Goliath, a ring-H2 mitochondrial protein, regulated by luteinizing hormone/human chorionic gonadotropin in rat leydig cells.

We have cloned the rat homologue of the ring-H2 protein Goliath involved in Drosophila development. The rat Goliath mRNA (1.85 kb) was translated as a major ubiquitous protein species of 28-kDa and three larger isoforms (50, 46, and 36 kDa) expressed mainly in liver, lung, stomach, heart, and thymus and barely detectable in other tissues (kidney, skeletal muscle, brain, testis, intestine, and spleen). By immunohistochemistry on rat testis sections, we localized the protein in interstitial tissue and seminiferous tubules. In tubules, Goliath was expressed mainly in postmeiotic germ cells and to a much lesser extent in Sertoli cells. In the interstitium, Goliath was exclusively present in Leydig cells. Using a series of immunolabeling, cellular fractionation, and electron microscopy experiments, we established that Goliath is present in mitochondria of the R2C Leydig cell line. Using short-term hypophysectomized animals, we showed that Goliath is regulated by LH/hCG in Leydig cells but not in germ cells. This regulation in Leydig cells concerned only the 50-kDa isoform. This report is the first description of a differential regulation of the Goliath protein between germ cells and Leydig cells.

[Recent approaches to the pathogenesis of minimal change nephrotic syndrome]. / Nouvelles approaches dans l'étude de la pathogénie de la néphrose lipoïdique.

Clinical and experimental observations suggest that lipoid nephrosis (Minimal change nephrotic syndrome) results from T cell dysfunction due to still unknown mechanisms. By substractive screening library, we identified 84 transcripts, of which 42 correspond to known genes, 12 match with proteins of yet unknown function and 30 are unknown clones. Among the 42 known transcripts, at least 18 are closely involved in the T-Cell Receptor mediated signaling cascades. This includes genes encoding components of the T-Cell Receptor and proteins associated with the cytoskeleton scaffold, as well as transcription factors. During the relapse phase, we have detected very low levels of IL12R beta 2 mRNA suggesting that the T-cell activation evolves toward a Th2 phenotype. Thus, the combination of substractive cloning and differential screening constitutes an efficient approach to identify genes likely involved in the pathophysiology of MCNS.

Meiotic human sperm cells express a leucine-rich homologue of Caenorhabditis elegans early embryogenesis gene, Zyg-11.

We cloned a human protein (Hzyg) homologue to Caenorhabditis elegans Zyg-11, an essential protein for cell division at the initial developmental stages of this species, and to a Drosophila melanogaster gene product (Mei-1) which is likely to be involved in meiosis. Hzyg mRNA encodes a protein of 766 amino acids (88 kDa), 14% of which are leucine residues, with some being arranged in four leucine rich repeat motives usually involved in protein-protein interactions. Hzyg is encoded by a single gene, located on chromosome 9q32-q34.1, and transcribed as two mRNA: a 5 kb transcript strongly expressed in testis and skeletal muscle and barely detectable in other human tissues, and an abundant 3.1 kb mRNA detected only in the testis. By using in-situ hybridization and immunohistochemistry, we clearly established the presence of Hzyg expression in pachytene spermatocytes (stage V) and spermatids (stage I and/or II) around the time of meiosis. The cell specific expression of Hzyg transcripts in testis, and the conservation of this gene among distant species, suggest that this protein may have an important role during meiosis.

Sporadic testicular germ cell cancers do not exhibit specific alteration in CAG/CTG repeats containing genes expressed in human testis.

CAG/CTG repeat expansions in genomic DNA of testicular tumour cell lines, and germline DNA from members of families predisposed to this malignancy, have been previously described. In order to identify genes possibly concerned by this alteration, we attempted to clone all possible human testis cDNA containing at least five CAG/CTG repeats. Thirty-four different transcripts were identified. By using PCR and non denaturing gel electrophoresis, we determined the size of their repeats, as well as their polymorphisms in a collection of human testicular germ cell tumours and the normal surrounding tissues. For all tested genes, we detected the presence of several species of the same mRNA for each person. Nine genes exhibited specific patterns of expression among different groups of individuals, indicative of polymorphism. None of these polymorphisms was related to human testicular tumours.

hH-Rev107, a class II tumor suppressor gene, is expressed by post-meiotic testicular germ cells and CIS cells but not by human testicular germ cell tumors.

