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The Biomolecular Engineering Commission considers that the knowledge acquired in the last three years has provided significant information in reply to the points raised in its review dated 16 February 2001.

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.

Cloacal exstrophy is a rare malformation, belonging to a spectrum of birth defects, which, in order of severity, includes phallic separation with epispadias, pubic diastasis, bladder exstrophy, and cloacal exstrophy. This malformation overlaps the OEIS complex (O = omphalocele, E = bladder exstrophy, I = imperforate anus, S = spinal defects). The etiology of cloacal exstrophy is unknown to date. It may result from either a single defect of early blastogenesis or a defect of mesodermal migration during the primitive streak period. We report an infant with cloacal exstrophy, exomphalos, right kidney agenesis, ambiguous external genitalia, and axial hypotonia. The karyotype showed a de novo unbalanced translocation between the long arm of chromosome 9 and the long arm of chromosome Y resulting in a 9q34.1-qter deletion. Reviewing the literature, we did not find any observation of cloacal exstrophy associated with a structural chromosomal abnormality. The steroidogenic factor 1 (SF1) gene, included in the deleted region, was a good candidate gene but no pathogenic mutation was found by direct sequencing. We hypothesize that another gene, expressed early in embryogenesis and responsible for cloacal exstrophy, is present in the 9q34.1-qter region. © 2003 Wiley-Liss, Inc.

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.

Among farm animals, two species present an intersex condition at a relatively high frequency: pig and goat. Both are known to contain XX sex-reversed individuals which are genetically female but with a true hermaphrodite or male phenotype. It has been clearly demonstrated that the SRY gene is not involved in these phenotypes. Consequently, autosomal or X-linked mutations in the sex-determining pathway may explain these sex-reversed phenotypes. A mutation referred to as “polled” has been characterized in goats by the suppression of horn formation and abnormal sexual differentiation. The Polled Intersex Syndrome locus (PIS) was initially located in the distal region of goat chromosome 1. The homologous human region has been precisely identified as an HSA 3q23 DNA segment containing the Blepharophimosis Ptosis Epicanthus locus (BPES), a syndrome combining Premature Ovarian Failure (POF) and an excess of epidermis of the eyelids. In order to isolate genes involved in pig intersexuality, a similar genetic approach was attempted in pigs using genome scanning of resource families. Genetic analyses suggest that pig intersexuality is controlled multigenically. Parallel to this work, gonads of fetal intersex animals have been studied during development by light and electron microscopy. The development of testicular tissue and reduction of germ cell number by apoptosis, which simultaneously occurs as soon as 50 days post coïtum, also suggests that several separate genes could be involved in pig intersexuality. J. Exp. Zool. 290:700–708, 2001. © 2001 Wiley-Liss, Inc.

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.

Sex determination in mammals is controlled by the SRY gene located on the Y chromosome. It encodes a protein containing a DNA-binding and DNA-bending domain. In spite of recent advances in the identification of the mechanisms that regulate male sex determination in mammals, the expression profile of the SRY protein in normal and sex-reversed human tissues is not well established. In order to localize the SRY protein and determine its cellular distribution and expression at different stages of development, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One of these antibodies, LSRY1.1, recognizes a protein of 27 kDa in total lysates of HeLa SRYB3, a human cell line transfected with the SRY gene under the control of the SV40 promoter. Immunocytochemical analysis in the cell lines shows nuclear localization of the SRY protein. We have studied SRY protein expression in human tissues at different stage of fetal development until adult life and have demonstrated that the SRY protein is located in the nuclei of somatic cells and germ cells in the genital ridge during testis development. After testis determination, it can be detected until the adult stage in both germ cells and Sertoli cells. The presence of the SRY protein was also analyzed in biopsies of gonadal tissues of sex-reversal patients such as SRY-positive 46,XX males or SRY-positive 46,XX true hermaphrodites. SRY protein is detected in the nuclei of Sertoli cells of the testis and in the nuclei of granulosa cells in the ovotestis in these patients and in the nuclei of germ cells of both tissue types. These results suggest a common cellular origin for both Sertoli cells and granulosa cells. J. Exp. Zool. 290:607–615, 2001. © 2001 Wiley-Liss, Inc.

