the membrane proximal neck domains in the mouse genes are much shorter than their counterparts in human DC-SIGN and DC-SIGNR/L-SIGN. This family of mouse C-type lectins is therefore complex, but only one of the new genes, DC-SIGN, is juxtaposed to CD23 and is expressed at high levels in DC.

The Mail (molecule possessing ankyrin repeats induced by lipopolysaccharide) protein is a member of the IkappaB family. It has six ankyrin repeats that are conserved in other IkappaB proteins, such as IkappaB-alpha and Bcl-3. Mail mRNA expression is induced rapidly following lipopolysaccharide (LPS) injection, most notably in the spleen, lung, and lymph nodes of mice, where immune cells, such as lymphocytes and macrophages, are abundant. In this study, we cloned and characterized the Mail gene. The isolated genomic clones span approximately 30 kb and encompass the entire gene. Comparisons with Mail cDNA revealed that the Mail gene consists of 14 exons. Several splice junctions encoding ankyrin repeats are conserved among Mail and other IkappaB family genes. Southern hybridization showed that Mail is a single-copy gene. Using fluorescence in situ hybridization analysis, mouse and rat Mail genes were mapped to Chromosome (Chr) 16C1.2-C1.3 and Chr 11q21.1, respectively. Primer extension determined the transcription start site of Mail. Sequence analysis of the proximal promoter region revealed the presence of a TATA box and putative transcription factor-binding sites, such as those for NF-kappaB and NF-IL6. This region is sufficient to drive high-level reporter gene expression in LPS-stimulated transfected cells.

The direct R-banding fluorescence in situ hybridization (FISH) method was used to map 18S-28S ribosomal RNA genes and 10 human cDNA clones on the chromosomes of the musk shrew (Suncus murinus). The chromosomal locations of 18S-28S ribosomal RNA genes were examined in the five laboratory lines and wild animals captured in the Philippines and Vietnam, and the genes were found on chromosomes 5, 6, 9, and 13 with geographic variation. The comparative mapping of 10 cDNA clones of human chromosome 1 demonstrated that human chromosome 1 consisted of at least three segments homologous to Suncus chromosomes (chromosomes 7, 10, and 14). This approach with the direct R-banding FISH method is useful for constructing comparative maps between human and insectivore species and for explicating the process of chromosomal rearrangements during the evolution of mammals.

Forty-one cDNA clones of human functional genes were newly mapped to chromosomes of the musk shrew (Suncus murinus, Insectivora) by fluorescence in situ hybridization, and a comparative cytogenetic map of 51 genes, including 10 genes reported in our previous study, was constructed between human (HSA) and musk shrew (SMU) chromosomes. In this comparative map, the 51 genes localized to human autosomes, except HSA 8, 16, and 20, were mapped to 15 shrew autosomes, except SMU 4, 16, 17 and 18. Twelve conserved segments were identified between human and shrew chromosomes, and six segments among the musk shrew, human, and mouse. Our results defined the presence of at least one inversion and several interchromosomal rearrangements that occurred during evolution after the two species diverged from a common ancestor. Localization of three major histocompatibility complex (MHC) genes to shrew chromosome 3 suggested that the MHC genes of the musk shrew are located in a cluster on chromosome 3. The cytogenetic map constructed in this study is the first cytogenetic map with many functional genes in insectivore species. This approach provides clues for clarifying the chromosomal evolution in this order.