By systematic analysis of a human testis library, we have isolated the hH-Rev107-3 cDNA, identical to hH-Rev107-1 cDNA, which was previously described as a class II tumor suppressor gene. In this study, two transcripts (1 and 0.8 kb) were detected by Northern blot in all human tissues, excepted in thymus. The strongest expression was found in testis, skeletal muscle and heart. These two mRNA are probably transcribed from only one gene that we mapped to the q12-q13 region of the chromosome 11. In human testis, hH-Rev107 gene expression was localized, by in situ hybridization, within the round spermatids. To investigate a possible role for hH-Rev107 protein in testicular malignant growth, we examined the expression of this gene in germ cell tumors. A strong hH-Rev107 gene expression was observed in normal testis as well as in samples with preinvasive carcinoma in situ but was completely absent in overt tumors, both seminomas and non-seminomas. By in situ hybridization, CIS was found hH-Rev107 positive and tumor negative. A semi-quantitative assessment of hH-Rev107 mRNA level in testicular germ cell tumors, by RT-PCR, exhibited a ninefold decrease in the gene expression. No gross structural aberrations of hH-Rev107 gene were detected in these human primary tumors. The results suggest that down-regulation of hH-Rev107 may be associated with invasive progression of testicular germ cell tumors.

Human testis expresses a specific poly(A)-binding protein.

In testis mRNA stability and translation initiation are extensively under the control of poly(A)-binding proteins (PABP). Here we have cloned a new human testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical residues to the ubiquitous PABP1. A northern blot of multiple human tissues hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2 kb) was present in all tested tissues. In human adult testis, PABP3 mRNA expression was restricted to round spermatids, whereas PABP1 was expressed in these cells as well as in pachytene spermatocytes. PABP3-specific antibodies identified a protein of 70 kDa in human testis extracts. This protein binds poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3 gene is intronless with a transcription start site 61 nt upstream from the initiation codon. A sequence of 256 bp upstream from the transcription start site drives the promoter activity of PABP3 and its tissue-specific expression. The expression of PABP3 might be a way to bypass PABP1 translational repression and to produce the amount of PABP needed for active mRNA translation in spermatids.

Expression of the TAR RNA binding protein in human testis.

In testis, several RNA binding proteins have been shown to play a role in the translational regulation of specific transcripts. The human protein TRBP (TAR RNA binding protein) is the homologue of the mouse Prbp (Prm-1 RNA binding protein) involved in the protamine mRNA translational delay. TRBP is known to activate the HIV-1 long terminal repeat but this protein has never been investigated during spermatogenesis. The aim of this work was to analyse the TRBP expression in human testis. By Northern blot analysis, we demonstrated a major 1.5 kb transcript present at a high level in human testis and, to a lesser extent, in some other tissues. In-situ hybridization revealed that this transcript was present only in elongating spermatids. Antibodies raised against a 27 amino acid TRBP-specific peptide revealed a single protein of 43 kDa expressed in the cytoplasm of elongated spermatids. At the ultrastructural level, quantitative analysis of both TRBP mRNA and protein, using electron microscopy in-situ hybridization and immunocytochemistry, showed that TRBP is expressed mainly in spermatids at steps 3-4 of spermiogenesis. These results are in agreement with the probable role of TRBP in the control of human protamine mRNA translation.

Structure of the 5' sequences of the human gamma-glutamyltransferase gene.

