Search

Nucleoporins are the main components of nuclear pore complexes (NPCs) involved in nucleo-cytoplasmic transport. Starting with an expressed DNA fragment retrieved by exon trapping from pooled human BAC clones mapped to the short arm of chromosome 5, we identified a human nucleoporin cDNA sequence by PCR from a human testis cDNA library. The coding sequence showed high homology to that of the rat nucleoporin 155 (Nup155) cDNA. FISH analysis with the human BAC clone as probe localized the human NUP155 gene to chromosome band 5p13. Northern analysis showed that the human NUP155 gene was expressed at different levels in all tissues tested. Two species of transcripts were observed with estimated lengths of 5.4 and 4.7 kb, respectively, in concordance with the finding of two alternative polyadenylation sites in the cDNA. The genomic location of the human NUP155 gene suggests a possible role in the mental and developmental retardation associated with hemizygous deletions of the 5p13 region.

The cDNA sequence of the human chondroadherin gene was cloned using PCR-based techniques. The gene encodes a protein of 359 amino acids, of which the first 21 amino acids represent a putative signal peptide sequence and which possesses 11 leucine-rich repeats flanked by cysteine-rich regions. The cDNA possesses a 5' untranslated region of 149 bp, a coding region of 1080 bp including the stop codon, and a 3' untranslated region of 561 bp terminating in a poly(A) tail. The cDNA hybridizes with a single messenger RNA of 1.9 kb, which is present in chondrocytes at all ages. Analysis of genomic DNA revealed that the chondroadherin gene possesses two introns, both of which reside within the coding region. The first intron has a length of about 2.3 kb and separates the codons for lysine(258) and phenylalanine(259). The second intron has a length of about 0.5 kb and splits the codon for tryptophan(314). This genomic organization results in exon 1 encoding the signal peptide, the amino-terminal cysteine-rich region, and the first 9 leucine-rich repeats; exon 2 encoding the last 2 leucine-rich repeats and part of the carboxy-terminal cysteine-rich region; and exon 3 encoding the remainder of the carboxy-terminal cysteine-rich region. The gene does not possess a TATA box prior to its transcription start site. Isolation of a cosmid clone spanning the chondroadherin gene enabled its chromosome location to be established. The gene was shown to reside at chromosome 17q21.33.

Neogenin was first identified in the chick embryo, and like a number of cell surface proteins of the immunoglobulin (Ig) superfamily, including N-CAM and L1 (generally called cell adhesion molecules or CAMs), it is expressed on growing nerve cells in the developing nervous system of vertebrate embryos. Neogenin is also expressed in other embryonic tissues, suggesting a more general role in developmental processes such as tissue growth regulation, cell-cell recognition, and cell migration. Neogenin, unlike the CAMs, is closely related to a unique tumor suppressor candidate molecule, deleted in colorectal carcinoma (DCC). Like DCC, the neogenin protein consists of four immunoglobulin-like (Ig-like) domains followed by six fibronectin type III domains, a transmembrane domain, and an intracellular domain. We now report the cloning and sequencing of cDNA clones coding for the human neogenin protein. Human neogenin shares 87% identity with its chicken homolog, and like its chicken counterpart it is expressed in at least two different isoforms derived from alternative splicing in the intracellular domain. Northern blot analysis revealed two mRNA species of about 5 and 7 kb. The chromosomal location of the human neogenin gene (HGMW-approved symbol NEO1) was determined as 15q22.3-q23, using fluorescence in situ hybridization. The gene therefore maps in the vicinity of a locus associated with Bardet-Biedl syndrome. The identification of human neogenin and its chromosomal location provides a basis for studying its involvement in genetic disorders or diseases.

