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Striatin is an intracellular protein characterized by four protein-protein interaction domains, a caveolin-binding motif, a coiled-coil structure, a calmodulin-binding domain, and a WD repeat domain, suggesting that it is a signaling or a scaffold protein. Down-regulation of striatin, which is expressed in a few subsets of neurons, impairs the growth of dendrites as well as rat locomotor activity (Bartoli, M., Ternaux, J. P., Forni, C., Portalier, P., Salin, P., Amalric, M., and Monneron, A. (1999) J. Neurobiol. 40, 234-243). Zinedin, a "novel" protein described here, and SG2NA share with striatin identical protein-protein interaction domains and the same overall domain structure. A phylogenetic analysis supports the hypothesis that they constitute a multigenic family deriving from an ancestral gene. DNA probes and antibodies raised against specific domains of each protein showed that zinedin is mainly expressed in the central nervous system, whereas SG2NA, of more widespread occurrence, is mainly expressed in the brain and muscle. All three proteins are both cytosolic and membrane-bound. All three bind calmodulin in the presence of Ca(2+). In rat brain, SG2NA and striatin are generally not found in the same neurons. Both localize to the soma and dendrites, suggesting that they share a similar type of addressing and closely related functions.

International audience; The mouse heat shock factor 2 (HSF2) cDNA was previously cloned by homology to HSF1, the heat shock factor involved in the cellular response to stress [Sarge, K.D., Zimarino, V., Holm, K., Wu, C., Morimoto, R.I., Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability. Genes Dev. 5 (1991) 1902-1911]. HSF2 is active in restricted cell types during pre- and post-implantation stages of development, and only in male germ cells of adult mice. However, the function of this factor remains elusive. We report here the cloning of the mouse Hsf2 gene and its genomic structure. We show that the gene is composed of 13 exons of variable sizes spanning at least 43kb in the genome. The transcription start site has been determined, and upstream sequences with promoter activity have been identified by their ability to direct the expression of a luciferase reporter gene in transfected cells. A preliminary analysis of the proximal promoter sequence determined that the TATA box is absent, but that a GC-rich region with several potential binding sites for transcription factors is present. The gene has been mapped to mouse chromosome 10 by in-situ hybridization on metaphase chromosomes.

D52 proteins are emerging as signalling molecules which may be regulators of cell proliferation. Having previously reported the existence of the human D52 gene family, comprising the hD52 and hD53 genes expressed in human breast carcinoma, we report the identification of a novel human gene hD54 ( TPD52L2 ), which represents a third D52 gene family member. In situ mapping placed the hD54 gene on human chromosome 20q13.2–q13.3, a localization distinct from those of both hD52 and hD53 genes. The identified hD54 cDNAs predicted three hD54 isoforms, suggesting that alternatively-spliced transcripts may be produced from D52-like genes. This was confirmed by directly sequencing reverse transcriptase–polymerase chain reaction (RT–PCR) products amplified from D52-like gene transcripts expressed in developing and adult rat tissues, and by performing sequence analyses of the expressed sequence tag divisions of nucleotide databases. Alternative splicing of sequences encoding two regions, termed ins2 and ins3, was identified in one or more D52-like genes, with these alternative splicing events being differentially regulated. The functional consequences of alternative splicing were examined by characterizing the protein–protein interactions mediated by a truncated hD53 isoform within the yeast two-hybrid system. This hD53 isoform displayed altered interaction capabilities with respect to those of full-length hD53, suggesting that alternative splicing within the D52 gene family functions in part to alter the protein–protein interaction capabilities of encoded isoforms.

We report here the identification of two distinct human cDNAs, called VNN1 and VNN2, related to the recently described mouse Vanin-1 molecule involved in lymphocyte migration (M. Aurrand-Lions et al., 1996, Immunity 5: 391-405). Tissue distribution of the expression of these two human Vanin-like genes is differential. Since Vanin-1 shares significant homologies with human biotinidase (BTD), we describe here a new family of related genes including at least four members: mouse Vanin-1, VNN1, VNN2, and BTD. We have mapped the murine locus encompassing the Vanin-1 gene on mouse chromosome 10 in position A2B1. The two human Vanin-like genes are closely linked, since they were found on the same YAC clone and colocalized on human chromosome 6q23-q24 known to contain several genetic alterations linked to the progression of metastatic human cancers.

We have identified a novel lysosome-associated membrane glycoprotein localized on chromosome 3q26.3-q27, DC-LAMP, which is homologous to CD68. DC-LAMP mRNA is present only in lymphoid organs and DC. A specific MAb detects the protein exclusively in interdigitating dendritic cells. Expression of DC-LAMP increases progressively during in vitro DC differentiation, but sharply upon activation with LPS, TNFα, or CD40L. Confocal microscopy confirmed the lysosomal distribution of the protein. Furthermore, DC-LAMP was found in the MHC class II compartment immediately before the translocation of MHC class II molecules to the cell surface, after which it concentrates into perinuclear lysosomes. This suggests that DC-LAMP might change the lysosome function after the transfer of peptide–MHC class II molecules to the surface of DC.

The ADAM (A Disintegrin And Metalloprotease) family of cell-surface proteins may have an important role in cellular interactions and in modulating cellular responses. In this report we describe a novel, secreted form of human ADAM 12 (meltrin alpha), designated ADAM 12-S (S for short), and a larger, membrane-bound form designated ADAM 12-L (L for long form). These two forms arise by alternative splicing of a single gene located on chromosome 10q26. Northern blotting demonstrated that mRNAs of both forms are abundant in human term placenta and are also present in some tumor cell lines. The ADAM 12-L transcript can also be detected in normal human adult skeletal, cardiac, and smooth muscle. Human A204 embryonal rhabdomyosarcoma cells that do not differentiate into muscle cells and do not express any form of ADAM 12 were stably transfected with an ADAM 12-S minigene encoding the disintegrin domain, the cysteine-rich domain, and the unique 34 amino acid carboxyl terminus. Nude mouse tumors derived from these transfected cells contained ectopic muscle cells of apparent mouse origin as shown by species-specific markers. These results may have potential applications in the development of muscle-directed gene and cell therapies.

