Characterization of the human common fragile site FRA2G

Common fragile sites are nonrandom loci that show gaps and breaks when cells are exposed to specific compounds. They are preferentially involved in recombination, chromosomal rearrangements, and foreign DNA integration. These sites have been suggested to play a role in chromosome instability observed in cancer. In this work we used a FISH-based approach to identify a BAC contig that spans the FRA2G fragile site located at the 2q31 region. Our observations indicate that a very fragile region spanning at least 450 kb is present within a large fragile region that extends over 1 Mb. At least seven genes are mapped in the fragile region. One of these seems to be a good candidate as a potential tumor suppressor gene impaired by the recurrent deletions observed at the 2q31 region in some neoplasms. In the fragile region, a considerable number of regions of high flexibility that may be related to the fragility are present.     

The human mineralocorticoid receptor gene (MLR) is located on chromosome 4 at q31.2

The gene for the human mineralocorticoid receptor (MLR) was previously localized to chromosome 4. Here, we have localized this gene to 4q31.2 by in situ hybridization. This precise mapping of MLR will assist in the linkage analysis and genetic characterization of pseudohypoaldosteronism, an autosomal recessive disorder which likely results from a defect in the MLR gene.     

Identification of the human cortactin-binding protein-2 gene from the autism candidate region at 7q31.

Human chromosome 7q31 contains putative susceptibility loci for autism (AUTS1) and speech and language disorder (SPCH1). We report here the identification and characterization of a novel gene encoding cortactin-binding protein-2 (CORTBP2), which is located 45 kb telomeric to the cystic fibrosis transmembrane conductance regulator gene (CFTR) at 7q31.3. The full-length (5975-bp) gene was isolated and found to be composed of 23 exons encompassing 170 kb of DNA. In addition to being a positional candidate for AUTS1, CORTBP2 was expressed at highest levels in the brain, as shown by northern blot analysis. Subsequent mutation analysis of CORTBP2 in 90 autistic patients identified two polymorphisms, including a leucine to valine change caused by a T to G substitution in exon 15. However, comparison of allele frequencies between autistic and control populations (n=96) showed no significant difference, suggesting that this variant is not a susceptibility factor for autism.     

Cytogenetic mapping of five YAC clones to human chromosome region 2q31 --> q32.1 in relation to the FRA2G common fragile site

In this work, five YAC clones have been mapped by fluorescent in situ hybridization (FISH) to human chromosome region 2q31 q32.1 and ordered in relation to each other and to the FRA2G common fragile site. YAC clones that span the fragile site have been identified. Moreover a deleted HOXD 13 gene has been identified on the 942D2 YAC.     

The human aldose reductase gene maps to chromosome region 7q35

Summary The human aldose reductase (AR) gene has been mapped to chromosome 7 using the polymerase chain reaction to specifically amplify the human AR sequence in hamster/human hybrid DNA and also in mouse/ human monochromosome hybrids. The assignment to chromosome 7 was confirmed by in situ hybridisation to human metaphase chromosomes using a novel, rapid hybridisation, method giving a regional localisation at 7q35.     

Chromosomal localization of a human band 3-like gene to region 7q35----7q36.

Band 3, the major transmembrane protein of erythrocytes, mediates the exchange of anions across the membrane and anchors the erythroid membrane skeleton. Proteins immunologically related to Band 3 have been detected in a variety of nonerythroid cells. We have isolated a human cDNA clone that encodes a protein related to but distinct from the erythroid form of Band 3, based on the comparison of the amino acid sequence for the two proteins. The presence of the gene for the Band 3-like protein in a panel of mouse-human somatic cell hybrids containing subsets of human chromosomes correlated with the presence of human chromosome 7. In situ hybridization analysis using the c-DNA for this nonerythroid Band 3 gene further localized the gene to region 7q35----7q36 of human metaphase chromosomes.     

Molecular cloning of human lysyl oxidase and assignment of the gene to chromosome 5q23.3-31.2.