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.

Anterior-posterior (A-P) patterning is a key element in early embryonic development. Polycomb group (PcG) genes act as transcriptional repressors to regulate A-P patterning by either directly or indirectly controlling the coordinated expression of the HOM/Hox homeobox (Curr. Opin. Genet. Dev. 7 (1997) 488; Trends Genet. 13 (1997) 167). We describe the isolation and characterization of a novel mammalian PcG gene, termed Nervous System Polycomb-1 (NSPc1). Human and mouse NSPc1genes encode proteins with an N-terminal RING finger domain and share homology with Drosophila melanogaster lethal(3)73Ahand the mammalian Mel18and Bmi1genes. Transcripts are observed at 10 dpc in the otic vesicle, urogenital bud and dorsal root ganglia. At 11.5 dpc, transcripts are present in a subset of neural crest cell derivatives of the peripheral nervous system, and in the neural tube. NSPc1expression is ubiquitous in adult tissue.

“Functional wasteland,” “Nonrecombining desert” and “Gene-poor chromosome” are only some examples of the different definitions given to the Y chromosome in the last decade. In comparison to the other chromosomes, the Y is poor in genes, being more than 50% of its sequence composed of repeated elements. Moreover, the Y genes are in continuous decay probably due to the lack of recombination of this chromosome. But the human Y chromosome, at the same time, plays a central role in human biology. The presence or absence of this chromosome determines gonadal sex. Thus, mammalian embryos with a Y chromosome develop testes, while those without it develop ovaries (Polani [38]). What is responsible for the male phenotype is the testis-determining SRY gene (Sinclair [52]) which remains the most distinguishing characteristic of this chromosome. In addition to SRY, the presence of other genes with important functions has been reported, including a region associated to Turner estigmata, a gene related to the development of gonadoblastoma and, most important, genes related to germ cell development and maintenance and then, related with male fertility (Lahn and Page [31]). This paper reviews the structure and the biological functions of this peculiar chromosome.

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 MIC2 gene, 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 XG expression 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 XG mRNA 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,XG transcripts were detected in erythroid tissues and several nonerythroid tissues.

Deletions of 9p have been associated with 46,XY gonadal dysgenesis, and the smallest region of overlap has been mapped to the tip of chromosome 9. Two candidate genes (DMRT1and 2) have been found in the region. Despite intensive mutation searches, no mutations have been detected in these genes. To gain insights into the genomics of the region and to isolate other candidate genes for the phenotype, we have constructed a P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC) contig spanning over 500 kb and covering the consensus critical region. We have analyzed the expression pattern of several ESTs mapped or sublocalized within the framework of the contig. In addition, a sample shotgun sequencing of a PAC containing the mentioned DM genes led to the detection of novel transcripts displaying an expression pattern specific to testis and kidney, consistent with a role in the development of the urogenital system. One of them, expressed in adult testis and human embryos aged 4–5 weeks, encodes a potential polypeptide and is located immediately downstream of a sequence capable of encoding a novel DM domain. The region was partially screened for mutations in sex-reversed patients by Southern blot, sequencing, and FISH. No mutations were found. Our results suggest that the critical region on 9p involved in male-to-female sex reversal displays greater gene density and genomic complexity than previously anticipated. Future investigations will include functional and mutational studies of the novel transcripts mapped or sublocalized within the critical region by this study as well as cloning efforts to isolate additional candidate genes.

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.

Although SRY was first identified 10 years ago, we still know remarkably little about its mode of action or downstream target genes. Recently, potential protein partners have been identified and there has been considerable activity to understand the roles of WT1, SF-1, DAX-1 and SOX9 in gonadogenesis. The emerging picture is one of complex interactions, involving both positive and negative regulatory signals that, depending on the cellular and promoter context, drive the expression of male-specific genes. Despite recent advances, however, we are still unable to explain the genetic cause of most cases of 46,XY gonadal dysgenesis or even a single case of Y-chromosome-negative 46,XX maleness. Am. J. Med. Genet. (Semin. Med. Genet.) 89:176–185, 1999. © 2000 Wiley-Liss, Inc.