The murine Sycp1 gene is expressed at the early stages of meiosis. We show that it is composed of a number of small exons and localized on mouse chromosome 3. In the laboratory strains, two retrogenes were also identified. The first one (Sycp1-ps1), on chromosome 7, has accumulated point mutations and deletions and is not transcribed. A second retrogene (Sycp1-ps2), on chromosome 8, is inserted within the continuity of a moderately repeated element, in an intron of another gene (Cad11). The two retroposition events can be dated to distinct periods in the evolution of the Muridae. Sycp1-ps2 has kept features indicative of a relatively recent origin, namely a nearly intact coding region, a poly(A) tail, and 14-bp terminal repeats. Its recent origin was confirmed by the fact that it is found in all the laboratory strains of mice, but neither in a recent isolate from Mus musculus domesticus wild stocks nor in the closely related subspecies M. musculus musculus, M. m. molossinus, M. m. castaneus, and M. m. bactrianus. Appearance of the more ancient Sycp1-ps1 retrogene is concomitant with the radiation of the genus. It is present in various Mus species (M. spretus, M. spicilegus, M. macedonicus, and M. cookii), but neither in the rat nor in the more closely related Pyromis genus. Transposition of retrotranscripts during meiosis and their hereditary establishment thus appear to occur relatively frequently. They may, therefore, play a significant role in the evolutionary process.

Secretory phospholipases A2(sPLA2s) represent a rapidly expanding family of structurally related enzymes found in mammals as well as in insect and snake venoms. In this report, a cDNA coding for a novel sPLA2 has been isolated from human fetal lung, and its gene has been mapped to chromosome 16p13.1-p12. The mature sPLA2protein has a molecular mass of 13.6 kDa, is acidic (pI 5.3), and made up of 123 amino acids. Key structural features of the sPLA2include: (i) a long prepropeptide ending with an arginine doublet, (ii) 16 cysteines located at positions that are characteristic of both group I and group II sPLA2s, (iii) a C-terminal extension typical of group II sPLA2s, (iv) and the absence of elapid and pancreatic loops that are characteristic of group I sPLA2s. Based on these structural properties, this sPLA2 appears as a first member of a new group of sPLA2s, called group X. A 1.5-kilobase transcript coding for the human group X (hGX) sPLA2 was found in spleen, thymus, and peripheral blood leukocytes, while a less abundant 0.8-kilobase transcript was detected in the pancreas, lung, and colon. When the hGX sPLA2cDNA was expressed in COS cells, sPLA2 activity preferentially accumulated in the culture medium, indicating that hGX sPLA2 is an actively secreted enzyme. It is maximally active at physiological pH and with 10 mm Ca2+. hGX sPLA2 prefers phosphatidylethanolamine and phosphatidylcholine liposomes to those of phosphatidylserine.

A subtractive cloning procedure was used to characterize the molecular changes involved in transformation of normal myoblasts to rhabdomyosarcoma (RMS) cells. Here we describe the cloning of DRAL, a novel LIM-domain protein expressed in primary myoblasts but down-regulated in the RMS cell line RD. DRAL is a LIM-only protein with five LIM domains whereby one LIM domain consists only of the second half of the consensus motif. Interestingly, down-regulation of DRAL was not confined to the RD RMS cells, but was a phenomenon extended to other RMS cell lines of both embryonal and alveolar subtype, and to some breast cancer cell lines. Analysis of the expression pattern in normal human tissues revealed that DRAL is expressed at high levels in the heart, suggesting an important function in the specification of the terminally differentiated phenotype of heart muscle cells. Immunofluorescence studies using an antibody directed against recombinant DRAL localized the protein predominantly in the nucleus of cultured cells. On the basis of these results, we conclude that down-regulation of DRAL correlates with the tumor phenotype of RMS cells.

Several lines of evidence indicate a crucial role for unconventional myosins in the function of the sensory hair cells of the inner ear. We report here the characterization of the cDNAs encoding two unconventional type I myosins from a mouse cochlear cDNA library. The first cDNA encodes a putative protein named Myo1c, which is likely to be the murine orthologue of the bullfrog myosin I{beta} and which may be involved in the gating of the mechanotransduction channel of the sensory hair cells. This myosin belongs to the group of short-tailed myosins I, with its tail ending shortly after a polybasic, TH-1-like domain. The second cDNA encodes a novel type I myosin Myo1f which displays three regions: a head domain with the conserved ATP- and actin-binding sites, a neck domain with a single IQ motif, and a tail domain with the tripartite structure initially described in protozoan myosins I. The tail of Myo1f includes (1) a TH-1 region rich in basic residues, which may interact with anionic membrane phospholipids; (2) a TH-2 proline-rich region, expected to contain an ATP-insensitive actin-binding site; and (3) an SH-3 domain found in a variety of cytoskeletal and signaling proteins. Northern blot analysis indicated that themore » genes encoding Myo1c and Myo1f display a widespread tissue expression in the adult mouse. Myo1c and Myo1f were mapped by in situ hybridization to the chromosomal regions 11D-11E and 17B-17C, respectively. The human orthologuous genes MYO1C and MYO1F were also characterized, and mapped to the human chromosomal regions 17p13 and 19p13.2- 19p1.3.3, respectively. 45 refs., 5 figs., 2 tabs.« less

Prader-Willi syndrome (PWS) is a neurogenetic disorder resulting from the loss of paternal expression of gene(s) localized in the 15q11-q12 region. A new human gene encoding a putative protein with high homology to the mouse NECDIN protein has recently been characterized and mapped to chromosome 15q11-q12. It is expressed from the paternal allele only, suggesting its potential involvement in PWS. We now report the localization of the mouse Necdin gene in a region of conserved synteny to the human PWS region. We demonstrate the paternal specific expression of Necdin in the mouse central nervous system, and show that parental alleles display a differential methylation profile in the coding region. Finally, fluorescence in situ hybridization analysis reveals an asynchronous pattern of replication at the Necdin locus. These results clearly demonstrate imprinting of the mouse Necdin gene. Mouse models will be powerful tools in the study of human PWS phenotype and imprinting mechanisms.

