RESUMEN
Deletions of the AZFc (azoospermia factor c) region of the Y chromosome are the most common known cause of spermatogenic failure. We determined the complete nucleotide sequence of AZFc by identifying and distinguishing between near-identical amplicons (massive repeat units) using an iterative mapping-sequencing process. A complex of three palindromes, the largest spanning 3 Mb with 99.97% identity between its arms, encompasses the AZFc region. The palindromes are constructed from six distinct families of amplicons, with unit lengths of 115-678 kb, and may have resulted from tandem duplication and inversion during primate evolution. The palindromic complex contains 11 families of transcription units, all expressed in testis. Deletions of AZFc that cause infertility are remarkably uniform, spanning a 3.5-Mb segment and bounded by 229-kb direct repeats that probably served as substrates for homologous recombination.
Asunto(s)
Deleción Cromosómica , Infertilidad Masculina/genética , Cromosoma Y/genética , Secuencia de Bases , Inversión Cromosómica , Cromosomas Humanos Par 3/genética , Proteína 1 Delecionada en la Azoospermia , Evolución Molecular , Duplicación de Gen , Humanos , Masculino , Modelos Genéticos , Datos de Secuencia Molecular , Oligospermia/genética , Especificidad de Órganos , Mapeo Físico de Cromosoma , Proteínas de Unión al ARN/genética , Recombinación Genética/genética , Análisis de Secuencia de ADN , Eliminación de Secuencia/genética , Homología de Secuencia de Ácido Nucleico , Espermatozoides/metabolismo , Secuencias Repetidas en Tándem/genética , Testículo/metabolismo , Transcripción Genética/genéticaRESUMEN
The non-recombining region of the human Y chromosome (NRY), which comprises 95% of the chromosome, does not undergo sexual recombination and is present only in males. An understanding of its biological functions has begun to emerge from DNA studies of individuals with partial Y chromosomes, coupled with molecular characterization of genes implicated in gonadal sex reversal, Turner syndrome, graft rejection and spermatogenic failure. But mapping strategies applied successfully elsewhere in the genome have faltered in the NRY, where there is no meiotic recombination map and intrachromosomal repetitive sequences are abundant. Here we report a high-resolution physical map of the euchromatic, centromeric and heterochromatic regions of the NRY and its construction by unusual methods, including genomic clone subtraction and dissection of sequence family variants. Of the map's 758 DNA markers, 136 have multiple locations in the NRY, reflecting its unusually repetitive sequence composition. The markers anchor 1,038 bacterial artificial chromosome clones, 199 of which form a tiling path for sequencing.
Asunto(s)
Mapeo Físico de Cromosoma , Cromosoma Y , Cromosomas Artificiales Bacterianos , Eucromatina , Amplificación de Genes , Genoma Humano , Heterocromatina , Humanos , Masculino , Mapeo Físico de Cromosoma/métodos , Mapeo de Híbrido por Radiación , Lugares Marcados de SecuenciaRESUMEN
Deletion of any of three regions of the human Y chromosome results in spermatogenic failure and infertility. We previously sequenced one of these regions, azoospermia factor a (AZFa) and found that it spanned approximately 800 kb. By sequence-tagged site (STS) content mapping, we roughly defined deletion breakpoints in two unrelated, azoospermic men with AZFa deletions. The positions of proximal and distal breakpoints were similar in the two men. Hypothesizing that the deletions might have been generated by homologous recombination, we searched electronically for DNA sequence similarities between the proximal and distal breakpoint regions. These comparisons revealed the most striking sequence similarities anywhere along or near the AZFa region. In the proximal breakpoint region, we identified a 10 kb provirus of the recently defined HERV15 class of endogenous retroviruses. In the distal breakpoint region, we found a second HERV15 provirus, 94% identical in DNA sequence to the first and in the same orientation. (A partial LINE insertion in this distal provirus proved to be the basis of the previously described DYS11/p12f polymorphism.) The AZFa deletions in the two men differed slightly, but all breakpoints fell within the HERV15 proviruses. Indeed, sequencing of deletion junctions from the two men revealed that homologous recombination had occurred within large domains of absolute sequence identity between the proximal and distal proviruses. When combined with published STS mapping data for other AZFa-deleted men, our findings suggest that recombination between these two HERV15 proviruses could account for most AZFa deletions.