Chromosome 21 is a model for the study of human chromosomal aneuploidy, and the construction of its physical and transcriptional maps is a necessary step in understanding the molecular basis of aneuploidy-dependent phenotypes. To identify the gene(s) responsible for Down syndrome congenital heart disease (DS-CHD), we constructed a physical map of the D21S55 to MX1 region. A bacterial artificial chromosome (BAC) library was screened using several YACs spanning the interval, and a P1-derived artificial chromosome (PAC) library was screened using radiolabeled STS PCR products and whole BACs in gap-filling initiatives. FISH confirmed the location of all BAC and PAC clones to 21q22.2-q22.3. Overlaps were established using clone-to-clone Southerns and 24 new STSs, generated from the direct sequencing of BAC and PAC ends, along with 35 preexisting STSs. Approximately 3.5 Mb of the 4- to 5-Mb D21S55 to MX1 interval is covered in 85 BACs and 24 PACs, representing fourfold coverage within the contigs. These BAC and PAC contigs are valuable reagents for isolating the genes for DS-CHD.

The gene corresponding to a 55-kDa matrix protein previously described in adult human articular cartilage was characterized by sequencing of genomic clones. The deduced protein sequence corresponds to the recently described matrix protein PRELP. The protein was encoded by messages of 1.7, 4.6, and 6.7 kb, whose relative abundance increased as their size decreased. The message heterogeneity appears to originate from variation in the length of the 3'-untranslated region, with the smallest message being contained within the reported sequence and the larger messages having extended 3'-untranslated regions. Two introns were identified within the genomic sequence encoding the smallest message. The first intron of about 6.7 kb resides 16 nucleotides prior to the translation initiation codon, and the second intron of about 2.6 kb resides 173 nucleotides prior to the translation termination codon. The gene, which encompasses at least 16 kb of genomic DNA, was shown to reside on chromosome 1q32. Primer extension techniques were used to establish that the coding sequence commences 199 bp downstream from the major transcription start site. Analysis of the DNA sequence upstream from the transcription start site reveals the presence of numerous potential transcription factor binding sites, but no CAAT or TATA box. At the message level, gene expression was at a high level in juvenile and adult cartilage, but not in the fetus or neonate. The presence of protein in the cartilage matrix was also much lower in the neonate than in the adult. In noncartilagenous tissues appreciable message levels were observed only in the adult lung.

The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Ig domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescence in situ hybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1-q31.2. This chromosomal locus has been previously identified as containing a tumor suppressor candidate gene commonly deleted in certain human cancer tissues.

We have created a resource for chromosome 22 consisting of 96 unique, well-characterized Fosmids. The Fosmid vector permits efficient cloning of DNA fragments averaging 40 kb in a single-copy vector based on the F factor of Escherichia coli. We have found that Fosmid clones from human chromosome 22 show remarkable stability and are useful for a wide variety of applications in genome analysis. These 96 clones have been localized by FISH, using high-resolution fluorescent banding and multicolor mapping techniques, and their position on the chromosome was correlated with their content of a number of common repeated sequence elements. We identified a subset of clones likely to contain genes by restriction analysis using the enzymes NotI, MluI, SacII, and BssHII. This collection of cytogenetically anchored clones, representing nearly 7% of the chromosome, is of immediate value for detecting chromosomal rearrangements, for use in gene isolation, and as a framework for physical mapping.

The gene encoding the tight junction (zonula occludens) protein, TJP1, was mapped to human chromosome 15q13 by fluorescence in situ hybridization (FISH) using a cDNA probe. The Jackson Laboratory backcross DNA panel derived from the cross (C57BL/6JEi X SPRET/Ei) F1 females X SPRET/Ei males was used to map the mouse Tjp1 to chromosome 7 near position 30 on the Chromosome Committee Map, a region with conserved homology to human chromosome 15q13. FISH studies on metaphases from patients with the Prader-Willi (PWS) or the Angelman syndrome (AS) showed that TJP1 maps close but distal to the PWS/AS chromosome region. 13 refs., 2 figs.