The genetic and cytogenetic map around the chromosome 1 region shown to be linked with polledness and intersexuality (PIS) in the domestic goat (Capra hircus) was refined. For this purpose, a goat BAC library was systematically screened with primers from human coding sequences, scraped chromosome 1 DNA, bovine microsatellites from the region, and BAC ends. All the BACs (n= 30) were mapped by fluorescencein situhybridization (FISH) on goat chromosome 1q41–q45. The genetic mapping of 30 new goat polymorphic markers, isolated from these BACs, made it possible to reduce the PIS interval to a region of less than 1 cM on goat chromosome 1q43. The PIS locus is now located between the two genesATP1BandCOP,which both map to 3q23 in humans. Genetic, cytogenetic, and comparative data suggest that the PIS region is now probably circumscribed to an ∼1-Mb DNA segment for which construction of a BAC contig is in progress. In addition, a human YAC contig encompassing the blepharophimosis-ptosis-epicanthus-inversus region was mapped by FISH to goat chromosome 1q43. This human disease, mapped to HSA 3q23 and affecting the development and maintenance of ovarian function, could be a potential candidate for goat PIS.

Interferons produce a variety of biological effects on cells. They induce resistance to virus proliferation1,2, inhibit cell growth3, modify cell structure and differentiation3–5, stimulate some immune functions and inhibit others6. However, the different interferon (IFN) species may vary in their mechanism of action and, hence, in their relative efficiency for inducing each of the effects. IFN-γ (type II) appears to show stronger immunoregulatory7–9and growth inhibitory effects10–12than antiviral effects13, but this conclusion has been challenged in other reports14. The aim of the present work is to compare the action of IFN-γ and other (type I) interferons on the induction of (2′–5′) oligo(A) synthetase which is probably part of the antiviral response15,16and the induction of the histocompatibility HLA-A,-B,-C antigens17–19. We have shown previously that the induction of both proteins is regulated by interferons at the mRNA level20,21, but show here that IFN-γ from stimulated human lymphocytes22and from monkey cells transfected by cloned human IFN-γ cDNA23induced the HLA-A,-B,-C and β2-microglobulin mRNAs or proteins at concentrations over 100 times lower than those needed to induce the (2′–5′)oligo(A) synthetase and the antiviral state. This difference was not found with IFN-α and -β (type I).

A 45,X complement was found in lymphocyte and fibroblast cultures of a male infant with severe growth and mental retardation and mild dysmorphism. Lymphocyte DNA from this patient was found to contain Yp chromosome sequences. In situ hybridization (ISH) with the 50f2 probe led to a clear assignment of euchromatic material on the short arm of chromosome 1. This observation and others from the literature argue in favour of the conclusion that all 45.X males are probably either the result of undetected mosaicism or are carriers of Y translocated material.

We have studied nine patients aged 1 month to 16 years with 46, XX karyotypes and testicular tissue. Some of these patients were followed through puberty. Phenotypically, two presented normal and seven abnormal external genitalia (AG). Among this latter group, four showed hypospadias and three true hermaphroditism (TH). The endocrine data were similar in all three groups: testosterone levels were within normal limits during puberty, decreasing in adulthood; gonadotrophin levels were above the control values at mid puberty. Histologies of the two sub groups of AG patients were identical up to 5 years of age and presented differences when compared with controls, regardless of the ovarian part of the ovotestis. However, in patients older than 8 years, germ cells disappeared and dysgenesis became obvious. In one patient, the ovarian zone of the gonad was detected only after complete serial sections of the removed gonad were examined. Southern blot analysis with Y-DNA probes displayed Y-specific material for the classic 46 XX males and a lack of such sequences for all patients with AG and TH. Based on these findings, we postulate that 46, XX males with AG and 46, XX TH may represent altenative manifestations of the same genetic defect. These data together with those concerning familial cases of 46, XX males with AG and 46, XX TH suggest an autosomally (or pseudoautosomally) determined mechanism.