International audience; Integrated hepatitis B virus (HBV) DNA is found in the great majority of human hepatocellular carcinomas, suggesting that these viral integrations may be implicated in liver oncogenesis. Besides the insertional mutagenesis characterized in a few selected cases and the contribution of viral transactivators to cell transformation to malignancy, HBV has been shown to generate gross chromosomal rearrangements potentially involved in carcinogenesis. Here, we report a t(3;8) chromosomal translocation present in a hepatocellular carcinoma developed in noncirrhotic liver tissue. One side of the translocation, in 8p23, is shown to be in the vicinity of the carboxypeptidase N gene, a locus that is heavily transcribed in liver tissue and frequently deleted in hepatocellular carcinomas and other epithelial tumors. The other side of the translocation, in 3q27-29, is widely implicated in several types of translocations occurring in different malignancies, such as large-cell lymphomas. The present data strongly support a model in which HBV-induced chromosomal rearrangements play a key role during multistep liver oncogenesis.

The genomic structure of the mouse gene encoding the CDEBP protein has been established. The protein was initially identified on the basis of its ability to bind the CDEI motif (GTCACATG). The same locus has been independently described under the name APLP2, on the basis of sequence similarities with the Amyloid Precursor Protein (APP). The exon-intron distribution of Cdebp appears strikingly similar to that of the App gene in the regions encoding the conserved domains, with a divergent structure in the other parts. The transcription start site has been localized, and sequences with promoter activity have been identified immediately upstream of it by their ability to direct the expression of a reporter luciferase gene in transfected cells. This region is devoid of either TATA or CAAT boxes. The gene has been mapped to mouse chromosome 9 by in situ hybridization on metaphase chromosomes.

With anti-hnRNP monoclonal antibody 6D12 we previously showed in HeLa cells that as early as 10 min after the onset of a heat shock at 45 degrees C, a 72.5-74 kDa antigen doublet leaves the hnRNPs and strongly associates with the nuclear matrix, the effect being reversed after a 6 h recovery at 37 degrees C. cDNA cloning and sequencing enabled us to identify these antigens as hnRNP-M proteins and further to show that the correct sequence differs by an 11 amino acid stretch from the originally published sequence. We also show that monoclonal antibodies raised against synthetic hnRNP-M peptides can directly inhibit in vitro splicing. Furthermore, stressing cells at 45 degrees C for 10 min is sufficient to abolish the splicing capacity of subsequently prepared nuclear extracts which, interestingly, do not contain the hnRNP-M proteins any more. Taken together, our data suggest that these proteins are involved in splicing as well as in early stress-induced splicing arrest. Further in situ hybridization assays located the hnRNP-M encoding gene on human chromosome 19.

2–5A-Dependent RNase (RNase L), an important component of the 2–5A pathway, is directly implicated in the molecular mechanism of interferon action. We have cloned and sequenced following immunoscreening, a full-length cDNA that encodes the RNase L inhibitor (RLI). Northern blot analysis from a variety of human tissues revealed that two transcript forms (3.8 kb and 2.4 kb) are ubiquitously expressed but differences in levels of expression suggest a tissue-specific regulation. The RLI gene was localized to locus 4q31 by in situ hybridization indicating that this gene and other enzymes of the 2–5A pathway are not organized in cluster in the human genome.

Using a reverse transcription coupled to PCR amplification strategy, with degenerated primers localized in highly conserved domains of known glutathione peroxidase (GPX) proteins, we have generated, from mouse epididymal RNA, a cDNA fragment which was subsequently used to isolate a genomic clone encoding mouse plasma GPX (GPX3). GPX3 is a major enzyme in reducing lipid hydroperoxides and hydrogen peroxide in plasma. We confirm here that the mouse epididymis is a new site of expression of GPX3 and report, together with the sequence, the structural analysis and the chromosomal localization of the mouse GPX3 single-copy gene to chromosome 11.

We have cloned two novel homeo☐ genes which are the mouse ( Lbx1 ) and human ( LBX1 ) homologs of the Drosophila lady bird genes. They are highly related not only within the coding region but also in 5′ and 3′ untranslated regions. Several amino acid residues inside and around the homeodomain, have been conserved between the mammalian Lbx genes and their Drosophila counterparts. The mouse Lbx1 gene is located on chromosome 19 (region D) and the human LBX1 gene maps to the related q24 region of chromosome 10, known as a breakpoint region in translocations t(7;10) and t(10;14) involved in T-cell leukemias. Thus, LBX1 and the protooncogene HOX11 map to a common chromosomal region, as do their Drosophila counterparts, the lady bird and 93Bal genes. The mouse Lbx1 gene is specifically expressed during embryogenesis. From 10.5 days of gestation, Lbx1 expression is detected in the central nervous system and some developing muscles. In the CNS, Lbx1 transcripts are expressed in the dorsal part of the mantle layer of the spinal cord and hindbrain, up to a sharp boundary within the developing metencephalon. Thus, Lbx1 may be involved in spinal cord and hindbrain differentiation and/or patterning, and its restricted expression pattern could depend upon evolutionarily conserved inductive signals involving some mammalian Wnt and Pax genes, as is the case for Drosophila lady bird genes and wingless or gooseberry .