The genomic organization and nucleotide structure of the human cone photoreceptor cGMP phosphodiesterase alpha'-subunit (alpha'-PDE) gene (PDEA2) as well as its chromosomal localization have been determined. This gene, which spans about 48 kb, consists of 22 exons and codes for an 858-amino-acid protein. The alpha'-PDE gene maps to human chromosome 10q24. Its coding region shows about 90% nucleotide identity and 93% amino acid identity with the corresponding region of the bovine gene. The intron-exon organization of the genes encoding human cone alpha'-PDE and rod beta-PDE are very similar, suggesting that these proteins have a close phylogenetic relationship and probably a common origin.

We have constructed a human chromosome 2-specific bacterial artificial chromosome (BAC) library using DNA from the somatic cell hybrid GM10826. The average size of the clones is about 63 kb. The coverage and distribution of the library were estimated by screening with known polymorphic genetic markers and fluorescence in situ hybridization (FISH). Twenty-one markers tested positive when DNA pools prepared from approximately one-sixth of the library were screened with 33 known markers. This is consistent with the theoretical calculation of 63% coverage at one genomic equivalent. This suggested that the coverage of the library is approximately 5-6x. FISH analysis with 54 BACs revealed single site hybridization to chromosome 2, and the clones were distributed randomly on the chromosome. We have also performed direct sequencing of the BAC insert ends to generate sequence-tagged sites suitable for mapping and chromosome walking. This is the first reported human chromosome 2-specific BAC library and should provide a resource for physical mapping and disease searching for this chromosome.

A genomic clone encoding the Shaker-related potassium channel gene, Kcna4/mKv1.4, was isolated from mice. Its coding region is contained in a single exon, encodes a protein of 654 amino acids, and shares approximately 91% nucleotide sequence identity with human KCNA4/hKv1.4. We show that 0.8 kb of the 5' noncoding region (NCR), the entire protein coding region (approximately 2.0 kb), and all of the known 3' NCR (approximately 1.1 kb) are contained within a single exon; the remaining 0.5 kb of the 5' NCR is separated from this exon by a 3.4-kb intron. The sequenced genomic region thus accounts for essentially all of the longest known transcript (4.5 kb), although the precise ends of this transcript have not been defined. The 3' NCR contains several ATTTA and ATTTG motifs that are thought to destabilize mRNAs, and these are also present in rat, bovine, and human Kcna4/Kv1.4 cDNAs. It also contains three conserved polyadenylation signals, alternate utilization of which could generate mRNAs of differing stabilities. The 5' NCR of Kcna4/mKv1.4 may also serve to regulate channel expression. This region is approximately 85% identical to KCNA4/hKv1.4 and contains eight consensus translation start sites [(G, A)NNATG] that, based on the 5'-3' scanning model, would lead to a lowering of translational efficiency. The shortest Kcna4/Kv1.4 transcript (2.4 kb) can contain at most 400 bp of NCR and should lack the 3' ATTTAs and most of the 5' ATGs; this transcript might therefore exhibit increased stability and translational efficiency. The Kcna4/mKv1.4 channel exhibited biophysical and pharmacological properties indistinguishable from its rat and human homologues. Kcna4/mKv1.4 lies on mouse chromosome 2, near the Fshb locus, and in humans on the proximal half of chromosome 11p14 near human FSHB. Another K+ channel gene, Kcnc1/mKv3.1, lies approximately 1.8 cM from the Myod-1 gene on mouse chromosome 7, and in situ hybridization localizes KCNC1/hKv3.1 to the homologous region on human chromosome 11p14.3-p15.2. A third gene, KCNA1/hKv1.1, was mapped to human 12p13.

