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Background and objective: Mutations in bone morphogenic protein 15 (BMP15) and growth/differentiation factor 9 (GDF9) lead to altered fertility in animal models. In the human, a heterozygous point mutation of BMP15has been associated with premature ovarian failure (POF).Subject and methods: We have directly sequenced both genes in a cohort of 203 POF patients presenting with primary or secondary amenorrhea and high FSH levels and in a control population including 54 women with regular menstrual cycles who had at least one child.Results: We have identified several heterozygous variants. One alteration in GDF9 (S186Y) and one in BMP15 (L148P) may have pathogenic effects as both positions are conserved in vertebrate species, ranging from the chicken to mammals. These variants were absent in the control samples. We also found synonymous and neutral substitutions.Conclusions: We propose that although mutations in BMP15and GDF9are not a major cause of ovarian insufficiency, they may be involved in POF.

FOXL2is a forkhead transcription factor involved in ovarian development and function. Here, we have studied the evolution and pattern of expression of the FOXL2gene and its paralogs in fish. We found well conserved FoxL2sequences (FoxL2a) and divergent genes, whose forkhead domains belonged to the class L2 and were shown to be paralogs of the FoxL2asequences (named FoxL2b). In the rainbow trout, FoxL2aand FoxL2bwere specifically expressed in the ovary, but displayed different temporal patterns of expression. FoxL2aexpression correlated with the level of aromatase, the key enzyme in estrogen production, and an estrogen treatment used to feminize genetically male individuals elicited the up-regulation of both paralogs. Conversely, androgens or an aromatase inhibitor down-regulated FoxL2aand FoxL2bin females. We speculate that there is a direct link between estrogens and FoxL2expression in fish, at least during the period where the identity of the gonad is sensitive to hormonal treatments.

MHC class II expression defects have been evidenced in several human tumor cell lines originating from lung cancers or retinoblastoma. Accordingly, the mouse adenocarcinoma and fibrosarcoma cell lines, RAG and L(tk-), do not express I-A and I-E molecules even when treated with IFN-gamma. Here we show that fusion of both cell lines restores the inducible expression of MHC class II, thereby demonstrating that they present different and recessive alterations outside the MHC class II locus. CIITA, the MHC class II transactivator, controls the tissue-specific expression of MHC class II genes and creates the architecture of the transcriptional complex that binds to the MHC class II gene promoters. In L(tk-) cells, C2ta transcripts, expressed from the gene encoding CIITA, were indeed detected in severely limited amounts, with a defect in C2ta transcription initiation. In agreement we show here that the L(tk-) cell line does not express the CIITA protein. In contrast, in the RAG cell line, C2ta transcripts were expressed at normal levels, from the proper initiation site. The nucleotide sequencing of the CIITA cDNA from RAG did not reveal any mutation. However, the CIITA protein was not detected. These data evidence a new type of defect in a MHC class II-defective tumor cell line, as we show here that the alteration in the RAG cells occurs downstream of C2ta transcription. The RAG mutation might therefore reside in the C2ta transcript nuclear export or translation, or in the stability of the CIITA protein.

Capture of cellular mRNA by mobile elements has been an evolutionary catalyst for the spread of genes and a cause of cancer development. Here we present evidence that an orphan gene, FAM8A1(family with sequence similarity 8), was captured by a retrovirus, followed by multiple retrotransposition events, during primate evolution between 45 and 58 million years ago. This represents the first record of cellular mRNA transduction in humans. The human gene is localized on chromosome 6p23 with five related pseudogenes (FAM8A2P–A6P), each inserted within a human endogenous retrovirus (HERV). Only the functional FAM8A1gene is expressed and displays a ubiquitous mRNA and a testis-specific transcript present in the haploid phase of spermatogenesis.The structural features of the FAM8A1pseudogenes include two short sequences of similarity between the FAM8A1mRNA and the HERV sequences at both the 5′ and 3′ integration sites. These hallmarks suggest an alternative model to account for the capture of FAM8A1cellular mRNA by HERV-K, involving illegitimate recombination events at the two sites of sequence similarity during reverse transcription. Unlike previous models, which assume at least one step of retroviral integration in the genome, our model is consistent with in vitroobservations showing that multiple template switches occur among packaged viral transcripts. This leads to the speculation that, in some cases, cellular mRNAs may have been captured through similar processes involved in the retroviral life cycle.