The Landsteiner-Wiener (LW) blood group antigens reside on a 42-kD erythrocyte membrane glycoprotein that has recently been cloned. Here, we found that the molecular basis for the LWa/LWb polymorphism is determined by a single base pair mutation (A308G) that correlates with a Pvu II restriction site and results in a Gln70Arg amino acid substitution. COS-7 cells transfected with LWa or LWb cDNAs reacted with human anti-LWa and anti-LWb sera, respectively, as well as with a murine monoclonal anti-LWab antibody, as shown by flow cytometry analysis. Moreover, a 42-kD protein was immunoprecipitated from the transfected cells with the monoclonal anti-LWab antibody. These findings indicate that LWa and LWb are alleles of the LW blood group locus as defined also by a monoclonal anti-LWab of nonhuman origin. In addition, the LW locus has been assigned to chromosome 19p13.3 by in situ hybridization. Study by Southern blot analysis indicated also that the LW locus is composed of a single gene that was not grossly rearranged in rare LW(a-b-) and Rhnull individuals deficient for LW antigens. In addition, Pvu II restriction fragment-length polymorphism analysis indicated that these variants were all homozygous for a phenotypically silent LWa allele.

Human DNA-binding proteins, dbpA and dbpB (YB-1), are members of a protein family containing a cold-shock domain, and are regarded as transcriptional regulators. Here, we isolated genomic fragments of these genes and characterized their transcriptional regulation. Analysis of lambda phage genomic clones revealed that the dbpA gene consists of 10 exons spanning a 24-kb genomic region. The cold-shock domain, composed of about 70 amino acid residues, is encoded separately by exons 2-5. The exon 6, encoding 69 amino acid residues, was found to be an alternative exon. Northern-blot analysis showed that both genes were highly expressed in skeletal muscle and heart compared with in other tissues. The dbpA gene contains no typical TATA box or CAAT box at the immediate 5' region, but a sequence similar to an initiator consensus sequence was revealed at a major transcription-start site. A transient expression assay using the chloramphenicol acetyltransferase reporter gene revealed that the sequence located at positions -17 to +70 relative to the major transcription-start site was critical for promoter function. Within this region, the consensus sequence for serum-response element, CC(A/T)6GG, is present at positions -13 to -4 in addition to the initiator sequence. Immunofluorescence showed the cellular localization of dbpA to be both in the cytoplasm and nucleus, particularly at the perinuclear region. In situ hybridization demonstrated the localization of the dbpA gene on chromosome 12 band p13.1, whereas dbpB-(YB-1)-related genes were dispersed on many chromosomes with strongest hybridization signals on chromosome 1. All 16 dbpB (YB-1) clones, isolated from the same genomic library used for dbpA genomic cloning, were processed genes because of their intronless structures and multiple mutations. One of these processed genes possesses an open reading frame, which encodes most of the amino acid residues of dbpB (YB-1). These results indicate that dbpA and dbpB (YB-1) genes evolved in different fashions after deviation from a common ancestral gene.

The human poliovirus (PV) receptor (PVR) is a member of the immunoglobulin (Ig) superfamily with unknown cellular function. We have isolated a human PVR-related (PRR) cDNA. The deduced amino acid (aa) sequence of PRR showed, in the extracellular region, 51.7 and 54.3% similarity with human PVR and with the murine PVR homolog, respectively. The cDNA coding sequence is 1.6-kb long and encodes a deduced 57-kDa protein; this protein has a structural organization analogous to that of PVR, that is, one V- and two C-set Ig domains, with a conserved number of aa. Northern blot analysis indicated that a major 5.9-kb transcript is present in all normal human tissues tested. In situ hybridization showed that the PRR gene is located at bands q23-q24 of human chromosome 11.

The murine homologue of the human NFE2L1 basic leucine-zipper gene was isolated from an early embryo library. The deduced amino acid sequence shows 97% identity between the two proteins. Significant sequence similarity is also seen with the p45 subunit of NF-E2 and with the Drosophila CNC protein. Murine Nfe2l1 maps to chromosome 11DE with similar sequences at 7D1-7F1 and 2E4-2G. (C) 1995 Academic Press, Inc.

The ubiquitously expressed upstream stimulatory factor (USF) involved in the transcription of a wide variety of cellular genes is defined as dimers of c-myc-related proteins, composed of a basic helix-loop-helix/leucine zipper region. The USF family consists of different members that split into two groups: MLTF or USF1 and USF2 or FIP. We present here evidence that USF1 and USF2 are distinct closely related genes in human, rat, and mouse. Based on the recent cloning of rat and human new cDNAs, we have isolated genomic clones encompassing the murine USF2 gene, which consists of at least 10 exons spanning a minimum of 10 kb of genomic DNA. Unexpectedly, the organization of USF2 appears very split up by introns (0.08 to over 6 kb in size), compared to the myc gene structure. The entire gene (but the larger intron) and 1.6 kb of the 5' flanking region were sequenced. This 5' flanking region is GC-rich, contains several putative transcription binding sites, and has no apparent TATA box. Gene mapping of murine USF2 and USF1 has been determined by in situ hybridization, indicating the localization of USF2 on chromosome 7 and of USF1 on chromosomes 1 and 11.

Remodeling of the extracellular matrix (ECM) is an essential component of normal development and is also involved in the pathogenesis of arthritis and the spread of cancer. The matrix metalloproteinases and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), play an important role in this context. We have isolated mouse cDNA clones encoding a novel member of the TIMP family, designated TIMP-3. We have assigned the Timp-3 locus to the [C1-D1] region of mouse chromosome 10 using both genetic and cytogenetic methods. The conceptual translation product of the Timp-3 cDNA shows a high degree of similarity with ChIMP-3, a recently cloned chicken metalloproteinase inhibitor, as well as significant structural similarity with the amino acid sequences of the previously isolated members of this family, TIMP-1 and TIMP-2. The pattern of expression of Timp-3 in the developing mouse embryo is distinct from that previously reported for Timp-1. Timp-3 is expressed in cartilage and skeletal muscle, in myocardium, in the skin, oral and nasal epithelium, in the newborn mouse liver, in the epithelium of some tubular structures such as the developing bronchial tree, oesophagus, colon, urogenital sinus, bile duct, in the kidney, salivary glands, and in the choroid plexus of the brain. The patterns of Timp-3 expression in surface epithelia and in the epithelial lining of many tubular organs suggests that TIMP-3 may be involved in regulating ECM remodeling during the folding of epithelia and during the formation, branching, and expansion of epithelial tubes. In the mouse placenta, expression is seen in the trophoblast, raising the possibility that TIMP-3 may be involved in regulating trophoblastic invasion of the uterus. We propose a role for TIMP-3 in musculoskeletal and cardiac development, in the morphogenesis of certain epithelial structures, and placental implantation.