Lysyl oxidase (EC 1.4.3.13) initiates the crosslinking of collagens and elastin by catalyzing oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues. We report here on the isolation and characterization of cDNA clones for the enzyme from human placenta and rat aorta lambda gt11 cDNA libraries. A cDNA clone for human lysyl oxidase covers all the coding sequences, 230 nucleotides of the 5' and 299 nucleotides, of the 3' untranslated sequences, including a poly(A) tail of 23 nucleotides. This cDNA encodes a polypeptide of 417 amino acid residues, including a signal peptide of 21 amino acids. Sequencing of two rat lysyl oxidase cDNA clones indicated six differences between the present and the previously published sequence for the rat enzyme [Trackman et al. (1990) Biochemistry 29: 4863-4870], resulting in frameshifts in the translated sequence. The human lysyl oxidase sequence was found to be 78% identical to the revised rat sequence at the nucleotide level and 84% identical at the amino acid level, with the degree of identity unevenly distributed between various regions of the coded polypeptide. Northern blot analysis of human skin fibroblasts RNA indicated that the human lysyl oxidase cDNA hybridizes to at least four mRNA species; their sizes are about 5.5, 4.3, 2.4, and 2.0 kb. Analysis of a panel of 25 human x hamster cell hybrids by Southern blotting mapped the human lysyl oxidase gene to chromosome 5, and in situ hybridization mapped it to 5q23.3-31.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of human cytoglobin gene promoter region

Cytoglobin (CYGB) is a member of the vertebrate globin family together with hemoglobin, myoglobin and neuroglobin. Although the physiological function of CYGB is still unclear, spectroscopic studies show that CYGB contains a hexacoordinated heme pocket similar to other pentacoordinated globin proteins. CYGB shares a common phylogenetic ancestry with vertebrate myoglobin from which it diverged by duplication before the appearance of jawed vertebrates. The objective of this study is to identify the regulatory and promoter region of the human cytoglobin gene. 5' unidirectional deletion constructs demonstrated that the proximal promoter elements of human CYGB gene are located between -1113 to -10 relative to the translation start site. Site-directed mutagenesis showed that mutation of a c-Ets-1 motif at -1008 and Sp1 motifs at -400, -230 and -210 remarkably decreased the promoter activity. Gel shift assays confirmed the binding of DNA-nuclear proteins to these motifs. All these results indicate that CYGB gene expression can be up-regulated by c-Ets-1 and Sp1 motifs.     

Characterization of five novel human genes in the 11q13-q22 region

The redundancy of sequences in dbEST has approached a level where contiguous cDNA sequences of genes can be assembled, without the need to physically handle the clones from which the ESTs are derived. This is termed EST based in silico gene cloning. With the availability of sequence chromatogram files for a subset of ESTs, the quality of EST sequences can be ascertained accurately and used in contig assembly. In this report, we performed a study using this approach and isolated five novel human genes, C11orf1-C11orf5, in the 11q13-q22 region. The full open reading frames of these genes were determined by comparison with their orthologs, of which four mouse orthologs were isolated (c11orf1, c11orf2, c11orf3 and c11orf5). These genes were then analyzed using several proteomics tools. Both C11orf1 and C11orf2 are nuclear proteins with no other distinguishing features. C11orf3 is a cytoplasmic protein containing an ATP/GTP binding site, a signal peptide located in the N-terminus and a similarity to the C. elegans protein "Probable ARP 2/3 complex 20kD subunit." C11orf4 is a peptide which displays four putative transmembrane domains and is predicted to have a cytoplasmic localization. It contains signal peptides at the N- and C-termini. C11orf5 is a putative nuclear protein displaying a central coiled coil domain. Here, we propose that this purely EST-based cloning approach can be used by modestly sized laboratories to rapidly and accurately characterize and map a significant number of human genes without the need of further sequencing.     