Several studies have reported significant linkage for schizophrenia on 6p23, with a maximum lod score between D6S274 and D6S285. In this paper, we present a new human kinesin gene localized in this 2-cM interval. This gene, termed KIF13A, belongs to the unc-104/KIF1A kinesin subfamily and represents the orthologue of Drosophilakinesin-73. Several alternative transcripts are differentially expressed in human tissues, probably reflecting differences in cargo binding and transport of corresponding proteins. During early mouse development, its homologue (Kif13A) is expressed essentially in the central nervous system. In Caenorhabditis elegans,the unc-104 gene is involved in axonal anterograde transport, and null mutants present several behavioral defects. The putative function and genomic localization of KIF13A make this gene an interesting candidate for genetic predisposition to schizophrenia. We provide sequences of 20 single-nucleotide polymorphisms localized within KIF13A to test for association studies between this gene and schizophrenia.

Microdeletions of the long arm of the Y chromosome (Yq) are a common cause of male infertility. Since large structural rearrangements of the Y chromosome are commonly associated with a 45,XO/46,XY chromosomal mosaicism, we studied whether submicroscopic Yq deletions could also be associated with the development of 45,XO cell lines. We studied blood samples from 14 infertile men carrying a Yq microdeletion as revealed by polymerase chain reaction (PCR). Patients were divided into two groups: group 1 (n = 6), in which karyotype analysis demonstrated a 45,X/46,XY mosaicism, and group 2 (n = 8) with apparently a normal 46,XY karyotype. 45,XO cells were identified by fluorescence in-situ hybridization (FISH) using X and Y centromeric probes. Lymphocytes from 11 fertile men were studied as controls. In addition, sperm cells were studied in three oligozoospermic patients in group 2. Our results showed that large and submicroscopic Yq deletions were associated with significantly increased percentages of 45,XO cells in lymphocytes and of sperm cells nullisomic for gonosomes, especially for the Y chromosome. Moreover, two isodicentric Y chromosomes, classified as normal by cytogenetic methods, were detected. Therefore, Yq microdeletions may be associated with Y chromosomal instability leading to the formation of 45,XO cell lines.

Abstract: During a study of deletions of Y-chromosomal DNA in infertile males, sY116, a Y-linked STS, showed different electrophoretic mobilities in three males, two infertile and one fertile. A study of this STS among 35 other normal males showed that this locus is polymorphic. sY1 16 has a polyA-rich stretch whose instability appears to be the most likely cause of this polymorphism. The possible usefulness of sY116 polymorphism in the detection of subtle genome-wide instabilities in some types of cancer is discussed.

CD99, the product of the MIC2gene, exhibits an erythroid-specific quantitative polymorphism coregulated with the polymorphism of the XG blood group gene. As a preliminary study of this phenomenon, human XG and CD99 recombinant proteins were expressed in murine RAG cells and analyzed by flow cytometry. Both proteins were expressed independently and at a similar level in single and double transfectants. Immunoprecipitation and Western blot analysis, using the murine monoclonal antibodies NBL-1 and 12E7, revealed species of 26 kd (XG) and 32 kd (CD99), respectively. A putative 28-kd intracellular precursor of CD99 was also detected, as was a 26-kd species after neuraminidase treatment of CD99-expressing cells. No evidence of association or complex formation between XG and CD99 proteins could be proven, either on transfected RAG cells or on human erythrocytes. These results were confirmed using somatic hybrids between single transfectants. These findings suggest that the phenotypic relationship between XG and CD99 is mostly regulated at the transcriptional level, but they do not formally exclude some posttranscriptional effect. Studies on the tissue specificity of XGexpression showed that surface expression of the XG protein could not be restored in somatic hybrids between B-lymphoblastoid cell lines from Xg(a+) persons and fibroblasts (RAG) or erythroid (MEL) cells. RT-PCR analysis of the transcripts revealed the existence of an XGmRNA in each cell line, suggesting that the tissue-specific regulation of cell surface XG expression occurs either at a quantitative transcriptional level or is a posttranscriptional event. By Northern blot analysis,XGtranscripts were detected in erythroid tissues and several nonerythroid tissues.