In humans, five distinct mRNAs code for gamma-glutamyltransferase (GGT). Their coding regions are identical and their 5' untranslated regions exhibit both common and type-specific sequences. To elucidate the mecanisms that generate these different mRNAs, we cloned and determined the structure of the 5' region of the human GGT gene. The common regions of the 5' UTR are encoded by five exons, localized within a 2.4-kb region of the genomic DNA. Three of them are separated only by intron-donor or intron-acceptor sites at their boundaries. Alternative splicing of these exons may determine the unique pattern of the different GGT mRNA 5' UTRs in a tissue-specific manner. In addition, we have isolated a genomic fragment containing the most distal 5' sequences of the major GGT mRNA in HepG2 cells. Primer extension analysis revealed one major transcription initiation site while 5' RACE indicated that one more distal initiation site could be present. In the putative promoter sequence neither classical TATA or CAAT boxes were found. However, sites for AP1, AP2, CREB, GRE and SP1 transcription factors were identified. Chimeric plasmids, containing this genomic region fused to the luciferase gene, were transiently expressed in three cell lines of different origin: HeLa cells, ovarian carcinoma A2780 cells and V79 lung fibroblasts. The significant promoter activities obtained indicate a transcription start within this region. However, differences in the level of expression were found between the different cell lines used. These data suggest that the human GGT gene employs regulatory sequences and alternative splicing, and gene expression may therefore be regulated in tissue specific and cell-type-specific manners.

Chromosomal localization of three human poly(A)-binding protein genes and four related pseudogenes.

In humans, the poly(A)-binding proteins (PABPs) comprise a small nuclear isoform and a conserved gene family that displays at least three functional proteins: PABP1, inducible PABP (iPABP), and PABP3, plus four pseudogenes (1, 2, 3, and PABP4). In situ hybridization of PABP3 cDNA as the probe on metaphasic chromosomes have revealed five possible loci for this gene family at 2q21-q22, 13q11-q12, 12q13.3-q15, 8q22, and 3q24-q25. Amplifications of specific DNA fragments from a human-rodent somatic cell hybrid panel have allowed us to associate PABP1 and PABP3 with 8q22 and 13q11-q12, respectively. The iPABP gene has been assigned to chromosome 1. This result, compared with radiation hybrid database information, strengthens the location of this gene to 1p32-p36. The pseudogenes PABP4, 1, and 2 have been assigned to chromosomes 15, 4, and 14, respectively. Three loci detected on chromosome spreads are not associated with any amplified fragment. They might represent other related PABP genes not yet identified.

Gamma-glutamyl transpeptidase gene organization and expression: a comparative analysis in rat, mouse, pig and human species.

Gamma-glutamyl transpeptidase (GGT) is an enzyme located at the external surface of epithelial cells. It initiates extracellular glutathione (GSH) breakdown, provides cells with a local cysteine supply and contributes to maintain intracellular GSH level. GGT expression, highly sensitive to oxidative stress, is a part of the cell antioxidant defense mechanisms. We describe recent advances in GGT gene structure and expression knowledge and put emphasis on the complex transcriptional organization of that gene and its conservation among different species. GGT gene structure has been elucidated in rat and mouse where a single gene is transcribed from multiple promoters into several transcripts which finally yield a unique polypeptidic chain. Analysis of rat, mouse, human and pig cDNA and gene sequences reveals a large conservation of the transcriptional organization of that gene. This complex structure provides flexibility in GGT expression controlled at the promoter level, through multiple regulatory sites, and at RNA level by alternate 5' untranslated sequences which may create a diversity in the stability and translational efficiency of the different transcripts. In conclusion, transcription of the GGT gene from several promoters offers multiple DNA and RNA targets for various oxidative stimuli and contributes to a broad antioxidant cell defense through GGT induction and subsequent cysteine supply from extracellular glutathione.

Contrin, the human homologue of a germ-cell Y-box-binding protein: cloning, expression, and chromosomal localization.