Pro-melanin-concentrating hormone gene (PMCH) is localized on human chromosome 12q and rat chromosome 7

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that may be involved in regulation of the stress response and food intake behavior in mammals. MCH and two other putative neuropeptides, NEI and NGE, are encoded by the same precursor, designated pro-melanin-concentrating hormone (PMCH). A panel of somatic cell hybrids segregating either human or rat chromosomes was used to determine the chromosomal localization of the PMCH locus. It was assigned to human chromosome 12q and to rat chromosome 7. This is the first neuropeptide-encoding gene found in this new synteny group conserved in rat and human.     

Replication timing of two human common fragile sites: FRA1H and FRA2G

The mammalian chromosomes present specific sites of gaps or breaks, the common fragile sites (CFSs), when the cells are exposed to DNA replication stress or to some DNA binding compounds. CFSs span hundreds or thousands of kilobases. The analysis of these sequences has not definitively clarified the causes of their fragility. There is considerable evidence that CFSs are regions of late or slowed replication in the presence of sequence elements that have the propensity to form secondary structures, and that the cytogenetic expression of CFSs may be due to unreplicated DNA. In order to analyse the relationship between DNA replication time and fragility, in this work we have investigated the timing of replication of sequences mapping within two CFSs (FRA1H and FRA2G), of syntenic non-fragile sequences and of early and late replicating control sequences by using fluorescent in situ hybridization on interphase nuclei, conventional fluorescence microscopy and confocal microscopy. Our results indicate that the fragile sequences are slow replicating and that they enter G2 phase unreplicated with very high frequency. Thus these regions could sometimes reach mitosis unreplicated or undercondensed and be expressed as chromosome gaps/breakages.     

The syntenic relationship of proximal mouse chromosome 7 and the myotonic dystrophy gene region on human chromosome 19q

The syntenic relationship of the myotonic dystrophy (DM) gene region on human chromosome 19q and proximal mouse chromosome 7 was examined using an interspecific backcross between C3H/HeJ-gld/gld mice and Mus spretus. Segregation analyses were used to order homologs of nine human loci linked with the DM gene. Their order from the centromere was Prkcg, [Apoe, Atpa-2, Ckmm, D19S19h, Ercc-2], Cyp2b, Mag, Lhb. Two other murine loci, D7Rp2 and Ngfg, were also positioned within this interval. Homologs for five human chromosome 11 and 15 loci (Calc, Fes, Hras-1, Igflr, Tyr) were localized within an 18-cM span telomeric to Lhb. Comparison of the gene orders indicates an inversion extending from Prkcg through the interval between Mag and Lhb. This study establishes a detailed map of proximal mouse chromosome 7 that will be useful in identifying and determining whether new human chromosome 19 probes are linked to the DM region.     

Owl monkey gene map: evidence for a homologous human chromosome 7q region near the cystic fibrosis locus

We have demonstrated the assignments of two gene loci (COLIA2, MET) and two noncoding DNA markers (D7S13, D7S8) to owl monkey chromosome 14 (K-VI) by hybridizing DNA probes from the cystic fibrosis (CF) region of human chromosome 7q21-32 to panels of rodent-owl monkey somatic cell hybrids. The assignments are substantiated by in situ chromosome hybridization of markers COLIA2, MET, and D7S13 to the distal long arm of chromosome 14 (K-VI). These results support genomic conservation of the human CF region, at least in the higher primates.     

A major imprinted gene involved in hydatidiform mole is not located in 2q31.2-qter or 5q34-qter

Introduction

Hydatidiform mole is an abnormal human pregnancy, characterised by absent or abnormal embryonic differentiation, vesicular chorionic villi and trophoblastic hyperplasia. Although the mole phenotype has hereto not been correlated to mutations in the molar genome, the aetiology for hydatidiform moles clearly is genetic: Most molar genomes analysed either have had a relative excess of paternal genome sets relative to maternal genome sets, or a global error in maternally imprinted genes, giving them a “paternal pattern”. However it remains yet to be specified which gene(s) in the molar genome actually causes the molar phenotype when present in a state of “paternal excess” or “maternal deficiency”.

