Isolation and chromosomal localization of the human glutathione peroxidase gene

We have isolated cDNA clones for the gene, termed GPX1, encoding the major human selenoprotein, glutathione peroxidase. Sequence analysis confirmed previous findings that the unusual amino acid seleno-cysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPX1 cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPX1 gene to a single site on chromosome 3, at region 3q11-13.1. Thus, three genomic sites bear sequence homology to the GPX1 cDNA, and the one most homologous maps to 3q11-13.1.     

Isolation and chromosomal localization of the human En-2 gene

By low stringency hybridization we have isolated from a human cosmid genomic library sequences homologous with a probe from the Drosophila engrailed gene. Partial nucleotide sequence analysis shows a consensus splice acceptor site followed by an open reading frame (ORF) that can encode 104 amino acids; the first 94 amino acids have 71% identity with the Drosophila engrailed protein. The shared region contains a homeo domain and is within the region of engrailed shared with the Drosophila invected gene and the mouse En-1 and En-2 genes. At the amino acid level, the human sequence is 85% identical with the mouse En-1 gene and 100% identical with the mouse En-2 gene. Hybridization against a panel of human-hamster somatic cell hybrids maps this human En-2 gene to chromosome 7, and regional mapping by in situ hybridization to human chromosomes localizes it to region 7q36 at the end of the long arm.     

Molecular cloning, structural characterization and chromosomal localization of human lipoyltransferase gene.

Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl-AMP to the lysine residue of the lipoate-dependent enzymes. We isolated human lipoyltransferase cDNA and genomic DNA. The cDNA insert contained a 1119-base pair open reading frame encoding a precursor peptide of 373 amino acids. Predicted amino acid sequence of the protein shares 88 and 31% identity with bovine lipoyltransferase and Escherichia coli lipoate-protein ligase A, respectively. Northern blot analyses of poly(A)+ RNA indicated a major species of about 1.5 kb. mRNA levels of lipoyltransferase were highest in skeletal muscle and heart, showing good correlation with those of dihydrolipoamide acyltransferase subunits of pyruvate, 2-oxoglutarate and branched-chain 2-oxo acid dehydrogenase complexes and H-protein of the glycine cleavage system which accept lipoic acid as a prosthetic group. The human lipoyltransferase gene is a single copy gene composed of four exons and three introns spanning approximately 8 kb of genomic DNA. Some alternatively spliced mRNA species were found by 5'-RACE analysis, and the most abundant species lacks the third exon. The human lipoyltransferase gene was localized to chromosome band 2q11.2 by fluorescence in situ hybridization.     

Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4.

We report here the full coding sequence of a novel mouse putative membrane-associated mucin containing three extracellular EGF-like motifs and a mucin-like domain consisting of at least 20 tandem repeats of 124-126 amino acids. Screening a cosmid and a BAC libraries allowed to isolate several genomic clones. Genomic and cDNA sequence comparisons showed that the gene consists of 25 exons and 24 introns covering a genomic region of approximately 52 kb. The first intron is approximately 16 kb in length and is followed by an unusually large exon (approximately 9.5 kb) encoding Ser/Thr-rich tandemly repeated sequences. Radiation hybrid mapping localized this new gene to a mouse region of chromosome 16, which is the orthologous region of human chromosome 3q29 encompassing the large membrane-anchored mucin MUC4. Contigs analysis of the Human Genome Project did not reveal any other mucin on chromosome 3q29 and, interestingly, our analysis allowed the determination of the genomic organization of the human MUC4 and showed that its exon/intron structure is identical to that of the mouse gene we cloned. Furthermore, the human MUC4 shares considerable homologies with the mouse gene. Based on these data, we concluded that we isolated the mouse ortholog of MUC4 we propose as Muc4. Expression studies showed that Muc4 is ubiquitous like SMC and MUC4, with highest levels of expression in trachea and intestinal tract.     

Isolation and chromosomal localization of a novel nonerythroid ankyrin gene.

Immunoreactive isoforms of erythrocyte ankyrin have been shown to be present in a variety of nonerythroid tissues. Isolation of the genes that encode these isoforms will clarify their relationship to erythrocyte ankyrin. Using an erythrocyte ankyrin cDNA clone as a hybridization probe, we screened a human genomic library and isolated a clone that hybridizes with the probe at low stringency but not at high stringency. Partial nucleotide sequence of the clone revealed the presence of a 99-bp segment that is homologous to an exon of the erythrocyte ankyrin gene. Northern analysis showed that a labeled fragment of the clone hybridized to a 7-kb message in RNA of fetal brain but not of erythroid cells, suggesting that this clone is part of a novel gene that is expressed predominantly in nonerythroid tissue. Comparison of the sequence of the genomic clone with that of a recently isolated cDNA clone for brain ankyrin (Otto et al., 1989) showed identity of 96 of 99 bp between the putative exon and a segment of the cDNA clone (V. Bennett, personal communication, 1991), suggesting that the genomic clone is part of a gene for nonerythroid ankyrin, which we have designated ANK2. By analysis of somatic cell hybrids and fluorescence in situ hybridization, we assigned ANK2 to human chromosome 4 at a position equivalent to bands 4q25-q27.     