Chromosome preparations from seven subjects with aberrations of sex chromosomes were utilized for in situ hybridization studies with the tritium-labeled Y-derived probe p50f. Two subjects had a pseudodicentric chromosome consisting of two copies of Yp and a portion of Y long arm; two were XX males [46,XX,t(Xp;Yp)], one was missing part of the Y short arm, and another had t(5p;Yq); in addition cells from an XYY male as well as a normal 46,XY male, and a 46,XX female, were hybridized with the same probe. The hybridization technique of Harper and Saunders (1981) was used. There was excess labeling of the Yp/paracentromeric regions in the cases with the normal Y, the XYY, the pseudodicentric Y, and the 5/Y translocation. No significant label was seen on metaphases from the normal 46,XX female or the female with the partially missing Y short arm. Excess label was present on the X short arm in the cases of the XX males; there were 8% and 9.5% of cells with label. The combined cytogenetic and hybridization data indicate that one X short arm in these XX males has undergone a translocation with Yp, and that genes for sex determination probably reside on the distal half of the Y short arm.

Abstract: In 46,XY individuals, testes are determined by the activity of the SRY gene (sex-determining region Y), located on the short arm of the Ychromosome. The other genetic components of the cascade that leads to testis formation are unknown and may be located on the Xchromosome or on the autosomes. Evidence for the existence of several loci associated with failure of male sexual development is indicated by reports of 46,XY gonadal dysgenesis associated with structural abnormalities of the Xchromosome or of autosomes (chromosomes9, 10, 11 and 17). In this report, we describe the investigation of a child presenting with multiple congenital abnormalities, mental retardation and partial testicular failure. The patient had a homogeneous de novo 46,XY,inv dup(9)(pter→p24.1::p21.1 →p23.3::p24.1→qter) chromosome complement. No deletion was found by either cytogenetic or molecular analysis. The SRY gene and DSS region showed no abnormalities. Southern blotting dosage analysis with 9p probes and fluorescent in situ hybridisation data indicated that the distal breakpoint of the duplicated fragment was located at 9p24.1, proximal to the SNF2 gene. We therefore suggest that a gene involved in normal testicular development and/or maintenance is present at this position on chromosome 9.

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.

Drosophila melanogaster embryos were injected before the blastoderm stage with conditioned media from several male Burkitt's lymphoma human cell lines and the Daudi cell line. Such injections do not have any effect on the male genital apparatus or on the female tract. The Daudi conditioned medium modifies the ovarian morphogenesis of the flies and the rudimentary ovaries obtained look like nymphal gonads. Moreover, they have a drastically reduced number of germ cells. The ovaries that looked functional contain numerous necrotic germ cells and the mean number of ovarioles per fly is significantly smaller than that of the controls. The abnormalities observed resemble the results of experimental and genetic lack of germ cells. They disappear at very high dilution (1×10-6).

Although true hermaphroditism (TH) accounts for less than 10% of intersex patients, it stands as a diagnostic challenge and has allowed a better understanding of the mechanisms involved in sexual differentiation. In this paper we review the clinical and laboratory data as well as molecular biology findings on 16 TH patients followed up at the Pediatric Endocrine Unit, Instituto da Criança, Hospital das Clínicas, São Paulo University Medical School. They were of a mean age of 3 years 8 months and nine of them were black. All the patients had ambiguous external genitalia as the main complaint. The 46,XX karyotype accounted for 50% of the cases and the ovotestis was the most frequent gonad found (59%). In the eight TH patients with a 46,XX karyotype, the sex-determining region of the Y chromosome (SRY) was negative, posing an intriguing question about the testicular differentiation mechanisms involved in these cases. In 7/19 ovotestes, the ovarian portion of the gonad has been preserved, keeping open the possibility of fertility. The female sex option was made in 10/16 cases (62·5%) and three patients exhibited spontaneous puberty. The mechanism through which testicular tissue develops without SRY has not yet been completely clarified, suggesting the involvement of the X chromosome as well as autosomal genes in the process.European Journal of Endocrinology136201–204

Fucose-labeled glycopeptides from four human teratoma cell lines of independent origin show similar elution profiles on Sephadex G-50 column chromatography. The fucosyl glycopeptides elute in two major regions: one near the void volume, the other in fractions corresponding to a molecular weight of 2500–3000. These elution profiles are very different from those obtained with the other human cell lines examined which included 3 lymphomas, 2 colon carcinomas, and HeLa. The elution profiles of the human teratomas, however, show remarkable similarities to those obtained with murine embryonal carcinoma cell culture and early mouse embryos. These results suggest that the excluded G-50 fraction may well contain glycopeptides playing a role in mammalian embryogenesis.