Material and methods

A molar pregnancy in a woman with a balanced translocation (t(2;5) was subjected to histopathological evaluation and genetic analyses of ploidy and parental origin of the genome.

Results

Morphology: Partial hydatidiform mole. Karyotyping of metaphase chromosomes: 69,XXY,der(5)t(2;5)(q23;q33)mat. SNP array analysis mapped the breakpoints to 2q31.2 (genome position 179 Mb) and 5q34 (genome position 165 Mb). DNA microsatellite marker analysis showed that for the regions not involved in the translocation, the conceptus had two paternal and one maternal allele(s). Telomeric to the breakpoint on chromosome 2, the mole had two paternal and two maternal alleles and telomeric to the breakpoint on chromosome 5 the mole had paternal alleles, exclusively.

Conclusions

If the molar phenotype is caused by paternal excess of one gene, only, it is unlikely that this gene is located telomeric to genome position 179 Mb on chromosome 2. And similarly, if the phenotype complete mole is caused by the presence of exclusively paternally imprinted alleles of one gene, this gene is not located telomeric to genome position 165 Mb on chromosome 5.     

Assignment of the human calpastatin gene (CAST) to chromosome 5 at region q14----q22

The human calpastatin gene (CAST) was assigned to chromosome 5 by spot-blot hybridization analysis with flow-sorted chromosomes, and it was further sublocalized to bands 5q14----q22 using in situ hybridization to metaphase chromosomes.     

Assignment of the human phosphoserine phosphatase gene (PSP) to the pter leads to q22 region of chromosome 7

Phosphoserine phosphatase (PSP) catalyzes the hydrolysis of phosphoserine to serine. PSP expression has been examined in human-mouse somatic cell hybrids retaining different combination of human chromosomes. Human PSP is expressed only when the pter leads to q22 segment of the human 7 and its enzyme marker beta-glucuronidase (GUSB) are retained in cell hybrids. The structural gene, PSP, is therefore assigned to this region of the human 7.     

Fine mapping of the human pentraxin gene region on chromosome 1q23

 The 1q21 to 25 region of human chromosome 1 contains genes which encode proteins with immune- and inflammation-associated functions. These include the pentraxin genes, for C-reactive protein (CRP), serum amyloid P (SAP) protein (APCS), and a CRP pseudogene (CRPP1). The region of chromosome 1 containing this cluster is syntenic with distal mouse chromosome 1. We constructed an approximately 1.4 megabase yeast artificial chromosome (YAC) contig with the pentraxin genes at its core. This four-YAC contig includes other genes with immune functions including the FCER1A gene, which encodes the α-subunit of the IgE high-affinity Fc receptor and the IFI-16 gene, an interferon-γ-induced gene. In addition, it contains the histone H3F2 and H4F2 genes and the gene for erythroid α-spectrin (SPTA1). The gene order is cen.-SPTA1-H4F2-H3F2-IFI-16-CRP-CRPP1-APCS-FCER1A- tel. The contig thus consists of a cluster of genes whose products either have immunological importance, bind DNA, or both. Received: 13 December 1995 / 6 February 1996     

Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q

The localization to 19q of the gene causing myotonic dystrophy (DM) has been defined more precisely by refinement of the physical location of several linked markers. A somatic cell hybrid mapping panel from cells with t(1;19), t(12;19), and t(X;19) translocation products was constructed to define five different intervals across 19q. In addition, we have derived a series of cell hybrids by irradiation of a der(19)-only hybrid to further subdivide the cen-q13.1 region. Using an array of 36 cloned genes, anonymous DNAs, and enzyme markers, we have tested the location of the panel breakpoints and refined the regional assignment of several of these markers. All markers tightly linked to DM are localized mainly within 19q13.2, thus suggesting that the DM gene is also close to this region.