Isolation, characterization, and chromosomal location of the mouse enamelysin gene

Mouse enamelysin (Mmp20), a member of the matrix metalloproteinase (MMP) family of extracellular matrix degrading enzymes, shows a high degree of homology with other MMPs, particularly those of the stromelysin/collagenase subfamilies. It is expressed exclusively in ameloblasts and odontoblasts. The mouse enamelysin gene (Mmp20) is made up of 10 exons spanning approximately 65 kb within the MMP gene cluster at the centromeric end of chromosome 9.     

Isolation, tissue distribution, and chromosomal localization of the human activation-induced cytidine deaminase (AID) gene

The gene encoding activation-induced cytidine deaminase (AID), a member of the cytidine deaminase family, was isolated from a murine B cell lymphoma line, CH12F3-2, induced by combined stimulation of TGF-beta, IL-4, and CD40L. We have isolated the human orthologue of mouse AID cDNA, which has an open reading frame of 198 residues containing a conserved cytidine deaminase motif. The amino acid sequence of human AID is 92% identical to that of mouse AID. RT-PCR analysis of 15 human tissues showed that AID mRNA is expressed strongly in lymph nodes and tonsils. The complete human AID gene consisting of five exons was isolated and mapped to chromosome 12p13 by fluorescence in situ hybridization.     

Isolation and characterization of a cDNA clone corresponding to bovine cellular retinoic-acid-binding protein and chromosomal localization of the corresponding human gene

A bovine adrenal cDNA library was constructed and a clone corresponding to cellular retinoic-acid-binding protein (CRABP) mRNA was isolated and sequenced. The insert of the clone corresponds to 75 bp of the 5' untranslated portion, the whole translated and the complete 3' untranslated portion of the bovine CRABP mRNA. A genomic Southern blot, probed with CRABP cDNA, indicated that only one copy of the gene is present in the human genome. Hybridizing bands in restricted chicken and fish DNA were also observed. Using the CRABP cDNA as probe we have located the human CRABP gene to chromosome 3 in hybridizations to mouse-human, hamster-human and rat-human cell hybrids. In situ hybridizations on rat testis cells probed with CRABP and cellular retinol-binding protein antisense mRNA indicate that both proteins are expressed in tubuli cells.     

Structural characterization and chromosomal localization of the MAGE-E1 gene

Genes of the melanoma-associated antigen (MAGE) family are characterized by the expression of tumor antigens on a malignant melanoma recognized by autologous cytolytic T lymphocytes. We have previously identified novel members of the MAGE gene family expressed in human glioma and named them MAGE-E1a-c. In the present study, we have revealed the genomic structure of MAGE-E1 by sequence analysis of a human chromosome bacterial artificial chromosome clone containing the MAGE-E1 gene. The MAGE-E1 gene is composed of 13 exons, and three of these (exon 2, exon 3 and exon 12) are alternatively spliced in each variant (E1a-c). The open reading frame encoding the MAGE-E1 peptides initiates in exon 2 and ends in exon 13. We have also demonstrated that the MAGE-E1 gene is located in Xp11 through the analysis of radiation hybrid panels. The genomic structure of MAGE-E1 is markedly similar to that of MAGE-D and its chromosomal locus is also identical to that of MAGE-D, but these features contrast with those of other MAGEs. These results suggest that MAGE-D and -E1 may be evolutionarily distant from other members of the MAGE family, and the two may be ancestral genes for the others.     

Structure and chromosomal localization of the human thrombospondin gene

Thrombospondin (THBS1) is a large modular glycoprotein component of the extracellular matrix and contains a variety of distinct domains, including three repeating subunits (types I, II, and III) that share homology to an assortment of other proteins. Determination of THBS1 gene structure has revealed that the type I repeat modules are encoded by symmetrical exons and that the heparin-binding domain is encoded by a single exon. To further elucidate the higher level organization of THBS1, the gene was localized to the q11-qter region of chromosome 15.     

Structure, expression and chromosomal localization of human p80-coilin gene.