Intense efforts are currently being pursued to identify autosomal genes associated with 46,XY male-to-female sex reversal. The genes DMRT1and 2are located on distal 9p, a region deleted in 46,XY sex-reversed patients. They are considered excellent candidates because of their homology to regulators of sex development in invertebrates. We present the genomic structure of DMRT2,showing that it generates several transcripts with distinct coding potential. In addition to the previously reported 226-amino-acid protein-encoding transcript, we describe other mRNA isoforms that are potentially bicistronic and are predicted to encode an additional 328-amino-acid polypeptide. Finally, a stop codon-containing exon (exon 4) can be skipped by alternative splicing and can generate a transcript that is predicted to encode a fusion protein. The latter shares 58% amino acid identity with a gene recently described in fish, termed terra.Differences in expression pattern exist for DMRT2mRNA isoforms among the human adult tissues tested, between adult tissues and human embryos, and between DMRT2and DMRT1during embryonic development. We failed to detect mutations by sequencing of DMRT2in a sample of 46,XY female patients. The interesting structure of DMRT2coupled to preliminary functional studies in fish showing that terrais involved in somitogenesis suggests that validation or exclusion of this gene as a cause of sex reversal will require more in-depth investigations.

Abstract:  In this study, various sets of somatic cell hybrids, generated by the fusion of epithelial cell lines with B-lymphoblastoid cell lines, were analyzed for the expression of major histocompatibility complex (MHC) class II antigens. We first demonstrate, in human and mouse intraspecies hybrids, the coordinate suppression of MHC class II, Ii (invariant chain) and HLA-DM gene transcription, and the release of the silencing by the addition of interferon gamma. Using interspecies hybrids, the segregation of human chromosomes allowed us to establish that MHC class II extinction is linked to the presence in the hybrids of the chromosomes from the epithelial fusion partner. Moreover, our data provide evidence that the expression pattern of MHC class II mRNA is correlated with that of the class II transactivator (CIITA), suggesting that CIITA is the actual target of the silencing. To gain further insight into the suppression phenomenon we performed luciferase assays which show that silencing affects the activity of the B-cell-specific promoter of CIITA. These results therefore demonstrate that the MHC class II gene silencing in somatic cell hybrids is due to an active suppression of one of the promoters of the CIITA gene, mediated by the epithelial cell fusion partner.

tyk2 belongs to the JAK family of nonreceptor protein tyrosine kinases recently found implicated in signaling through a large number of cytokine receptors. These proteins are characterized by a large amino-terminal region and two tandemly arranged kinase domains, a kinase-like and a tyrosine kinase domain. Genetic and biochemical evidence supports the requirement for tyk2 in interferon-α/β binding and signaling. To study the role of the distinct domains of tyk2, constructs lacking one or both kinase domains were stably transfected in recipient cells lacking the endogenous protein. Removal of either or both kinase domains resulted in loss of the in vitrokinase activity. The mutant form truncated of the tyrosine kinase domain was found to reconstitute binding of interferon-α8 and partial signaling. While no contribution of this protein toward interferon-β binding was evident, increased signaling could be measured. The mutant form lacking both kinase domains did not exhibit any detectable activity. Altogether, these results show that a sequential deletion of domains engenders a sequential loss of function and that the different domains of tyk2 have distinct functions, all essential for full interferon-α and -β binding and signaling.

The expression of class I transplantation antigens encoded in the major histocompatibility complex (H–2 in mouse, HLA in man) can be induced by α-, β- and γ-interferons1–5. Both transcriptional and post-transcriptional mechanisms have been postulated. Recently, a common sequence has been found in the promoter region of several human genes responsive to IFN-α (ref. 6). The promoters of H–2Kband several other mouse class I genes contain a similar interferon response sequence7. We show here, in a transient assay, that the H–2Kbpromoter can be induced by all three types of interferon and that the interferon response sequence is necessary for induction to occur. However, the response sequence is active only when associated with a functional enhancer sequence which we have recently identified in the promoter of H–2Kband other class I genes7. The combination of these two sequences can render a heterologous promoter responsive to interferon, irrespective of its orientation relative to the cap site.

We previously described RAG, a mouse adenocarcinoma cell line, as deficient for the induction of major histocompatibility (MHC) class II antigens by IFN-, but responding normally for MHC class I antigen stimulation and anti-viral protection. We had established that the fusion of RAG with various human cell lines restored the induction of MHC class II antigens, whenever the human chromosome 16 was present in somatic cell hybrids. Here we show that the RAG cell line does not exhibit any induction by IFN- of DMA, DMB, and the invariant chain (Ii) mRNAs, and that the induction is restored in somatic cell hybrids containing human chromosome 16. In order to define the gene (designated F16) affected in the RAG cells, we performed a complementation analysis by fusing RAG with previously described human cell lines defective for MHC class II antigen expression (e.g., BLS cell lines), and which belong to five different complementation groups. Our data show that the resulting somatic cell hybrids present an inducible expression of mouse MHC class II antigens, Ii, DMA, and DMB. Therefore, the RAG cell line represents a yet undescribed cellular mutant affected in the expression of MHC class II antigens. In addition, we demonstrate that MHC class II antigens can be constitutively expressed in the RAG cell line when transfected with the cDNA encoding human CIITA driven by the RSV LTR promoter. Since the complementation analysis assessed that F16 and CIITA are distinct, our data suggest that F16 is required for the expression of CIITA.