The B29 gene encodes a B-cell-specific membrane protein in the immunoglobulin antigen receptor complex. B29 is a crucial member of this receptor complex and is believed to function as an effector of signal transduction in a manner analogous to that of the CD3 components of the T cell antigen receptor. We have isolated a full-length human B29 cDNA clone by using a murine B29 cDNA probe. We show that there is an extremely high degree of evolutionary conservation between the human and mouse proteins, particularly in the transmembrane and intracytoplasmic regions, where the identity is 96%. In addition, the intracytoplasmic region in both proteins contains an identical peptide motif that is present in a number of molecules involved in lymphocyte activation. Genomic Southern blot analysis of human cell lines hybridized with both murine and human B29 cDNAs gives patterns consistent with a single-copy gene occupying a small region of the genomic sequence. Using human B29 cosmid DNA, we have localized the B29 gene to human chromosome 17q23 via fluorescence in situ hybridization. B29 is the first gene localized to this area of the genome. Interestingly, a subset of human B cell chronic lymphocytic leukemias (CLL) has translocations in this locus on chromosome 17.

For a better understanding of genes that potentially function in B lymphocyte cell signaling, we isolated the human genomic counterpart of the murine pp52 or LSP1 gene. We unexpectedly found that the human pp52 gene is one of four closely related loci. Representative cosmids from each of the four family members were isolated and chromosomally localized by fluorescence in situ hybridization. Nucleotide sequence was obtained from an exon common to each locus and demonstrated very close similarity among all four loci. Two of the four loci harbored dysfunctional frameshift mutations or premature translation stop sites. The exon of one locus was flanked by an 80-bp perfect inverted repeat, suggesting that it may have originated through a looped intermediate DNA structure. Through a series of cDNA hybridization studies and nucleotide sequence analyses we were able to unambiguously link the lymphocyte-expressed gene to the locus mapped to chromosome 11p15.5. This same chromosomal band has been involved in tumor-related chromosomal translocations found in chronic lymphocytic leukemia.

Two recent studies have suggested the involvement of serum amyloid A (SAA) and P (APCS) genes in familial Mediterranean fever (MEF). To test the role of SAA and APCS in MEF and MEF-amyloidosis, we studied 17 informative families (15 Armenians, 2 non-Ashkenazi Jews) and 8 MEF patients with amyloidosis using a candidate gene approach. No evidence for any MEF-associated polymorphism was found in any of the 41 Armenian and Jewish MEF patients tested. Our family studies allowed us to rule out tight linkage between SAA and MEF (lod score = -2.16, theta less than or equal to 0.06). For APCS we found that the allele frequency in the MEF-amyloidosis patients was similar to that in 18 unrelated MEF patients without amyloidosis and their 33 healthy parents. Finally, we excluded close genetic linkage between APCS and MEF at 8.5 cM or less (lod score = -2.2).

Oligodendrocyte-specific protein (OSP) is a recently described protein present only in myelin of the central nervous system. Several inherited disorders of myelin are caused by mutations in myelin genes but the etiology of many remain unknown. We mapped the location of the mouse OSP gene to the proximal region of chromosome 3 using two sets of multilocus crosses and to human chromosome 3 using somatic cell hybrids. Fine mapping with fluorescence in situ hybridization placed the OSP gene at human chromosome 3q26.2-q26.3. To date, there are no known inherited neurological disorders that localize to these regions.

This report describes the cloning of the 3{prime}-untranslated region of the human fibromodulin cDNA and its use to map the gene. For somatic cell hybrids, the generation of the PCR product was concordant with the presence of chromosome 1 and discordant with the presence of all other chromosomes, confirming that the fibromodulin gene is located within region q32 of chromosome 1. The physical mapping of genes is a critical step in the process of identifying which genes may be responsible for various inherited disorders. Specifically, the mapping of the fibromodulin gene now provides the information necessary to evaluate its potential role in genetic disorders of connective tissues. The analysis of previously reported diseases mapped to chromosome 1 reveals two genes located in the proximity of the fibromodulin locus. These are Usher syndrome type II, a recessive disorder characterized by hearing loss and retinitis pigmentosa, and Van der Woude syndrome, a dominant condition associated with abnormalities such as cleft lip and palate and hyperdontia. The genes for both of these disorders have been projected to be localized to 1q32 of a physical map that integrates available genetic linkage and physical data. However, it seems improbable that either of these disorders, exhibiting more » restricted tissue involvement, could be linked to the fibromodulin gene, given the wide tissue distribution of the encoded proteoglycan, although it remains possible that the relative importance of the quantity and function of the proteoglycan may avry between tissues. 11 refs., 1 fig. « less