Coiled bodies (CBs) are non-capsular nuclear bodies with a diameter of 0.3-1 micron and appear to be composed of coiled fibrils. Human autoantibodies to CBs recognize an 80-kD nuclear protein highly enriched in CBs, and this protein has been named p80-coilin. CBs are known to assemble and disassemble during the cell cycle, with the highest number of CBs occurring at mid to late G1 where p80-coilin is assembled into several small nuclear body-like structures. In S and G2 phases, CBs become larger and their number decreases and often they are undetectable during mitosis. Using a human autoantibody as a probe for expression cloning, we initially isolated a partial cDNA encoding p80-coilin. In this report, the 5' end of the complete cDNA for p80-coilin was obtained using the 5'-RACE (rapid amplification of cDNA ends) methodology. The size of the reconstructed full-length cDNA corresponds to the 2.7-kb mRNA detected in Northern blot analysis. The complete p80-coilin protein consists of 576 amino acids with a predicted molecular mass of 62,608. A putative p80-coilin pseudogene was also detected during the rescreening of p80-coilin cDNA. To confirm the validity of the cDNA sequence, three overlapping genomic DNA clones representing the human p80-coilin gene were selected for further analysis. The complete gene for p80-coilin contains 7 exons spanning approximately 25kb. Sequence analysis of exons 1 and 2 in genomic DNA clones confirmed the accuracy of the 5' cDNA sequence derived from the 5'-RACE procedure. Furthermore, the human p80-coilin gene was localized to chromosome 17q22-23 by fluorescence in situ hybridization.     

Isolation, characterization and chromosomal mapping of the mouse tyrosine aminotransferase gene

The tyrosine aminotransferase (TAT) gene is expressed in a tissue and developmental-specific manner. In addition, this gene is regulated by glucocorticoid and polypeptide hormones and its expression is affected when a regulatory region near the albino locus of the mouse is deleted. In order to allow studies of the molecular effects of these deletion mutations we have isolated and characterized the mouse TAT gene. The gene is 9.2 x 10(3) bases in length and consists of 12 exons which give rise to a 2.3 x 10(3) base long messenger RNA. The DNA sequence at the 5' end of the gene was determined and compared with the corresponding sequence of the rat tyrosine aminotransferase gene. The sequence comparison showed extensive homology over the entire region sequenced. In addition, DNA: DNA heteroduplex studies between the mouse and rat tyrosine aminotransferase genes revealed that this homology extends over the entire gene and its flanking sequences. The mouse tyrosine aminotransferase gene has been mapped distal to the serum esterase-1 locus on mouse chromosome 8, using a restriction fragment length polymorphism between two mouse species. Since the albino deletions are located on mouse chromosome 7, the assignment of the TAT gene to chromosome 8 suggests that a regulatory factor(s) affecting TAT gene expression acts in trans.     

Isolation, chromosomal localization, and nucleotide sequence of the human HOX 1.4 homeobox

We have isolated a 14-kb DNA sequence containing a single homeobox from a low-stringency screen of a human genomic phage library by using heterologous homeobox sequences as probes. Chromosomal mapping of this clone using in situ hybridization to metaphase chromosomes and a panel of mouse x human somatic cell hybrids localized it to human chromosome 7p13-p15 in the region of the HOX 1 locus. We have sequenced the homeobox and show it has 100% identity to the deduced amino acid sequence of the mouse Hox-1.4 homeobox. We detect no restriction fragment length polymorphisms with the 14-kb clone, which is devoid of any moderately repetitive DNA sequences. This implies an inability of this region to tolerate change in sequence, consistent with a function highly conserved throughout evolution. The regions in the human genome where homeobox-containing loci reside share patterns of organization and sequence and have other gene loci in common, implying evolutionary constraints over these regions and providing clues on how they may have evolved.     

Placental expression and chromosomal localization of the human Gcm 1 gene.

Although gcm was first recognized for its role in specifying glial cell fate in Drosophila melanogaster, its mammalian counterparts are expressed predominantly in non-neural tissues. Here we demonstrate expression of the mouse and human GCM 1 proteins in placenta. We have prepared a highly specific antibody that recognizes the GCM 1 protein and have used it to assess the temporal and spatial expression profile of the protein. In both mouse and human placenta, the protein is associated with cells that are involved with exchange between maternal and fetal blood supplies: the labyrinthine cells of the mouse placenta and the syncytio- and cytotrophoblasts of the human placenta. Using the full-length hGcm 1 cDNA as a probe, we have mapped the gene on human chromosome 6p12 by fluorescent in situ hybridization.     

Genomic organization and chromosomal localization of the human CD27 gene.

CD27 is a lymphocyte-specific member of a recently identified receptor family with at least 10 members that includes the receptors for nerve growth factor and TNF, CD40, and Fas. Several members of this family play a role in cell differentiation, proliferation, and survival. Within the amino terminal ligand binding domain of these receptors, repeat motifs have been identified. These repeats contain many cysteine residues in a conserved pattern, characteristic of this family. We have isolated and characterized the human CD27 gene to gain insight into the evolution of this type of receptor domain. The gene was localized on chromosome 12, band 12p13. Sequence analysis showed no correlation between the intron/exon organization and the subdivision of the protein into distinct domains. Structural information for the cysteine-rich domain is contained within three exons. In addition, the splice sites in the CD27 gene are located in a different position from those in the related nerve growth factor receptor gene. However, a comparison of the splice sites within the regions encoding the respective ligand-binding domains of the CD27 and nerve growth factor receptor genes identifies the archetypal cysteine-rich building blocks, from which the members of this family may have arisen during the course of evolution. From this observation, we propose a new organization of the repeat motifs.