Human a and ß interferons increase the amount of class I human histocompatibility messenger RNA HLA-A, B, C and ß2-microglobulin in most human cells studied to date. This report concerns the effect of interferons on the Burkitt lymphoma-derived cell line Daudi, which does not express HLA-A, B, C antigens or ß2-microglobulin on its membrane. HLA-A, B, C messenger RNA present in Daudi cells is increased by both a and ß interferons. Furthermore, we have shown that although it was not possible to detect mature ß2-microglobulin protein in the cytoplasm or on the cell membrane of Daudi cells, a poly A+ messenger RNA is present in Daudi cells, which hybridizes with a cDNA clone specific for human ß2-microglobulin. This abnormal messenger RNA is, however, increased normally by interferon. These effects were also observed with human interferon ß on a variant of Daudi cells characterized by a markedly reduced sensitivity to anti-proliferative and anti-cellular effects of human interferon a.[/p]

Abstract:  The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex (MHC) class II antigens. The tissular patterns of CIITA and MHC class II gene expression are tightly correlated: CIITA mRNA is highly expressed in B cells, and is induced by interferon gamma (IFN-γ) in macrophage and epithelial cell lines. We first isolated two overlapping cosmids encoding human CIITA which, when co-transfected, are able to restore MHC class II expression in a B-lymphoblastoid cell line (B-LCL) defective for CIITA. Subsequently, a 1.8 kilobase (kb) fragment of the CIITA promoter was isolated and sequenced. A motif presenting a strong similarity to an initiator was detected, as well as putative binding sites for Sp1, GATA-2, LyF-1, ets-1, AP1, and MZF1 transcription factors, and two GAS motifs. When introduced in front of a luciferase reporter gene, this promoter is able to direct a high luciferase activity in a human B-LCL. In contrast, luciferase expression was not stimulated after IFN-γ treatment when the construct was transfected in macrophage or in epithelial cell lines. However, an induction of the human CIITA gene was observed in mouse macrophage and fibrosarcoma cell lines, when the cells were transfected with a cosmid containing the human CIITA gene, but lacking the 1.8 kb promoter described above. Taken together, these data suggest the existence of an intragenic promoter driving an IFN-γ-inducible expression of CIITA.

The cross-reactivity of several monoclonal antibodies recognizing monomorphic determinants of human HLA-A, B, C, and DR antigens and human ß2-microglobulin (ß2m) has been studied on peripheral blood leukocytes in 24 different species. An monoclonal HLA-A-, B-, and C-specific antibody and four monoclonal HLA-DR-specific antibodies cross-reacted with cells from all the primate species tested. Furthermore, antibodies HLA-DR-specific were positive with peripheral blood leukocytes (PBL) from cows, goats, sheep, horses, and dogs. Two monoclonal ß2m-specific antibodies, which were positive with PBL from certain primates, also reacted with cells from cows, goats, sheep, horses, and dogs. Two other #2m-specific antibodies reacted only with PBL from chimpanzees. No reaction could be detected with all our reagents in other classes tested (birds, reptiles, amphibians, and Teleostei).

Numerous colonies of hybrids between PCC4-aza 1 teratocarcinoma cells and fibroblasts of the heteroploid Cl.1D cell line were examined. All of the hybrids were fibroblasts showing extinction of the multiple developmental potentialities of the teratocarcinoma cell parent, irrespective of whether the teratocarcinoma parent was diploid or tetraploid. The hybrids did not show loss of any specific chromosome contributed by the PCC4-aza 1 cell parent. In contrast with the PCC4 parental cells which carry F9 antigens and do not express H-2b, the hybrids do not express F9 antigens and carry H-2 alloantigens of both parental specificities. These results suggest that in hybrids whose phenotype is that of the Cl.1D parent, a change may occur in the genetic program of the teratocarcinoma cells.