Cytokine signaling involves the activation of the Janus kinase (JAK) family of tyrosine kinases. These enzymes are physically associated with cytokine receptor components. Here, we sought to define the molecular basis of the interaction between Tyk2 and IFNAR1, a component of the interferon α/β receptor, by delimiting a minimal IFNAR1 binding region in the Tyk2 protein. Using an in vitroassay system, we narrowed down the interaction domain to a region comprising the JH7 and part of the JH6 homology boxes (amino acids 22–221). When expressed in Tyk2-negative cells, the JH7-6 region was unable to stabilize IFNAR1 protein levels, a critical function that we previously attributed to the N region (amino acids 1–591) of Tyk2. Moreover, substitution of the JH7-JH6 domain in JAK1 with that of Tyk2 did not restore IFNAR1 level nor interferon α signaling in Tyk2-negative cells. Thus, the major interaction surface lies within JH7-6, but additional JH regions (JH5-4-3) contribute in a specific manner to the in vivoassembly of Tyk2 and IFNAR1. Evidence is also provided of the lack of specificity of the Tyk2 kinase-like and tyrosine kinase domains in interferon α/β receptor signaling.

Frasier syndrome (FS) is a rare disease defined by male pseudo-hermaphroditism and progressive glomerulopathy1–3. Patients present with normal female external genitalia, streak gonads and XY karyotype4and frequently develop gonadoblastoma1,2,5,6. Glomerular symptoms consist of childhood proteinuria and nephrotic syndrome, characterized by unspecific focal and segmental glomerular sclerosis, progressing to end-stage renal failure in adolescence or early adulthood4. No case of Wilms′ tumour has been reported, even in patients with extended follow-up1–5. In contrast with FS patients, most individuals with Denys-Drash syndrome (DOS; refs 6,7) have ambiguous genitalia or a female phenotype, an XY karyotype and dysgenetic gonads. Renal symptoms are characterized by diffuse mesangial sclerosis, usually before the age of one year, and patients frequently develop Wilms′ tumour8–9. Mutations of the Wilms′-tumour gene, WT1, cause different pathologies of the urogenital system, including DDS10–12. WT1 is composed of ten exons and encodes a protein with four zinc-finger motifs and transcriptional and tumoursuppressor activities13–15. Alternative splicing generates four isoforms: the fifth exon may or may not be present, and an alternative splice site in intron 9 allows the addition of three amino acids (KTS) between the third and fourth zinc fingers of WT1 (ref. 17). Here we demonstrate that FS is caused by mutations in the donor splice site in intron 9 of WT1, with the predicted loss of the +KTS isoform. Examination of WT1 transcripts indeed showed a diminution of the +KTS/-KTS isoform ratio in patients with FS.

Monosomy of distal 9p is associated in rare cases with abnormalities of testicular determination, which can lead to male to female sex reversal in a 46,XY genetic background. We present two 46,XY individuals partially monosomic for 9p who were raised as females. Definition of the breakpoints using somatic cell hybrids containing only the rearranged chromosome 9 indicated that in the first patient the breakpoint was located between markers D9S256 and D9S144 and in the second patient, the breakpoint was distal to the marker D9S144. In both cases this corresponds to the cytogenetic position 9p23.3–p24.1. Analysis of highly polymorphic microsatellite markers demonstrated a paternal origin of the rearranged chromosome 9 in both patients. These studies define the minimum region associated with male to female sex reversal as 9p24.1–pter.

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.

