The sex-determining zinc finger sequences in XY females of Akodon azarae (Rodentia, Cricetidae)

Wild populations of Akodon azarae comprise females with a karyotype indistinguishable from that of males. These individuals were formerly assumed to be Xx, the x being an X chromosome with a deletion of most of its long arm. By using a DNA probe derived from the testis-determining region of the human Y chromosome (comprising a candidate gene for the testis-determining factor, Y-linked zinc finger [ZFY]), we demonstrate that A. azarae gonosomally variant females are XY and not Xx. The ZFY sequences in A. azarae are amplified and located in two different families of EcoRI fragments derived from Y-chromosome DNA. No rearrangement or change in the state of methylation of ZFY or ZFX (X-linked zinc finger) sequences were found in XY females. We propose that sex reversal in A. azarae may be mediated by a gene or genes other than ZFX or ZFY.     

Evolution of the X-linked Zinc Finger Gene and the Y-linked Zinc Finger Gene in Primates

We have sequenced the partial exon of the zinc finger genes (ZFX and ZFY) in 5 hominoids, 2 Old World monkeys, 1 New World monkey, and 1 prosimian. Among these primate species, the percentage similarities of the nucleotide sequence of the ZFX gene were 96-100% and 91.2-99.7% for the ZFY gene. Of 397 sites in the ZFX and ZFY gene sequences, 20 for ZFX gene and 42 for ZFY gene were found to be variable. Substitution causes 1 amino acid change in ZFX, and 5 in ZFY, among 132 amino acids. The numbers of synonymous substitutions per site (Ks) between human and the chimpanzee, gorilla and orangutan for ZFY gene were 0.026, 0.033, and 0.085, respectively. In contrast, the Ks value between human and hominoid primates for the ZFX gene was 0.008 for each comparison. Comparison of the ZFX and ZFY genes revealed that the synonymous substitution levels were higher in hominoids than in other primates. The rates of synonymous substitution per site per year were higher in the ZFY exon than in the SRY exon, and higher in the ZFY exon than in the ZFY intron, in hominoid primates.     

毛冠鹿ZFY、ZFX基因片段的克隆与性别鉴定

根据人和鼠性别分化相关的ZFY、ZFX基因序列设计引物,以雌雄毛冠鹿的基因组DNA为模板进行PCR扩增,将扩增产物克隆到pMD18T上,获得ZFY、ZFX重组克隆,并测定了ZFY、ZFX基因片段的序列,序列比较显示两者同源性达 91%,仅在少数位点有差异,以此确定AvaⅡ为ZFX上特异酶切位点,通过PCR扩增和AvaⅡ特异酶切对毛冠鹿性别进行鉴定。 Abstract: According to the human sex differentiation related ZFY and ZFX genes, a pair of primers were designed , and fragments were amplified from the genomic DNA of male or female tufted deer. Subsequently the amplified fragments were cloned into the vector pMD18T and were sequenced. It is found that the sequences of ZFY gene and ZFX gene have 91% homology. Based on the different nucleotides, restriction site of AvaⅡ was found to be specific to ZFX gene. The results show that the combination of PCR with AvaⅡ digestion is a simple and sensitive way to identify the tufted deer sex.     

Cloning and comparative analysis of the bovine, porcine, and equine sex chromosome genes ZFX and ZFY.

A growing body of evidence suggests the involvement of sex chromosome genes in mammalian development. We report the cloning and characterization of the complete coding regions of the bovine Y chromosome ZFY and X chromosome ZFX genes, and partial coding regions of porcine and equine ZFX and ZFY genes. Bovine ZFY and ZFX are highly similar to each other and to ZFX and ZFY from other species. While bovine and human ZFY proteins are both 801 amino acids long, bovine ZFX is 5 amino acids shorter than human ZFX. Like in humans, both bovine ZFY and ZFX contain 13 zinc finger motifs and belong to the Krueppel family of C2H2-type zinc finger proteins. The internal exon-intron organization of the bovine, porcine and equine ZFX and ZFY genes has been determined and compared. Within this region, the exon lengths and the positions of the splice sites are conserved, further suggesting a high evolutionary conservation of the ZFX and ZFY genes. Additionally, new alternatively spliced forms of human ZFX have been identified.     

A rapid sex-identification test for the forest musk deer (Moschus berezovskii) based on the ZFX/ZFY gene

We describe a rapid sex-identification method for the forest musk deer (Moschus berezovskii) using PCR based on zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes. Fragments of the ZFX and ZFY genes were amplified and sequenced. The ZFX and ZFY fragments were identical in length and 94% similar in nucleotide sequence. Specific primers for forest musk deer sex identification were designed on the basis of sequence differences between ZFX and ZFY. All the primers were multiplexed in single-tube PCR. Both male and female forest musk deer showed amplification bands of 447 bp and 212 bp separated in agarose gels. A sex-specific 278-bp band was amplified only from males. These results show that testing by PCR for the presence of the 278-bp sequence is a rapid and reliable method for sex identification.     