The spinocerebellar ataxia type 2 (SCA2) gene has been localized to chromosome 12q24.1. To characterize this region and to aid in the identification of the SCA2 gene, we have constructed a 3.9-Mb physical map, which covers markers D12S1328 and D12S1329 known to flank the gene. The map comprises a contig of 84 overlapping yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) onto which we placed 82 PCR markers. We localized eight genes and expressed sequence tags on this map, many of which had not been precisely mapped before. In contrast to YACs, which showed a high degree of chimerism and deletions in this region, PACs and BACs were stable. Only 1 in 65 PACs contained a small deletion, and 2 in 18 BACs were chimeric. The high-resolution physical map, which was used in the identification of the SCA2 gene, will be useful for the positional cloning of other disease genes mapped to this region.

We have isolated and sequenced SUPT6H and Supt6h, the human and murine homologues of the Saccharomyces cerevisiae and Caenorhabditis elegans genes SPT6 (P using 1603 aa = 6.7 e-95) and emb-5 (P using 1603 aa = 7.0 e-288), respectively. The human and murine SPT6 homologues are virtually identical, as they share98% identity and99% similarity at the protein level. The derived amino acid sequences of these two genes predict a 1603-aa protein (human) and a 1726-bp protein (mouse), respectively. There were several known features, including a highly acidic 5'-region, a degenerate SH2 domain, and a leucine zipper. These features are consistent with a nuclear protein that regulates transcription, whose extreme conservation underscores the likely importance of this gene in mammalian development. Expression of human and murine SPT6 homologues was analyzed by Northern blotting, which revealed a 7. 0-kb transcript that was expressed constitutively. The SPT6 homologue was mapped to chromosome 17q11.2 in human by somatic cell hybrid analysis and in situ hybridization. These data indicate that SUPT6H and Supt6h are functionally analogous to SPT6 and emb-5 and may therefore regulate transcription through establishment or maintenance of chromatin structure.

Advances in the Human Genome Project are shaping the strategies for identifying the 50,000-100,000 human genes. High-resolution genetic maps of the human genome combined with sequencing herald an era of rapid regional definition of disease genes. However, only once their chromosome band location is known will the systematic partial sequencing of thousands of random cDNA clones provide the reagents for teh rapid assessment of the genes responsible for the inherited disorders. We now present an approach to the rapid determination of map position and therefore to the creation of a transcribed map of the human genome. Sensitive fluorescence in situ hybridization has been combined with high-resolution chromosome banding and random cDNA sequencing to map 41 cDNAs with an average insert size of

Mutation of the Saccharomyces cerevisiae RAD52 epistasis group gene, MRE11, blocks meiotic recombination, confers profound sensitivity to double-strand break damage, and has a hyperrecombinational phenotype in mitotic cells. We isolated a highly conserved human MRE11 homologue using a two-hybrid screen for DNA ligase I-interacting proteins. Human MRE11 shares approximately 50% identity with its yeast counterpart over the N-terminal half of the protein. MRE11 is expressed at the highest levels in proliferating tissues, but is also observed in other tissues. The MRE11 locus maps to human chromosome 11q21 in a region frequently associated with cancer-related chromosomal abnormalities. A MRE11-related locus was found on chromosome 7q11.2-q11.3.