Determination of mammalian sex depends on the presence or absence of a functional testis. Testes are determined by the activity of the testis determining factor encoded by the sex determining gene, Y (SRY) located on the Y chromosome. Considerable evidence suggests that the SRY gene is the only gene on the Y chromosome that is both necessary and sufficient to initiate testis determination. Other steps in the mammalian sex determining pathway are unknown, although recent advances have shown that mutations in X chromosome and autosomal loci are also associated with sex reversal, suggesting the presence of at least one other sex determining gene. Duplications of sequences on the short arm of the human X chromosome, including the DAX-1 (DSS-AHC critical region on the X chromosome, gene 1) gene, are occasionally associated with XY male-to-female sex reversal. In addition, mutations in the SRY-related gene SOX9 (SRY-related box) are associated with a failure of human testicular determination. Furthermore, the occurrence of inherited sex reversed conditions in both mice and men indicate the presence of at least one other sex determining gene. Breeding the Y chromosome from certain Mus musculus domesticus strains into the laboratory mouse strain C57BL/6J results in XY male-to-female sex reversal. This suggests both allelic variation of the Sry gene and the presence of autosomal sex determining genes. In humans, familial cases of SRY-negative XX males occur. Analysis of the transmission of the trait indicates the segregation of an autosomal or X-linked recessive mutation. The mutation may be in a gene whose wild-type function is to inhibit male sex determination. SRY may trigger male sex determination by repressing or functionally antagonizing the product of this gene.Heredity (1995) 75, 599–611; doi:10.1038/hdy.1995.179

A case of a true hermaphrodite presenting with a karyotype of 46,X,del(X)(p21.1?pter) is described. The testis-determining gene, SRY, was not detected in DNA prepared from either peripheral blood lymphocytes or from a gonad biopsy. The patient also presented with a series of discrete somatic abnormalities, including abnormal skin and retinal pigmentation, and mental retardation. The extent of the Xp deletion was mapped by Southern blotting. X chromosome replication studies of lymphoblast cells prepared from the patient indicated that the deleted X chromosome was inactivated in all cells examined. It is suggested that the phenotype of the patient is caused by the unmasking of a recessive allele(s) on the grossly intact X chromosome. The relationship between the Xp deletion, the intersex phenotype, and the possible role of an Xp locus involved in human sex determination is discussed.

A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.

Four patients with 46,XX true hermaphroditism and one patient with 46,XY pure gonadal dysgenesis (Swyer syndrome) were analyzed with a Y chromosome-derived probe that detects a specific fragment on the short arm of the Y chromosome in the putative testicle-determining region and also a fragment on the short arm of the X chromosome. Normal males and females, an individual with Turner syndrome, and patients with various causes of anomalous gonadal differentiation accompanied by cytogenetically present Y chromosome were used as controls. The Y-specific fragment was not detected in any of the persons with 46,XX true hermaphroditism. However, this fragment was positive in the 46,XY female and in all Y-bearing patients. Cytogenetic and molecular absence of the ZFY sequence in 46,XX true hermaphrodites calls for explanations other than the classic embryogenie theory. The absence of testicular differentiation in the ZFY-positive XY female evidences functionally altered sex determination or, alternatively, defective gonadal receptors.

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]

Various methods are now available to identify the molecular partners of the component of a signal transduction pathway. Some interactions, however, can be technically difficult to detect because they depend upon transient tyrosine phosphorylation. Here, we present a simple affinity chromatography approach based on synthetic phosphopeptides to purify potential partners of phosphotyrosine-containing proteins. With this approach, we confirm the previously characterized interaction between Grb2 and the EGF receptor, and we identify novel partners of the IGF-1 receptor and of the JAK proteins. Methenyltetrahydrofolate synthetase (MTHFS) was identified as a potential mediator of IGF-1R dependent transformation. P85α, the regulatory subunit of PI3 kinase, was identified as one of four proteins recruited by a phosphopeptide mimicking a motif conserved in all JAK family members.

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).

The Daudi cell line, established from a Burkitt lymphoma, has recently been found to be HLA- and?2-microglobulin-negative, although it expresses B lymphocyte alloantigens. This report is concerned with the reexpression of HLA-A10, B38, and B17 on the Daudi cell, after cell fusion with another human cell line (Raji) or with mouse fibroblasts. In the latter fusion, the same HLA specificities are re-expressed, but not human?2-microglobulin while mouse?2-microglobulin andH-2 could be detected. No such reexpression was observed when Daudi was fused with the F9 mouse teratocarcinoma, which lacks mouse?2-m andH-2. No HLA activity (alloantigenic and xenogenic activity) was detected in the membrane or cytoplasm of Daudi, using salt extraction and sonication. Therefore we postulate that?2-microglobulin could be necessary for the expression and possible synthesis of the HLA antigen.

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.