Identification of sex in Cetaceans by multiplexing with three ZFX and ZFY specific primers

We sequenced 540 nucleotides of the last exon in the ZFY/ZFX gene in two males and two females for eight cetacean species; four odontocetes (toothed whales) and four mysticetes (baleen whales). Based upon the obtained nucleotide sequences, we designed two sets of oligonucleotide primers for specific amplification of the ZFX and the ZFY sequence in odontocetes and mysticetes, respectively. Each primer set consisted of three oligonucleotides; one forward-orientated primer, which anneals to the ZFY as well as the ZFX sequence, and two reverse-orientated primers that anneal to either the ZFX or the ZFY sequence. The resulting two amplification products (specific for the ZFY and ZFX sequences) can be distinguished by gel-electrophoresis through 2% NuSieve™. The accuracy of the technique was tested by determination of gender in 214 individuals of known sex. Finally we applied the technique to determine the sex of 3570 cetacean specimens; 2284 humpback whales, 315 fin whales, 37 blue whales, 7 minke whales, as well as 592 belugas, 335 narwhals and 25 harbour porpoises.     

Comparative molecular phylogeny and evolution of sex chromosome DNA sequences in the family Canidae (Mammalia: Carnivora)

To investigate the molecular phylogeny and evolution of the family Canidae, nucleotide sequences of the zinc-finger-protein gene on the Y chromosome (ZFY, 924-1146 bp) and its homologous gene on the X chromosome (ZFX, 834-839 bp) for twelve canid species were determined. The phylogenetic relationships among species reconstructed by the paternal ZFY sequences closely agreed with those by mtDNA and autosomal DNA trees in previous reports, and strongly supported the phylogenetic affinity between the wolf-like canids clade and the South American canids clade. However, the branching order of some species differed between phylogenies of ZFY and ZFX genes: Cuon alpinus and Canis mesomelas were included in the wolf-like canid clades in the ZFY tree, whereas both species were clustered in a group of Chrysocyon brachyurus and Speothos venaticus in the ZFX tree. The topology difference between ZFY and ZFX trees may have resulted from the two-times higher substitution rate of the former than the latter, which was clarified in the present study. In addition, two types of transposable element sequence (SINE-I and SINE-II) were found to occur in the ZFY final intron of the twelve canid species examined. Because the SINE-I sequences were shared by all the species, they may have been inserted into the ZFY of the common ancestor before species radiation in Canidae. By contract, SINE-II found in only Canis aureus could have been inserted into ZFY independently after the speciation. The molecular diversity of SINE sequences of Canidae reflects evolutionary history of the species radiation.     

Sex chromosomal transposable element accumulation and male-driven substitutional evolution in humans

We sequenced the genomic region containing the human Y-linkedzinc finger gene (ZFY). Comparison of ZFY to the related regionon the X chromosome (ZFX) and to autosomal sequences revealsa significant accumulation of transposable elements on the sexchromosomes. In addition, five times as many retroviruslikeelements (RLEs) are present in the ZFY region as in the ZFXregion. Thus, transposable elements accumulate more rapidlyon the sex chromosomes, and the insertion of RLEs may occurmore frequently in the male than in the female germ line. Whenthe accumulation of substitutions in Alu elements was analyzed,it was found that the Alu elements at the Y-chromosomal locusdiverged significantly faster than those at the X-chromosomallocus, whereas the divergence of autosomal Alu elements wasintermediate. The male-to-female mutation rate ratio was estimatedto be 2.5.     

牦牛与其他物种ZFX/ZFY基因片段间的进化关系

利用PCR扩增、克隆和序列分析法对牦牛ZFX/ZFY基因第11外显子部分片段进行了研究,并同来自于NCBI GenBank中人、猩猩、普通牛等9个物种的ZFX/ZFY基因核苷酸及其氨基酸序列进行了进化分析.结果表明,牦牛ZFX、ZFY基因间核苷酸序列同源性为94.1%,显示同一物种同源基因ZFX/ZFY间存在变异;比较的10个物种间ZFX基因核苷酸序列同源性为87.7%、ZFY基因为81.7%,相应ZFX、ZFY氨基酸同源性分别为96.6%、91.0%,ZFY基因的变异性大于ZFX基因,显示X染色体与Y染色体可能是独立进化.     

Interfamilial characterization of a region of the ZFX and ZFY genes facilitates sex determination in cetaceans and other mammals

Sequence polymorphism of homologues ZFX and ZFY, in a 604-base pair exon region, was examined in 10 known males and 10 known females across seven cetacean families and used to design a simple, highly sensitive and widely applicable fluorescent 5' exonuclease assay for gender determination in cetaceans. Multiplex amplification, cloning, and sequencing of these previously uncharacterized regions revealed (i) eight fixed differences between ZFX and ZFY, (ii) 29 variable sites between ZFX and ZFY and (iii) very low interspecific nucleotide diversity for both ZFX and ZFY across all families examined. We developed a 5' exonuclease assay that produces a small (105 bp) polymerase chain reaction (PCR) product from both the X and the Y chromosome orthologs, and used double-labelled fluorescent probes to distinguish between the two genes in a real-time PCR assay that is highly reproducible and sensitive. We demonstrated sex specificity for 33 cetacean species in nine families. Given the availability of conserved primers and sequence information for many mammalian species, this approach to designing sexing assays for a wide range of species is both practical and efficient.     