Analysis of cDNA clones that cross-hybridized with the fork head domain of the rat HNF-3 gene family revealed 10 cDNAs from human fetal brain and human testis cDNA libraries containing this highly conserved DNA-binding domain. Three of these cDNAs (HFK1, HFK2, and HFK3) were further analyzed. The cDNA HFK1 has a length of 2557 nucleotides and shows strong homology at the nucleotide level (91.2%) to brain factor 1 (BF-1) from rat. The HFK1 cDNA codes for a putative 476 amino acid protein. The homology to BF-1 from rat in the coding region at the amino acid level is 87.5%. The fork head homologous region includes 111 amino acids starting at amino acid 160 and has a 97.5% homology to BF-1. Southern hybridization revealed that HFK1 is highly conserved among mammalian species and possibly birds. Northern analysis with total RNA from human tissues and poly(A)-rich RNA from mouse revealed a 3.2-kb transcript that is present in human and mouse fetal brain and in adult mouse brain. In situ hybridization with sections of mouse embryo and human fetal brain reveals that HFK1 expression is restricted to the neuronal cells in the telencephalon, with strong expression being observed in the developing dentate gyrus and hippocampus. HFK1 was chromosomally localized by in situ hybridization to 14q12. The cDNA clones HFK2 and HFK3 were analyzed by restriction analysis and sequencing. HFK2 and HFK3 were found to be closely related but different from HFK1. Therefore, it would appear that HFK1, HFK2, HFK3, and BF-1 form a new fork head related subfamily.

Leydig insulin-like protein (LEY I-L) is a member of the insulin-like hormone superfamily. The LEY I-L gene (designated INSL3) is expressed exclusively in prenatal and postnatal Leydig cells. We report here the cloning and nucleotide sequence of porcine and human LEY I-L genes including the 5′ regions. Both genes consist of two exons and one intron. The organization of the LEY I-L gene is similar to that of insulin and relaxin. The transcription start site in the porcine and human LEY I-L gene is localized 13 and 14 bp upstream of the translation start site, respectively. Alignment of the 5′ flanking regions of both genes reveals that the first 107 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 80%. This conserved region contains a consensus TATAA box, a CAAT-like element (GAAT), and a consensus SP1 sequence (GGGCGG) at equivalent positions in both genes and therefore may play a role in regulation of expression of the LEY I-L gene. The porcine and human genome contains a single copy of the LEY I-L gene. By in situ hybridization, the human gene was assigned to bands p13.2-p12 of the short arm of chromosome 19.

A cDNA encoding a N-type Ca2+ channel has been cloned from the murine neuroblastoma cell line N1A103. The open reading frame encodes a protein of 2,289 amino acids (257 kDa). Analysis of different clones provided evidence for the existence of distinct isoforms of N-type channels. High levels of mRNA were found in the pyramidal cell layers CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus, in the cortex layers 2 and 4, in the subiculum and the habenula. The N-type Ca2+ channel gene has been localized on the chromosome 2, band A.

The tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of the matrix metalloproteinases, a group of zinc-binding endopeptidases involved in the degradation of the extracellular matrix. We have isolated overlapping cDNAs encoding a novel human TIMP, TIMP-3. The cDNAs contain a 591-bp-long open reading frame encoding 9 amino acid residues of the signal peptide and 188 residues of the mature TIMP-3 polypeptide. Both the nucleotide sequence and the conceptual translation product of the human TIMP3 cDNA have a high degree of similarity to ChIMP-3, a recently cloned metalloproteinase inhibitor in the chicken, and to the TIMP1 and TIMP2 gene products, including 12 conserved cysteinyl residues at the same relative positions. The TIMP3 gene is expressed in many tissues, with highest expression in the placenta. Upon hybridization with a panel of human-hamster somatic cell hybrid DNAs, the TIMP3 gene segregated with clones containing chromosome 22. Using in situ hybridization to human metaphase chromosomes, we have assigned the locus for the TIMP3 gene to the q12.1-q13.2 region of human chromosome 22.

Using both chromosomal in situ hybridization and molecular techniques, we report the genetic localization of the gene coding for the alpha 1 subunit of the skeletal slow Ca2+ current channel/DHP receptor gene (Cchl1a3) on human Chromosome (Chr) 1 (1q31-1q32 region) and on mouse Chr 1 (region (F-G)). On the basis of single-strand conformation polymorphism (SSCP-PCR) analysis in an interspecific backcross, we have determined that the Cchl1a3 = mdg (muscular dysgenesis) locus is very closely linked to the myogenin (Myog) locus.

Human glycophorin A, B, and E (GPA, GPB, and GPE) genes belong to a gene family located at the long arm of chromosome 4. These three genes are homologous from the 5'-flanking sequence to the Alu sequence, which is 1 kb downstream from the exon encoding the transmembrane domain. Analysis of the Alu sequence and flanking direct repeat sequences suggested that the GPA gene most closely resembles the ancestral gene, whereas the GPB and GPE genes arose by homologous recombination within the Alu sequence, acquiring 3' sequences from an unrelated precursor genomic segment. Here we describe the identification of this putative precursor genomic segment. A human genomic library was screened by using the sequence of the 3' region of the GPB gene as a probe. The genomic clones isolated were found to contain an Alu sequence that appeared to be involved in the recombination. Downstream from the Alu sequence, the nucleotide sequence of the precursor genomic segment is almost identical to that of the GPB or GPE gene. In contrast, the upstream sequence of the genomic segment differs entirely from that of the GPA, GPB, and GPE genes. Conservation of the direct repeats flanking the Alu sequence of the genomic segment strongly suggests that the sequence of this genomic segment has been maintained during evolution. This identified genomic segment was found to reside downstream from the GPA gene by both gene mapping and in situ chromosomal localization. The precursor genomic segment was also identified in the orangutan genome, which is known to lack GPB and GPE genes. These results indicate that one of the duplicated ancestral glycophorin genes acquired a unique 3' sequence by unequal crossing-over through its Alu sequence and the further downstream Alu sequence present in the duplicated gene. Further duplication and divergence of this gene yielded the GPB and GPE genes.