Asunto(s)
Retrovirus Endógenos/genética , Oligospermia/genética , Proteínas/genética , Recombinación Genética , Eliminación de Secuencia , Cromosoma Y/genética , Secuencia de Bases , Rotura Cromosómica , ARN Helicasas DEAD-box , Haplotipos , Humanos , Elementos de Nucleótido Esparcido Largo , Masculino , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Polimorfismo Genético , Alineación de Secuencia , Lugares Marcados de SecuenciaRESUMEN
The DAZ genes are candidate fertility factors that lie within the human Y chromosome's AZFc region, whose deletion is a common cause of spermatogenic failure. The number of DAZ genes has been difficult to determine, in part because the nucleotide sequences of the DAZ genes are nearly identical. Here, fluorescence in situ hybridization and characterization of BAC clones revealed four full-length DAZ genes on the human Y chromosome. They exist in two clusters, each comprising an inverted pair of DAZ genes (3' <-- 5'::5' --> 3'). Analysis of genomic sequences and testicular transcripts suggested that three or four DAZ genes are translated. Each gene contains at least seven tandem copies of a previously described, 2.4-kb repeat unit that encodes 24 amino acids. In addition, two DAZ genes contain tandem copies of a 10.8-kb repeat unit that encodes the RNA-binding domain, which appears to be multimerized in some DAZ proteins. Combining our present results with previous studies, we can reconstruct several steps in the evolution of the DAZ genes on the Y chromosome. In the ancestral Y-chromosomal DAZ gene, amplification of both intragenic repeats began before the human and cynomolgus (Old World) monkey lineages diverged. During subsequent evolution, an inverted duplication of this modified gene occurred. Finally, the resulting two-gene cluster was duplicated, generating the two-cluster/four-gene arrangement found on modern human Y chromosomes.
Asunto(s)
Familia de Multigenes , Proteínas de Unión al ARN/genética , Cromosoma Y/genética , Secuencia de Bases , Southern Blotting , Cromosomas Artificiales de Levadura , Cartilla de ADN/química , Proteína 1 Delecionada en la Azoospermia , Electroforesis en Gel de Campo Pulsado , Fibroblastos , Duplicación de Gen , Biblioteca Genómica , Humanos , Hibridación Fluorescente in Situ , Interfase , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas de Unión al ARN/química , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Secuencias Repetidas en TándemRESUMEN
In 1947, it was suggested that, in humans, the mutation rate is dramatically higher in the male germ line than in the female germ line. This hypothesis has been supported by the observation that, among primates, Y-linked genes evolved more rapidly than homologous X-linked genes. Based on these evolutionary studies, the ratio (alpha(m)) of male to female mutation rates in primates was estimated to be about 5. However, selection could have skewed sequence evolution in introns and exons. In addition, some of the X-Y gene pairs studied lie within chromosomal regions with substantially divergent nucleotide sequences. Here we directly compare human X and Y sequences within a large region with no known genes. Here the two chromosomes are 99% identical, and X-Y divergence began only three or four million years ago, during hominid evolution. In apes, homologous sequences exist only on the X chromosome. We sequenced and compared 38.6 kb of this region from human X, human Y, chimpanzee X and gorilla X chromosomes. We calculated alpha(m) to be 1.7 (95% confidence interval 1.15-2.87), significantly lower than previous estimates in primates. We infer that, in humans and their immediate ancestors, male and female mutation rates were far more similar than previously supposed.