Duplication of an Xp segment that includes the ZFX locus causes sex inversion in man

Summary Two 46,XY females with tandem duplications of an X short arm segment were studied by cytogenetic and Southern blot analysis. The results show that the duplicated segment in each case included the Xp21.2–Xp22.2 interval, resulting in a double dose of ZFX on the single active X chromosome. The results from our two cases, in conjunction with those reported by other workers, lead us to conclude that the duplication is the reason for the sex inversion. If ZFY and ZFX are indeed sex-determining gene loci, these findings favour a model of sex determination characterized by antagonistic interaction between these genes.     

Is selection responsible for the low level of variation in the last intron of the ZFY locus?

DNA variability was investigated in the last intron of the Y-chromosome-specific zinc finger gene, ZFY, and its X homolog on Xp21.3, ZFX. No polymorphisms were found in the 676-bp ZFY segment in a sample of 205 world-wide-distributed Y chromosomes, other than a solitary nucleotide variant in one individual (nucleotide diversity pi = 0.0014%). In contrast, 10 segregating sites (pi = 0.082%) were identified within 1,089 bp of the ZFX sequence in a sample of 336 X chromosomes. Four of these polymorphisms, which contributed most of the diversity, were located within an Alu insert disrupting the ZFY-ZFX homology (pi Alu = 0.24%). The diversity in the homologous portion of the ZFX intron, although higher than that in ZFY, was lower than that found in genomic segments believed to evolve neutrally; interspecies divergence in both segments was also reduced. Although this suggests that the evolution of both ZFY and ZFX homologs may not be entirely neutral, both Tajima and HKA tests did not reject neutrality. The lack of statistical significance may be attributed to a lack of power in these tests (the low divergence and variability values reduce the power of the HKA and Tajima tests, respectively); furthermore, Homo sapiens has recently undergone a rapid population growth, and selection is more difficult to detect in an expanding population. Therefore, the failure to reject neutrality does not necessarily indicate the absence of selection. In this context, the phylogenetic argument was given more weight in out interpretations. The high level of sequence identity in ZFY and ZFX segments, in spite of their separation 80-130 MYA, reflects a lower mutation rate as compared with other segments believed to undergo unconstrained evolution. Thus, the possibility of weak selection contributing to the low level of nucleotide diversity in the last ZFY intron cannot be excluded and should be kept in mind in the population genetics studies based on Y chromosome variability.     

Mapping the human ZFX locus to Xp21.3 by in situ hybridization

Summary In situ hybridization using a probe specific for the human ZFX and ZFY loci assigns the ZFX gene to Xp21.3 and the ZFY gene to Yp11.32.     

An ARMS-based technique for sex determination of red panda (Ailurus fulgens)

Molecular sexing is a key component in the investigation of wild populations. In this study, we developed a fast, accurate and reliable amplification refractory mutation system (ARMS) technique for sex determination of red panda based on the exon 4 of the ZFX/ZFY gene. The amplicons were distinguished simply by agarose gel electrophoresis, exhibiting one fragment in females (X: 300 bp) and two in males (X: 300 bp, Y: 166 bp). Robustness of this ARMS system was confirmed by testing both 43 captive red pandas using DNA samples with known-sex and 10 wild red pandas using faecal DNA samples with unknown sex.     

Sex determination in spotted hyena (Crocuta crocuta ) by restriction fragment length polymorphism of amplified ZFX/ZFY loci

We describe a quick and efficient method of determining the sex of DNA samples in the hyena. By choosing primers from sequences that are conserved between the human and bovine ZFY and ZFX genes, we amplified a 448 bp fragment from 1 male and 2 female hyenas. Using comparative sequencing, single base pair polymorphisms between the amplified ZFY and ZFX were established. Restriction fragment length polymorphism (RFLP) analysis with PstI and TaqI confirmed the sequence data and yielded specific banding patterns between the 2 sexes in the hyena.     

Regional assignments of the zinc finger Y-linked gene (ZFY) and related sequences on human and mouse chromosomes

Recent chromosome walking experiments have identified a candidate gene (ZFY) for the testis-determining factor on the human Y chromosome (Page et al., 1987). We report here the regional assignments of the ZFY gene and related sequences in the human and the mouse. By in situ hybridization, we assigned ZFX and ZFY to human chromosome bands Xp21 and Yp11.3, respectively. Although the mouse harbors two Zfy genes, only one site at band A1 of its Y chromosome was significantly labeled. The mouse Zfx gene and the Zfa gene on chromosome 10 were assigned to bands XD and 10B5, respectively. These assignments of the ZFX gene in human and mouse add another marker to the conserved syntenic group for evaluating the evolutionary relationship of the human and mouse X chromosomes.