We have characterized the human gene encoding the major peripheral myelin protein zero (P0) and assigned it, by in situ hybridization, to the q21.3-q23 region of human chromosome 1. This region is known to contain a cluster of interspersed genes coding for the related human leukocyte receptors of the Fc portion of the immunoglobulin G (Fc gamma RI, II, III). This colocalization was refined by the finding of a yeast artificial chromosome (YAC) of the Centre d'Etude du Polymorphisme Humain (CEPH) library, hybridizing to the P0 and Fc gamma RIIA genes, demonstrating their physical linkage. These data may have important implications in demyelinating diseases studies like Charcot-Marie-Tooth disease type 1B (CMT1B).

We have cloned a human cDNA from a new gene, spi-B, on the basis of its homology with the DNA-binding domain of the Spi-1/PU.1 putative oncogene product. spi-B codes for a protein of 262 amino acids presenting 43% overall identity with Spi-1. Its highly basic carboxy-terminal region exhibits 34% sequence identity with the DNA-binding domain of the Ets-1 protein. We showed that the Spi-B protein is able to bind the purine-rich sequence (PU box) recognized by Spi-1/PU.1 and to activate transcription of a reporter plasmid containing PU boxes. Chromosome in situ hybridization allowed us to map spi-B to the 19q13.3-19q13.4 region of the human genome. spi-B, like spi-1, was found to be expressed in various murine and human hematopoietic cell lines except T lymphoid cell lines.

A novel mouse homeobox-containing gene, Nkx-2.2, has been isolated. Nkx-2.2 is a member of a family of genes whose homeodomains are homologous to that of the Drosophila NK-2 gene. Nkx-2.2 transcripts are found in localized domains of the brain during mouse embryogenesis. Nkx-2.2 expression in the brain abuts and partially overlaps with the expression domains of two other related homeobox-containing genes, TTF-1 and Dlx. The expression domains of the three genes in the developing prosencephalon coincide with anatomical boundaries, particularly apparent in the diencephalon. This result raises the possibility that these genes may specify regional differentiation of the developing diencephalon into its anatomically and functionally defined subregions. Nkx-2.2 may be involved in specifying diencephalic neuromeric boundaries.

We have determined the genomic structure of the fosB gene and shown that it consists of 4 exons and 3 introns at positions also found in the c-fos gene. By deletion analysis we have characterized a region upstream of the TATA box which is the promoter region of the gene. Several consensus sequences have been identified, including an SRE and AP-1 binding site whose relative positions are identical to those in the 5' upstream region of the c-fos gene. We have also shown that FosB and c-Fos can downregulate the activity of the fosB promoter to a similar extent. The fosB gene is located in the [A1-B1] region of mouse chromosome 7.

Laser microdissection has been used to dissect material from the X-chromosome region involved in fragile-X-linked mental retardation. After dissection, single chromosome slices corresponding to this fragile site were subjected to DNA amplification using either a vector ligation method (to provide known anchor sequences) or primer oligonucleotides corresponding to the ubiquitous Alu sequences. Amplified material was then cloned or, alternately, used to screen a gridded cosmid library. Eight cosmid clones identified in this way were regionally mapped using a panel of hybrid cell lines and shown to originate from a narrow interval centered on the fragile X site. Two clones are included in the approximately 6-cM interval defined by probes RNI (DXS369, 5 cM proximal) and VK21 (DXS 296, 1-2 cM distal) and which includes the fragile site, and at least one clone contains sequences conserved across species suggestive of a gene. This method combines the focused approach of microdissection and the convenience of obtaining cosmid (rather than small-insert) clones; it may be useful for studies of other defined chromosomal regions.

Marfan syndrome (MFS), one of the most common genetic disorders of connective tissue, is characterized by skeletal, cardiovascular and ocular abnormalities. The incidence of the disease is about 1 in 20,000, with life expectancy severely reduced because of cardiovascular complications. As the underlying defect is unknown, MFS diagnosis is based solely on clinical criteria. Certain phenotypic features of MFS are also shared by other conditions, which may be genetically distinct entities although part of a clinical continuum. Immunohistochemical studies have implicated fibrillin, a major component of elastin-associated microfibrils, in MFS aetiology. Genetic linkage analysis with random probes has independently localized the MFS locus to chromosome 15. Here we report that these two experimental approaches converge with the cloning and mapping of the fibrillin gene to chromosome 15q15-21, and with the establishment of linkage to MFS. We also isolated a second fibrillin gene and mapped it to chromosome 5q23-31. We linked this novel gene to a condition, congenital contractural arachnodactyly, that shares some of the features of MFS. Thus, the cosegregation of two related genes with two related syndromes implies that fibrillin mutations are likely to be responsible for different MFS phenotypes.

Type VIII collagen molecules, expressed by corneal and vascular endothelial cells, appear to be heterotrimers composed of two genetically distinct polypeptides, alpha 1 (VIII) and alpha 2(VIII), in the ratio of 2:1. Characterization of the rabbit alpha 1(VIII) gene has demonstrated that it consists of only four exons, one of which is large and encodes the entire triple-helical and carboxyl non-triple-helical domains. A similar exon organization has been found for the chicken alpha 1(X) and the mouse and human alpha 2(VIII) collagen genes. The genes encoding alpha 1(VIII), alpha 2(VIII), and alpha 1(X) collagen chains are therefore homologous members of a unique class of genes within the collagen superfamily. In the present paper we describe for the first time the primary structure of the human alpha 1(VIII) collagen chain, based on isolation and sequencing of a genomic DNA fragment. The results indicate that the human alpha 1(VIII) chain has the same domain structure as the rabbit protein. We also demonstrate that the gene is localized on the long arm of the human chromosome 3. The availability of genomic DNA encoding the human alpha 1(VIII) collagen chain and information about its chromosomal location should make it possible to examine whether hereditary diseases are linked to abnormalities in the structure or expression of the alpha 1(VIII) gene.