DEC1 is a basic helix-loop-helix (bHLH) protein related to Drosophila Hairy, Enhancer of split and HES, and involved in the control of proliferation and/or differentiation of chondrocytes, neurons, etc. We report here the identification and characterization of human, mouse and rat DEC2, a novel member of the DEC subfamily. DEC2 had high (97%) and moderate (52%) similarities in the bHLH region and the Orange domain with DEC1, respectively. However, DEC2, but not DEC1, had alanine and glycine-rich regions in the C-terminal half. Unlike Hairy, Enhancer of split and HES, DEC2 lacked the WRPW motif for interaction with the corepressor Groucho. The DEC2 gene was mapped to human chromosome 12p11.23-p12.1, mouse chromosome 6 G2-G3 and rat chromosome 4q43 distal-q4, where the conserved linkage homology has been identified among these species. Unlike DEC1, which was broadly expressed in many tissues, DEC2 showed a more restricted pattern of mRNA expression. The DEC subfamily proteins may play an important role in tissue development.

We constructed comparative cytogenetic maps of the Chinese hamster to mouse, rat and human by fluorescence in-situ hybridization using 36 cDNA clones of mouse, rat, Syrian hamster, Chinese hamster and human functional genes. In this study, 30 out of the 36 genes were newly mapped to Chinese hamster chromosomes. The chromosomal homology of the Chinese hamster was identified and arranged in 19, 19 and 18 segments of conserved synteny in mouse, rat and human, respectively. Additionally, two of the 19 segments homologous to mouse chromosomes were initially identified in this study.

Robertsonian (Rb) karyotypic polymorphism in Apodemus speciosus has interested many researchers with particular referece to the genetic divergence between Rb and non-Rb populations. Failure to find morphologic, biochemical, or genetic differences in previous studies reveals the necessity of focusing on loci on Rb chromosomes, which can be characterized by FISH mapping with DNA probes. In an Rb heterozygote, DNA probes from laboratory mouse chromosomes (MMUs) 1 and 10 were simultaneously hybridized to the long arm of a metacentric and a medium-sized acrocentric chromosome and to the short arm of the metacentric and a small acrocentric chromosome, respectively. Four additional probes derived from each of MMUs 1 and 10 were mapped to the long and short arms, respectively, of the Rb chromosome identified by the above markers. Homologies between the long arm of the Rb chromosome and MMU 1 and between the short arm and MMU 10 were supported by all ten markers, which were dispersed along nearly the entire lengths of the Rb chromosomes. These results indicate that the long and short arms of the Rb chromosomes are homologous to Apodemus speciosus chromosomes 12 and 19 (defined in a previous study), respectively. This ten-marker series can be useful for detecting chromosome-specific divergence between the two karyotypic populations at the gene level.

The olfactory receptor (OR) genes represent the largest multigene family in the genome of terrestrial vertebrates. Here, the high-throughput next-generation sequencing (NGS) approach was applied to characterization of OR gene repertoires in the green anole lizard Anolis carolinensis and the Japanese four-lined ratsnake Elaphe quadrivirgata. Tagged polymerase chain reaction (PCR) products amplified from either genomic DNA or cDNA of the two species were used for parallel pyrosequencing, assembling, and screening for errors in PCR and pyrosequencing. Starting from the lizard genomic DNA, we accurately identified 56 of 136 OR genes that were identified from its draft genome sequence. These recovered genes were broadly distributed in the phylogenetic tree of vertebrate OR genes without severe biases toward particular OR families. Ninety-six OR genes were identified from the ratsnake genomic DNA, implying that the snake has more OR gene loci than the anole lizard in response to an increased need for the acuity of olfaction. This view is supported by the estimated number of OR genes in the Burmese python's draft genome (∼280), although squamates may generally have fewer OR genes than terrestrial mammals and amphibians. The OR gene repertoire of the python seems unique in that many class I OR genes are retained. The NGS approach also allowed us to identify candidates of highly expressed and silent OR gene copies in the lizard's olfactory epithelium. The approach will facilitate efficient and parallel characterization of considerable unbiased proportions of multigene family members and their transcripts from nonmodel organisms.

