Jonathan Wolfe

A Y chromosome gene family with RNA-binding protein homology : Candidates for the azoospermia factor AZF controlling human spermatogenesis

We have previously mapped the human azoospermia factor to a deletion in Y chromosome interval 6 (subinterval XII-XIV). We now report the isolation and characterization of a gene family located within this deletion. Analysis of the predicted protein products suggests a possible role in RNA processing or translational control during early spermatogenesis. The Y chromosome RNA recognition motif (YRRM) family includes a minimum of three members expressed specifically in the testis. Interphase in situ results and Southern blot analysis indicate that several further YRRM sequences map within interval 6. Several mammalian species show Y chromosome conservation of YRRM sequences. We have detected deletions of YRRM sequences in two oligospermic patients with no previously detectable mutation.

Comparative mapping ofYRRM- andTSPY-related cosmids in man and hominoid apes

Using chromosomalin situ hybridization it has been demonstrated that specific members of theYRRM and theTSPY families are multicopy and Y chromosome specific in hominoids. After hybridization with theYRRM-related cosmid A5F and theTSPY-related cosmids cos36 and cY91, a reverse and complementary pattern of main and seconary signals is detected on the Y chromosomes of the human, the pygmy chimpanzee and the gorilla, while the location of signals coincides on the Y chromosomes of the chimpanzee, both orang-utan subspecies and the white hand gibbon. This complementary distribution ofYRRM andTSPY sequences on the hominoid Y chromosomes possibly originates from a similar sequence motif that is shared by and evolutionarily conserved between certain members of both gene families and/or repeated elements flanking those genes. Otherwise this complementary distribution could go back to a common organization of these genes next to each other on an ancient Y chromosome which was disrupted by chromosomal rearrangements and amplification of one or other of the genes at each of the locations.

Tsga10 Encodes a 65-Kilodalton Protein That Is Processed to the 27-Kilodalton Fibrous Sheath Protein

Abstract We had previously reported the isolation of the testis-specific human gene Tsga10, which is not expressed in testes from two infertile patients. To study its role and function, we cloned the mouse homologue Mtsga10. Mtsga10 localizes to mouse chromosome 1, band B. This region is syntenic with human chromosome 2q11.2, where Tsga10 is located. We demonstrate that Mtsga10 mRNA is expressed in testis, but not in other adult tissues, and in several human fetal tissues and primary tumors. We uncovered that different species use different first exons and, consequently, different promoters. Using several antibodies, we discovered that, in mouse testis, Mtsga10 encodes a 65-kDa spermatid protein that appears to be processed to a 27-kDa protein of the fibrous sheath, a major sperm tail structure, in mature spermatozoa. Mtsga10 protein contains a putative myosin/Ezrin/radixin/moesin (ERM) domain. Transfection of fibroblasts with GFP-Mtsga10 fusion protein results in formation of short, thick filaments and deletion of the myosin/ERM domain abolished filament formation. Our results suggest the possibility that Tsga10 plays a role in the sperm tail fibrous sheath.

Identification and characterisation of a novel gene, TSGA10, expressed in testis

We describe the isolation of a novel gene, TSGA10, by differential mRNA display which is expressed solely in adult human testis. It seems likely that the gene is expressed during spermatogenesis possibly in spermatocytes. The gene is composed of 19 exons extending over more than 80 kb. The complete cDNA contains an open reading frame of 2094 nucleotides, which appears to encode a novel protein. It has been mapped by polymerase chain reaction on a panel of somatic cell hybrids and by fluorescence in situ hybridization to chromosome 2q11.2.

TSGA10 prevents nuclear localization of the hypoxia‐inducible factor (HIF)‐1α

The hypoxia‐inducible factor (HIF)‐1 is a transcriptional regulator of genes involved in oxygen homeostasis. We previously described testis‐specific isoforms of HIF‐1α (mHIF‐1αI.1 and hHIF‐1αTe). Using mHIF‐1α exon I.1 knock‐out mice we confirmed the specific expression of mHIF‐1αI.1 in the sperm tail. A protein–protein interaction between HIF‐1α and the testis specific gene antigen 10 (TSGA10) was identified by yeast two‐hybrid screening. TSGA10 is expressed in testis but also in other organs and malignant tissues. Immunofluorescence analysis indicated that the C‐terminal part of TSGA10 accumulates in the midpiece of spermatozoa, where it co‐localizes with HIF‐1α. HIF‐1α nuclear localization and HIF‐1 transcriptional activity were significantly affected by overexpressed TSGA10.

Mapping of RXRB to human chromosome 6p21. 3

Retinoid X Receptor beta (RXRB) is a member of the retinoid X receptor (RXR) family of nuclear receptors which are involved in mediating the effects of retinoic acid (RA). We have confirmed the localization of RXRB to chromosome 6 and we have mapped the gene to chromosome 6p21. 3‐p21.1 by PCR amplification of 5′ untranslated sequence in panels of rodent‐human somatic cell hybrids and to 6p21.3 by fluorescent in situ hybridization.

A physical map of the human PI and AACT genes

We have used probes from the human genes PI, PIL, and AACT (alpha 1-antitrypsin, alpha 1-antitrypsin-related sequence, and alpha 1-antichymotrypsin) to make a pulsed-field map of the surrounding region of 14q31-32. We have discovered that the PI-PIL gene cluster is only 220 kb away from the AACT gene and that it is orientated in the opposite direction. The comparatively short distance between the genes comes as a surprise given previous estimates of the level of genetic recombination between them.

The use of irradiation and fusion gene transfer (IFGT) hybrids to isolate DNA clones from human chromosome region 9q33-q34.

We have generated somatic cell hybrids containing fragments of human chromosome arm 9q by an irradiation and fusion technique. No selection for human material was imposed, but of 23 clones analyzed most contained human DNA sequences and many contained multiple fragments of the human chromosome arm. A hybrid that appears to contain only two small fragments of human DNA from the regions q33 and q34 has been used as a source from which to clone probes specific to those areas of the chromosome.

Advances in fluorescent in situ hybridisation

Recent advances in fluorescent in situ hybridisation included the generation of allele-specific probes, bar-coded chromosomes, and the visualisation of chromosome territories and genes within the nucleus. One major advance has been our ability to visualise and make precise and reproducible measurements from stretched DNA molecules prepared directly from human cells.

Cosmid contigs spanning 9q34 including the candidate region for TSC1.

The tuberous sclerosis disease gene TSC1 has been mapped to 9q34. However, its precise localisation has proved problematic because of conflicting recombination data. Therefore, we have attempted to clone the entire target area into cosmid contigs prior to gene isolation studies. We have used Alu-PCR from irradiation hybrids to produce complex probes from the target region which have identified 1,400 cosmids from a chromosome-specific library. These, along with cosmids obtained by other methods, have been assembled into contigs by a fingerprinting technique. We estimate that we have obtained most of the region in cosmid contigs. These cosmids are a resource for the isolation of expressed genes within the TSC1 interval. In addition, the cosmid contig assembly has demonstrated a number of previously unknown physical connections between genes and markers in 9q34.