We report the cloning of KIN17 cDNA, 1414 bp long with an ORF of 391 residues showing a zinc finger and nuclear localization signals. By recloning the cDNA into an appropriate vector, we produced kin17 protein in E. coli, purified it partially and shown that kin17 protein binds to double-stranded DNA. The KIN17 gene was localized by cytogenetic mapping in mouse chromosome 2, band A. Genomic sequences homologous to KIN17 cDNA were detected also in rat and human DNAs. KIN17 mRNA is highly expressed in rodent transformed AtT-20 neuroendocrine cells whereas it can be detected only in the total RNA of mouse embryos and various normal adult tissues by reverse transcription and PCR amplification. The mouse nuclear kin17 protein was identified by a local small structural similarity with E.coli recA protein. Kin17 and recA have only 39 amino acid residues in a region that might be involved in DNA-binding.

We have isolated a human cDNA (kup), encoding a new protein with two distantly spaced zinc fingers of the C2H2 type. This gene is highly conserved in mammals and is expressed mainly in hematopoietic cells and testis. Its expression was not higher in the various transformed cells tested than in the normal corresponding tissues. The kup gene is located in region q23-q24 of the long arm of human chromosome 14. The kup protein is 433 a.a. long, has a M.W. close to 50 kD and binds to DNA. Although the structure of the kup protein is unusual, the isolated fingers resemble closely those of the Kruppel family, suggesting that this protein is also a transcription factor. The precise function and DNA motif recognized by the kup protein remain to be determined.

HNF1 is a transcriptional activator, required for the liver-specific expression of a variety of genes, that binds to DNA as a dimer via the most diverged homeodomain known so far. We were interested to examine whether HNF1 is a unique homeoprotein example or whether it is the prototype of a new subfamily of homeodomain containing proteins. In this work we describe the isolation of a cDNA clone from a human liver library encoding a protein, highly homologous to HNF1 in three regions, including the homeo- and dimerization domains. We show that this protein can heterodimerize with human HNF1 in vitro. Sequence comparison of our clone with a rat variant HNF1 (vHNF1) clone, isolated in parallel in our laboratory from the dedifferentiated H5 hepatoma cell line, identified our cDNA as human vHNF1. vHNF1 is a nuclear protein recognizing the same binding site as HNF1 and previously thought to occur only in dedifferentiated hepatoma cells that fail to express most liver specific genes. Nevertheless, we show by Northern blot analysis that vHNF1 transcripts are present in differentiated human HepG2 hepatoma cells as well as in rat liver and that this transcript level is 10-20 fold lower than that of HNF1. We assigned the vHNF-1 gene to human chromosome 17 and murine chromosome 11. These chromosomal localizations differ from that of the HNF-1 gene indicating that both genes are not clustered on the genome.

Using a 796-basepair cDNA fragment obtained from a mouse pituitary library we have screened two mouse insulinoma libraries and isolated a full-length cDNA clone (2516 basepairs; 753 amino acids), designated mPC1. The cDNA sequence of mPC1 codes for a protein containing 753 amino acids and three potential N-glycosylation sites. This cDNA encodes a putative novel subtilisin-like proteinase, exhibiting within its presumed catalytic domain 64%, 55%, and 47% amino acid sequence identity to the recently characterized candidate prohormone convertases human Furin, mouse PC2, and yeast Kex2 gene products, respectively. An identical sequence to mPC1 was derived from a cDNA library of mouse corticotroph AtT-20 tumor cells. An ArgGlyAsp tripeptide identical to the recognition sequence of integrins was observed in the structures of the mammalian PC1, PC2, and Furin. In situ hybridization results demonstrated a distinct localization of the mPC1 and mPC2 transcripts in pituitary and brain. Thus, whereas both mPC1 and mPC2 are found in the intermediate lobe of the pituitary, only mPC1 is easily detected in the anterior lobe. In extrahypothalamic regions of the brain, including cortex, hippocampus, thalamus, and spinal cord, mPC2 transcripts predominate over mPC1. Both mRNAs are found in only a fraction of hypothalamic neurons, with greater abundance of mPC1 over mPC2 in the supraoptic nucleus. The genes coding for mPC1 and mPC2 map to the murine chromosomes 13 (band 13c) and 2 (2F3-2H2 region), respectively.

HNF1 is a transcription factor that is required for hepatocyte-specific expression of several genes, including albumin and fibrinogen. Rat HNF1-encoding cDNAs have recently been cloned, revealing that this factor is a distant member of the homeoprotein family. We have now isolated HNF1 clones from a human liver cDNA library by using a rat HNF1 cDNA-derived probe. The longest clone, HCL20, contains a sequence corresponding to the intact rat HNF1-coding region followed by a 3′ nontranslated region and a poly(A) tail, hence representing an almost full-length HNF1 cDNA. Alignment of the human and rat sequences shows that HNF1 is highly conserved between the two species. The HNF1 gene was mapped by in situ hybridization and by RFLP analysis of interspecific mouse backcrosses to chromosomes 12q24.3 and 5F in human and mouse, respectively, establishing a new segmental homology between these two chromosomes.