Inactivation of germ-cell-specific molecules essential for the production of functional spermatozoa could lead to attractive new means for male contraception. The mouse protein MSY2 is the mammalian homologue of a class of Xenopus DNA/RNA-binding proteins needed for the transcription of testis-specific genes and for translational repression (masking) of paternal mRNAs. In this report, we describe the human homologue for MSY2, Contrin. Sequence analysis of Contrin cDNAs predicts a protein highly similar to its mouse and Xenopus germ-cell Y-box protein homologues with a cold shock domain and four basic/aromatic islands. Contrin is highly basic and is rich in the amino acids arginine and proline. It contains seven putative casein kinase 2 phosphorylation sites and three putative protein kinase C phosphorylation sites, suggesting that Contrin could be highly phosphorylated in vivo. The predicted protein sequence contains two nuclear localization signals, consistent with its predicted role of shuttling between nucleus and cytoplasm. Contrin maps to human chromosome 17p11.2-13.1. By the criteria of northern and western blotting, Contrin appears to be testis specific and distinct from other mammalian Y-box-binding proteins. We predict that inactivation of Contrin function in mammalian germ cells would prevent the formation of functional male gametes.

A new human nm23 homologue (nm23-H5) specifically expressed in testis germinal cells.

We have identified a cDNA encoding a 212 amino acid protein (Nm23-H5) with 27-31% identity to the human members of the nm23/nucleoside diphosphate (NDP) kinase gene family. The nm23-H5 gene is located on chromosome 5q23-31 and is transcribed as one main transcript of 1.1 kb which is highly and specifically expressed in testis, in the spermatogonia and early spermatocytes. Nm23-H5 possesses most of the residues conserved among NDP kinases plus an additional COOH-terminus end of 55 amino acids unique to this protein. However, under usual assay conditions, Nm23-H5 expressed in Escherichia coli did not exhibit NDP kinase activity. These results suggest that Nm23-H5 is specifically involved in early stages of spermatogenesis.

An intronic promoter controls the expression of truncated human gamma-glutamyltransferase mRNAs.

We have identified and characterized a genomic DNA fragment containing the coding sequences corresponding to the human gamma-glutamyltransferase type 1 mRNA. The coding part of the gene spans over 16 kb and comprises 12 exons and 11 introns exhibiting a similar organization as for the mouse and rat GGT genes. The exons 1-7 encode the heavy subunit whereas exons 8-12 which encode the carboxy-terminal part of the heavy subunit (exon 8) and the light subunit are clustered in a 1.6-kb BglII fragment. Exons 7 and 8 are separated by a 3.9-kb intron containing in its 3' part the sequences corresponding to the 5'-UTRs of the truncated GGT mRNAs described for human lung. Sequence analysis upstream this transcribed region exhibited putative promoter sequences and after transient transfection significant promoter activities were measured in V79 lung fibroblasts and KYN-2 hepatoma cells but not in A2780 ovarian cells. This specificity disappeared when only 550 bp upstream the transcription start site were used as promoter. These results argue for a promoter of truncated GGT mRNAs in intron 7, specifically regulated in human tissues.

Identification and mapping of 26 human testis mRNAs containing CAG/CTG repeats.

Various types of pathologies, including neurodegenerative diseases, as well as different types of neoplasia, are related to genes exhibiting simple tandem repeat instabilities. In order to seek for new candidate genes for such disorders, we screened 4.10(6) human testis cDNAs for CAG- and CTG-containing clones. Among 910 positive clones, we characterized 109 cDNAs corresponding to 26 independent mRNAs. Fourteen of these mRNAs represent new genes. The corresponding clones contain between 3 and 19 consecutive CAG or CTG triplets. We assigned 15 out of these 26 genes to 14 different human chromosomes. These genes represent new potential candidates for diseases associated with CAG or CTG repeat mutations.

Identification and chromosomal localization of human genes containing CAG/CTG repeats expressed in testis and brain.