Glycoprotein 330 (gp330) is a member of a family of receptors with structural similarities to the low-density lipoprotein receptor. Gp330 is expressed by a number of specialized epithelia, including renal proximal tubules, where it can mediate endocytosis of ligands such as complexes of urokinase and the serpin, plasminogen activator inhibitor-1. Gp330 has also been shown to bind in vitro to lipoprotein lipase and apolipoprotein E-enriched {beta}VLDL, suggesting a role for this receptor in lipoprotein metabolism. The 39-kDa protein, referred to as receptor associated protein (RAP), binds to and copurifies with gp330 and antagonizes the ligand binding activity of gp330. In this paper, the authors report the use of homology-PCR cloning to isolate cDNAs encoding human gp330. Using gp330 cDNA and previously isolated human RAP cDNA probes, they performed fluorescence in situ hybridization to map the human chromosomal location of the genes for these proteins. The gene for gp330 was mapped at a single site on the long arm of human chromosome 2 on the border of bands 2q24-q31. The gene for RAP was mapped to the short arm of human chromosome 4 at position 4q16.3, which is in the region of the chromosomal deletion causing Wolf-Hirschhorn syndrome. The assignment ofmore » chromosomal map positions for gp330 and RAP genes will aid in the evaluation of their potential roles in human diseases such as Wolf-Hirschhorn syndrome and disorders of lipoprotein metabolism, such as atherosclerosis. 38 refs., 3 figs., 1 tab.« less

The zinc finger element is a conserved motif among a group of proteins involved in binding to nucleic acid. This motif has been detected in many regulatory factors and is highly represented in the human genome. To investigate the presence of zinc-finger-encoding genes on human chromosome 21, chromosome-specific libraries were screened with an oligodeoxynucleotide probe representing the conserved H-C link region between adjacent fingers. Three distinct genomic clones, designated ZF21-1, ZF21-2, and ZF21-3, were isolated and mapped to the long arm of chromosome 21 as well as to the heterochromatic short arm of several other chromosomes. DNA sequence analysis has shown that these genomic clones contain multiple zinc finger elements of the Kruppel type with only partial similarity to other known zinc finger genes. However, in each clone, few fingers were degenerated; they contain inframe stop codons and frameshifts that would preclude their translation. It seems therefore, that these chromosome 21 zinc finger sequences are not parts of functional genes. Nevertheless, the possibility that these domains are transcribed, and thus might have a regulatory role, is considered.

The large aggregating proteoglycan aggrecan is a major structural component of the extracellular matrix of articular cartilage. Recent cDNA cloning of the human aggrecan gene (AGC1) reveals a core protein of at least 2316 amino acids characterized by several distinct structural domains. Two globular domains, termed G1 and G2, are present at the amino terminus of the molecule and a third, termed G3, is present at the carboxy terminus. The G1 domain is homologous in structure to the cartilage link protein and accounts for the aggregating potential of aggrecan through its ability to interact with hyaluronic acid. The aggrecan gene is known to consist of 15 exons, with each exon encoding a distinct functional region of the mature protein. However, while the link protein gene is known to reside on chromosome 5 in the human, the location of the aggrecan gene is currently undetermined in any species. The probe (pAGG2) for the aggrecan gene was mapped on chromosome band 15q26, most likely in the subregion of 15q26.1, using fluorescence in situ hybridization. Clear signals were noted on both chromatids of chromosome band 15q26 in over 80% of the 300 metaphase cells examined in three independent experiments using pAGG2. No othermore » sites of hybridization were noted on both chromatids of any other chromosome band. The precise band location was identified by using chromsomes of about 650 bands and employing fluorescence reverse banding with chromomycin A3 and distamycin. 14 refs., 1 fig.« less

The Alzheimer amyloid protein precursor (APP) has previously been localized to human chromosome 21q11.2–q21. We have refined the regional localization to a small region including chromosome bands 21q21.105–q21.05 by using quantitative Southern blot analysis of cell lines aneuploid for parts of chromosome 21. Our findings exclude the APP gene from the minimal region producing the classical phenotypic features of Down syndrome. This further narrowing of the APP location will help in identifying the physical location of the gene causing familial Alzheimer disease.