The inflammatory response to bacteria and bacterial products, such as lipopolysaccharides (LPSs), is mediated by a variety of secreted factors, but cytotoxic effects of LPS have been ascribed to the tumor necrosis factor alpha (TNF-α) activity. TNF-α is probably the most pleiotropic cytokine and, given the deleterious effects to the host of this factor, it has been postulated that its expression must be tightly regulated. Our laboratory has recently isolated, cloned and characterized a novel human transcription factor named LITAF or LPS-induced TNF-alpha factor. The present study reports the isolation, cloning and characterization of the mouse LITAF cDNA. Chromosomal localization revealed that mouse LITAF mapped to mouse chromosome 16, in a region highly homologous with the area on which human LITAF was previously located. Northern blot analysis shows that mouse LITAF is already expressed at embryonic day 7 of development, and is highly expressed in adult liver, heart and kidney. Moreover, upon LPS stimulation, we show that: (i) LITAF expression is increased in a mouse monocyte/macrophage cell line; and (ii) TNF-α expression is reduced in ES cell-derived macrophages lacking one copy of LITAF gene. Taken together, these results highlight the important role of LITAF in the regulation of TNF-α gene expression and suggest a potential role of LITAF in mouse organogenesis.

We have cloned the cDNA encoding the mouse DAD1 (defender against apoptotic cell death) protein. While showing an expected high homology with the previously cloned human and Xenopus DAD1-encoding cDNAs, this sequence has striking homology to partial cDNA sequences reported from O. sativa (rice) and C. elegans (nematode), suggesting the existence of plant and invertebrate homologs of this highly conserved gene. The human and mouse DAD1 genes map to chromosome 14q11–q12 and chromosome 14, respectively. This mapping data supports and extends the previously reported similarities between human chromosome 14q and mouse chromosome 14.

The BAX gene is a member of the Bcl-2 gene family; it encodes a 21-kDa protein whose association with Bcl-2 is believed to play a critical role in regulating apoptosis. Through analysis of human-hamster somatic cell hybrid DNA and by in situ hybridization to metaphase chromosomes, we have determined that the human BAX gene is located in the q13.3-q13.4 region of human chromosome 19. We have also isolated a BAX cDNA clone in which that part of the mRNA encoded by exon 3 is absent. The skipping of exon 3 and the resultant splicing of exons 2 and 4 maintains the original reading frame and predicts the existence of an interstitially truncated form of the major Bax protein (Bax alpha), termed Bax delta. Unlike two previously described variant forms of Bax alpha (Bax beta and Bax tau), Bax delta retains the functionally critical C-terminal membrane anchor region as well as the Bcl-2 homology 1 and 2 (BH1 and BH2) domains.

The two cDNA clones pHGCH1 and pHSR1, harboring partial coding sequences for GTPCH and SR, respectively, were tritium-labeled by nick-translation and individually used as probes for in situ hybridization o metaphase cells. Metaphase chromosome spreads, prepared from lymphocytes cultured in the presence of 5-bromo-deoxyuridine as described, were hybridized, autoradiographed, Giemsa-stained, and photographed. Subsequently, the metaphase cells were R-banded by fluorochrome-photolysis-Giemsa, and rephotographed. After hybridization and autoradiography, the distribution of silver grains of the previously photographed chromosome spreads was analyzed. In the 100 metaphase cells examined after in situ hybridization with pHCGCH1, 23.4% of the silver grains from a total of 196 grains were located on chromosome 14. The distribution of grains on this chromosome was not random; i.e., 76.1% of them mapped to the q21-q22 region of the long arm of chromosome 14, with a maximum in the q21 band.

DC-LAMP, a member of the lysosomal-associated membrane protein (LAMP) family, is specifically expressed by human dendritic cells (DC) upon activation and therefore serves as marker of human DC maturation. DC-LAMP is detected first in activated human DC within MHC class II molecules-containing compartments just before the translocation of MHC class II-peptide complexes to the cell surface, suggesting a possible involvement in this process. The present study describes the cloning and characterization of mouse DC-LAMP, whose predicted protein sequence is over 50% identical to the human counterpart. The mouse DC-LAMP gene spans over 25 kb and shares syntenic chromosomal localization (16B2-B4 and 3q26) and conserved organization with the human DC-LAMP gene. Analysis of mouse DC-LAMP mRNA and protein revealed the expression in lung peripheral cells, but also its unexpected absence from mouse lymphoid organs and from mouse DC activated either in vitro or in vivo. In conclusion, mouse DC-LAMP is not a marker of mature mouse DC and this observation raises new questions regarding the role of human DC-LAMP in human DC.

During mouse B-cell development, Pax5 is an essential transcription factor that acts as an activator of B-cell-specific genes and as a repressor of alternative lineage fates. The repressive function is mediated by the recruitment of members of the Groucho co-repressor family. Using an RNA display approach, we have isolated a transcript, called QD, specifically expressed in human pro-B and pre-B cells, which is derived from the human Groucho TLE4 gene. The QD transcript contains the first four TLE4 exons and an intronic sequence 3' of exon 4, demonstrating that QD is a splice variant of TLE4. The putative resulting protein of 94 amino acids corresponds to approximately half of an N-terminal tetramerization domain. We also show specific expression of TLE4 transcripts in human B cells and of TLE4 proteins in B-cell nuclei. Moreover, we demonstrate that recombinant QD protein binds to the TLE4 Q domain and is able to abolish the TLE4/Pax5 interaction. Thus, QD could act as a negative regulator of TLE4 function, in early B-cell differentiation.

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-β. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe244 to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.

Ryanodine receptors (RyRs) are intracellular Ca[sup 2+] release channels responsible for the release of Ca[sup 2+] from intracellular stores following transduction of many different extracellular stimuli. The genes of the two RyRs, originally described in the sarcoplasmic reticulum of skeletal (RYR1) or cardiac (RYR2) muscles, have been cloned and mapped on chromosome 19q13.1 and chromosome 1, respectively, in humans. The RYR1 gene has been shown to be tightly linked to the locus for malignant hyperthermia susceptibility (MHS). A single-point mutation in RYR1 has been identified as the possible cause of MHS in swine and of at least some forms of MHS in human. Another MHS locus has been mapped on human chromosome 17q11-q24. We have recently cloned a third RyR (RYR3) from mink, which appears to be widely expressed. A RyR with 94% homology to mink cDNA has also been isolated from rabbit brain.