Human genes containing triplet repeats have been demonstrated to be involved in several neurodegenerative diseases by expansion of the repeat in succeeding generations. To identify novel genes involved in such pathologies, we have isolated transcripts containing (CAG/CTG)n repeats using two approaches. First, we screened 4 x 10(6) clones representing 10 copies of a human testis cDNA library using a (CAG)14 oligonucleotide probe. Among the 910 clones identified, the 243 clones with the strongest hybridization signal were sequenced partially from 3' or 5' ends. This provided us with 251 partial sequences that grouped into clusters corresponding to 39 genes, of which 19 represent unknown species. Second, we selected 203 additional ESTs containing (CAG/CTG)n repeats representing 121 clusters from the IMAGE consortium infant brain cDNA library. From these two series of sequences, we have localized 95 genes on human chromosomes using a panel of whole genome radiation hybrid (Genebridge 4). These genes are located on all of the chromosomes except for chromosome X, the highest density being observed on chromosome 19.

Molecular cloning and characterization of a mono(ADP-ribosyl)transferase from human testis.

A human homologue of the rodent T cell mono(ADP-ribosyl)transferase RT6 mRNA was identified by a systematic partial sequencing of human testis transcripts. This messenger encodes for a precursor protein of 367 aa (MW: 41.5 kDa) which exhibits a peptide signal, consensus domains for mono(ADP-ribosyl)transferase and a C terminal part which contains three repeated motives (GEKNQKLEDH) and a region characteristic of glycophosphatidyl inositol anchored proteins. This mRNA is transcribed from a gene localized in 4q13-q21. Surprisingly, it is not expressed in human white blood cells but it exhibits a very specific testis expression in which it is likely to correspond to a new ADP-ribosyl transferase.

High-level expression of functional human gamma-glutamyl transpeptidase using the baculovirus system.

The understanding of the structure and function of gamma-glutamyl transpeptidase (GGT) has been hindered by the difficulty of obtaining large quantities of functional enzyme. A recombinant baculovirus, encoding the human hepatoma cell (Hep G2) GGT, was easily purified using a histochemical procedure to reveal GGT activity. Infected insect cells synthesized a large amount of enzymatically active GGT representing up to 10% of the total cell extract protein. The GGT specific activity of the infected cells was 13 units per mg of protein which is the highest GGT expression level reported to date, 260-times more than in Hep G2 cells. The recombinant protein displayed an apparent molecular mass (M(r), 58,000 for the heavy subunit), immunoreactivity and catalytic features similar to those of the native protein. The high-level expression of functional GGT should provide an excellent tool to further study the structure-function relationships of the protein.

Human testis specifically expresses a homologue of the rodent T lymphocytes RT6 mRNA.

A human homologue of the rodent T cell mono ADP-ribosyl transferase RT6 mRNA was identified by a systematic analysis of human testis transcripts. This messenger encodes for a precursor protein of 367 aa (MW: 41.5 kDa) which exhibits a peptide signal, consensus domains for mono ADP-ribosyl transferase and a C-terminal part characteristic of glycophosphatidyl inositol anchored protein. This mRNA, transcribed from a gene localized in 4q13-q21, is not expressed in white blood cells but is specific for human testis in which it is likely to correspond to a new ADP-ribosyl transferase.

Identification of a cat eye syndrome using DNA sequence dosage analysis.

We report here the detection of a Cat Eye Syndrome (CES) in a woman who does not exhibit the related phenotype, due to intensive surgery. The analysis of her karyotype reveals a small supernumerary bisatellited chromosome likely to correspond to a fragment of chromosome 13, 15, 21 or 22 on banding analysis. Southern blot of genomic DNA of this patient and her parents hybridized with probes specific of these chromosomes, revealed a DNA amplification of the 22q11 region for the patient, likely to correspond to a CES.

Characterization of a large population of mRNAs from human testis.

We present the results of single-pass sequencing of 779 expressed sequence tags from normal human testis cDNA clones. Of the sequences generated, 319 (41%) appeared to be completely unknown and are likely to represent new genes, and 289 (37%) were identified based on exact or nonexact matches to sequences in public databases. In analyses of hybridization of four tissues, testis, brain, liver, and kidney, 6 of 12 cDNAs clones revealed testis-specific expression. This argues for the value of the combination of random sequencing and analysis of cellular expression for large-scale characterization of gene expression in the testis.