Comparative genome analysis of non-avian reptiles and amphibians provides important clues about the process of genome evolution in tetrapods. However, there is still only limited information available on the genome structures of these organisms. Consequently, the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes in tetrapods remain poorly understood. We constructed chromosome maps of functional genes for the Chinese soft-shelled turtle (Pelodiscus sinensis), the Siamese crocodile (Crocodylus siamensis), and the Western clawed frog (Xenopus tropicalis) and compared them with genome and/or chromosome maps of other tetrapod species (salamander, lizard, snake, chicken, and human). This is the first report on the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes inferred from comparative genomic analysis of vertebrates, which cover all major non-avian reptilian taxa (Squamata, Crocodilia, Testudines). The eight largest macrochromosomes of the turtle and chicken were equivalent, and 11 linkage groups had also remained intact in the crocodile. Linkage groups of the chicken macrochromosomes were also highly conserved in X. tropicalis, two squamates, and the salamander, but not in human. Chicken microchromosomal linkages were conserved in the squamates, which have fewer microchromosomes than chicken, and also in Xenopus and the salamander, which both lack microchromosomes; in the latter, the chicken microchromosomal segments have been integrated into macrochromosomes. Our present findings open up the possibility that the ancestral amniotes and tetrapods had at least 10 large genetic linkage groups and many microchromosomes, which corresponded to the chicken macro- and microchromosomes, respectively. The turtle and chicken might retain the microchromosomes of the amniote protokaryotype almost intact. The decrease in number and/or disappearance of microchromosomes by repeated chromosomal fusions probably occurred independently in the amphibian, squamate, crocodilian, and mammalian lineages.

In many vertebrates, steroid hormones are essential for ovarian differentiation during a critical developmental stage as well as promoting the growth and differentiation of the adult female reproductive system. Although studies have been extensively conducted in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens) action have been poorly examined in reptiles. Here, we evaluate hormone receptor and ligand interactions in two species of snake, the Okinawa habu (Protobothrops flavoviridis, Viperidae) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae) after the isolation of cDNAs encoding estrogen receptor α (ESR1) and estrogen receptor β (ESR2). Using a transient transfection assay with mammalian cells, the transcriptional activity of reptilian (Okinawa habu, Japanese four-striped rat snake, American alligator, and Florida red-belly freshwater turtle) ESR1 and ESR2 was examined. All ESR proteins displayed estrogen-dependent activation of transcription via an estrogen-response element-containing promoter; however, the responsiveness to various estrogens was different. Further, we determined the chromosomal locations of the snake steroid hormone receptor genes. ESR1 and ESR2 genes were localized to the short and long arms of chromosome 1, respectively, whereas androgen receptor was localized to a pair of microchromosomes in the two snake species examined. These data provide basic tools that allow future studies examining receptor-ligand interactions and steroid endocrinology in snakes and also expands our knowledge of sex steroid hormone receptor evolution.

The Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae, Testudines) has ZZ/ZW-type micro-sex chromosomes where the 18S-28S ribosomal RNA genes (18S-28S rDNA) are located. The W chromosome is morphologically differentiated from the Z chromosome by partial deletion and amplification of 18S-28S rDNA and W-specific repetitive sequences. We recently found a functional gene (TOP3B) mapped on the P. sinensis Z chromosome, which is located on chicken (Gallus gallus, GGA) chromosome 15. Then we cloned turtle homologues of 4 other GGA15-linked genes (GIT2, NF2, SBNO1, SF3A1) and localized them to P. sinensis chromosomes. The 4 genes all mapped on the Z chromosome, and 2 of them (SBNO1, SF3A1) were also localized to the W chromosome. Our mapping data suggest that at least one large inversion occurred between GGA15 and the P. sinensis Z chromosome, and that there are homologous regions in the distal portions of both the short and long arms between the Z and W chromosomes. W chromosomal differentiation in P. sinensis probably proceeded by the deletion of the proximal chromosomal region followed by 18S-28S rDNA amplification, after a paracentric inversion occurred at the breakpoints between the distal region of 18S-28S rDNA and the proximal region of SBNO1 on the Z chromosome.