Asunto(s)
Evolución Molecular , Hominidae/genética , Mutación , Caracteres Sexuales , Cromosoma X , Cromosoma Y , Animales , Secuencia de Bases , Gorilla gorilla , Humanos , Datos de Secuencia Molecular , Pan troglodytesRESUMEN
In humans, deletion of any one of three Y-chromosomal regions- AZFa, AZFb or AZFc-disrupts spermatogenesis, causing infertility in otherwise healthy men. Although candidate genes have been identified in all three regions, no case of spermatogenic failure has been traced to a point mutation in a Y-linked gene, or to a deletion of a single Y-linked gene. We sequenced the AZFa region of the Y chromosome and identified two functional genes previously described: USP9Y (also known as DFFRY) and DBY (refs 7,8). Screening of the two genes in 576 infertile and 96 fertile men revealed several sequence variants, most of which appear to be heritable and of little functional consequence. We found one de novo mutation in USP9Y: a 4-bp deletion in a splice-donor site, causing an exon to be skipped and protein truncation. This mutation was present in a man with nonobstructive azoospermia (that is, no sperm was detected in semen), but absent in his fertile brother, suggesting that the USP9Y mutation caused spermatogenic failure. We also identified a single-gene deletion associated with spermatogenic failure, again involving USP9Y, by re-analysing a published study.
Asunto(s)
Oligospermia/genética , Mutación Puntual , Cromosoma Y/genética , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/genética , Variación Genética , Humanos , Masculino , Datos de Secuencia Molecular , Oligospermia/patología , Linaje , Mapeo Físico de Cromosoma , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Espermatogénesis/genética , Testículo/patologíaRESUMEN
The DAZ gene cluster on the human Y chromosome is a candidate for the Azoospermia Factor (AZFc). According to the current evolutionary model, the DAZ cluster derived from the autosomal homolog DAZL1 through duplications and rearrangements and is confined to Old World monkeys, apes and humans. To study functional and evolutionary aspects of this gene family we have isolated from a cynomolgus (Old World) monkey testis cDNA library the Y chromosomal cynDAZ and the autosomal cynDAZL1 cDNA. cynDAZL1 contains one DAZ repeat and displays high homology to human DAZL1. cynDAZ comprises 11 repeats, each consisting of exons 7 and 8, whereas the human DAZ cDNA repeat units contain predominantly exon 7. Genomic studies revealed the same amplific- ation events of a 2.4 kb genomic unit encompassing exons 7 and 8 in both species, indicating that after splitting of the two lineages, in the human mainly exon 8 was converted to a pseudoexon by splice site mutations. The structural features of cynDAZ reveal a more detailed model for the sequence of events leading to the present form of human DAZ. Thus, in a monkey species DAZ is present in a form more ancestral than that of the human. Studies on the immunolocalization of cynDAZ / DAZL1 in cynomolgus monkey testis revealed a biphasic expression pattern with proteins being detectable in A-pale to B-spermatogonia, late spermatocytes and spermatids, but not in early spermatocytes and late spermatids. In contrast, in the marmoset monkey, an animal lacking DAZ, DAZL1 protein was only expressed in late spermatocytes and early spermatids. These findings point to an additional function of cynDAZ / cynDAZL1 during spermato- genesis in the Old World monkey not needed in the New World monkey.
Asunto(s)
Cercopithecidae/genética , Enfermedades de los Monos/genética , Oligospermia/veterinaria , Proteínas de Unión al ARN/genética , Cromosoma Y/genética , Animales , Secuencia de Bases , Callithrix/genética , Cromosomas Humanos Par 3/genética , ADN Complementario/genética , Proteína 1 Delecionada en la Azoospermia , Evolución Molecular , Exones/genética , Duplicación de Gen , Regulación del Desarrollo de la Expresión Génica , Genes , Humanos , Macaca fascicularis/genética , Masculino , Datos de Secuencia Molecular , Oligospermia/genética , Seudogenes/genética , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Espermátides/metabolismo , Espermatocitos/metabolismo , Espermatogénesis/genética , Testículo/metabolismoRESUMEN
It is widely believed that most or all Y-chromosomal genes were once shared with the X chromosome. The DAZ gene is a candidate for the human Y-chromosomal Azoospermia Factor (AZF). We report multiple copies of DAZ (> 99% identical in DNA sequence) clustered in the AZF region and a functional DAZ homologue (DAZH) on human chromosome 3. The entire gene family appears to be expressed in germ cells. Sequence analysis indicates that the Y-chromosomal DAZ cluster arose during primate evolution by (i) transposing the autosomal gene to the Y, (ii) amplifying and pruning exons within the transposed gene and (iii) amplifying the modified gene. These results challenge prevailing views of sex chromosome evolution, suggesting that acquisition of autosomal fertility genes is an important process in Y chromosome evolution.