Chromosome 7 long arm deletion breakpoints in preleukemia: mapping by pulsed field gel electrophoresis.

Chromosome 7 long arm deletions (7q-) are recurring chromosome abnormalities in leukemic bone marrow cells. In four patients we have previously localized the breakpoints in band 7q22 between the erythropoietin (EPO) and plasminogen activator inhibitor type 1 (PLANH1) genes that map 3 cM apart. The pro alpha 2(I) collagen (COL1A2, in band 7q22) and T cell receptor beta chain genes (TCRB, in band 7q35) have been found undeleted in one patient with an interstitial deletion. Pulsed field gel electrophoresis was used to map the breakpoints more accurately in two patients with a 7q- chromosome. The results suggested that lymphocytes and granulocytes give identical restriction patterns with several enzyme-probe combinations, and that a breakpoint possibly was within 195 kb of EPO in one patient but not in another. The gene order cen-COL1A2-EPO-breakpoint-tel was suggested but physical linkage between COL1A2 and EPO was not found. A new putative TCRB restriction fragment length polymorphism or inherited methylation site was detected.     

Physical mapping by PFGE localizes the COL3A1 and COL5A2 genes to a 35-kb region on human chromosome 2

The genes encoding the alpha 1 chain of Type III collagen (COL3A1) and the alpha 2 chain of Type V (COL5A2) collagen have been mapped to the long arm of human chromosome 2. Linkage analysis in CEPH families indicated that these two genes are close to each other, with no recombination in 37 informative meioses. In the present study, DNA probes from the 3' ends of each gene have been physically mapped by pulsed-field gel electrophoresis. The probes recognized 11 macrorestriction fragments in common, ranging from greater than 1000 kb MluI and NotI fragments to a 35-kb SfiI fragment. Therefore, the COL3A1 and COL5A2 genes appear to exist as a gene cluster on chromosome 2. This is the third example of a collagen gene cluster. Other examples include the COL4A1-COL4A2 genes on chromosome 13q and the COL6A1-COL6A2 genes on chromosome 21q. The physical proximity of these genes may indicate common evolution and/or regulation.     

Macrorestriction mapping of COL4A1 and COL4A2 collagen genes on human chromosome 13q34

The genes for the alpha-1 and alpha-2 chains of type IV collagen (COL4A1 and COL4A2) map to the same chromosomal band (13q34) and have a high degree of nucleotide homology. We have used pulsed field gel electrophoresis and cloned COL4A1 and COL4A2 DNA fragments as molecular probes to construct a 1200-kb macrorestriction map which encompasses both genes. The two genes are located within a 340-kb region with the 3' end of COL4A2 and the 5' region of COL4A1 separated by at least 100 kb but not more than 160 kb. These genes, therefore, are two members of a gene cluster on chromosome 13q34.     

Fine structure physical mapping of the region of mouse chromosome 10 homologous to human chromosome 21.

Comparative mapping of human and mouse DNA for regions of genetic homology between human Chromosome 21 and the mouse genome is of interest because of the possibility of developing mouse models of human trisomy 21 (Down syndrome), understanding chromosome evolution, and isolating novel sequences conserved between the two species. At least two mouse chromosomes are known to carry sequences homologous to those on human Chromosome 21: mouse Chromosome 16 (D21S16h, D21S13h, D21S52h, App, Sod-1, Mx-1, Ets-2, Prgs,Ifnar) and mouse Chromosome 17 (D21S56h, Crya-1, and Cbs). Recently, five additional genes have been mapped within region 21q22 of human Chromosome 21:PFKL, CD18, COL6A1, COL6A2, and S100B. To assign these sequences to specific mouse chromosomes, we used human cDNA probes for COL6A1, COL6A2, CD18, and PFKL and a rat brain cDNA probe for S100B in conjunction with a panel of seven Chinese hamster-mouse somatic cell hybrids segregating mouse chromosomes. The specific chromosome complements of the hybrid cell lines and the presence or absence of hybridizing mouse sequences in their DNAs allow us to assign all five sequences to mouse Chromosome 10, with the assignment of Pfkl reported here for the first time. Analysis of genomic mouse DNA fragments produced by digestion with rare-cutting restriction enzymes and separated using pulsed-field gel electrophoresis allows us to construct a fine-structure physical map of two segments of the region of Chromosome 10 containing these five markers. The five loci span at least 1900 kb of mouse DNA and are consistent with the human order: Pfkl-Cd-18-Col6a-1-Col6a-2-S100b.(ABSTRACT TRUNCATED AT 250 WORDS)     

Consistent linkage of dominantly inherited osteogenesis imperfecta to the type I collagen loci: COL1A1 and COL1A2

The segregation of COL1A1 and COL1A2, the two genes which encode the chains of type I collagen, was analyzed in 38 dominant osteogenesis imperfecta (OI) pedigrees by using polymorphic markers within or close to the genes. This was done in order to estimate the consistency of linkage of OI genes to these two loci. None of the 38 pedigrees showed evidence of recombination between the OI gene and both collagen loci, suggesting that the frequency of unlinked loci in the population must be low. From these results, approximate 95% confidence limits for the proportion of families linked to the type I collagen genes can be set between .91 and 1.00. This is high enough to base prenatal diagnosis of dominantly inherited OI on linkage to these genes even in families which are too small for the linkage to be independently confirmed to high levels of significance. When phenotypic features were compared with the concordant collagen locus, all eight pedigrees with Sillence OI type IV segregated with COL1A2. On the other hand, Sillence OI type I segregated with both COL1A1 (17 pedigrees) and COL1A2 (7 pedigrees). The concordant locus was uncertain in the remaining six OI type I pedigrees. Of several other features, the presence or absence of presenile hearing loss was the best predictor of the mutant locus in OI type I families, with 13 of the 17 COL1A1 segregants and none of the 7 COL1A2 segregants showing this feature.     

Localization of CYP2F1 by multipoint linkage analysis and pulsed-field gel electrophoresis

CYP2F1, which encodes a P450 enzyme capable of metabolizing several mono-oxygenase substrates with the highest activity toward ethoxycoumarin, has been mapped to human chromosome 19 by somatic cell hybrid studies. The CYP2A and CYP2B subfamilies are known to lie within 350 kb of each other on chromosome 19. To determine the locations of CYP2F1 with respect to CYP2A and CYP2B, multipoint linkage analysis and pulsed-field gel electrophoresis were performed. No recombinants were found between CYP2F1 and CYP2B in more than 50 meioses, and one recombinant was found between CYP2F1 and CYP2A (50 meioses). Pulsed-field gel electrophoresis showed the three loci to lie within a 240-kb region. Multipoint analysis of the haplotyped CYP cluster with four other chromosome 19 loci yielded the order D19S7-D19S9-APOC2-D19S8-CYP, although four other orders could not be excluded. The odds were 4.4 x 10(3) against the order proposed by the HGM10 consortium, D19S7-D19S9-CYP-D19S8-APOC2.     

Sequence and detailed organization of the human caveolin-1 and -2 genes located near the D7S522 locus (7q31.1). Methylation of a CpG island in the 5' promoter region of the caveolin-1 gene in human breast cancer cell lines

The CA microsatellite repeat marker, D7S522, is located at the center of a approximately 1000 kb smallest common deleted region that is lost in many forms of human cancer. It has been proposed that a putative tumor suppressor gene lies in close proximity to D7S522, within this smallest common deleted region. However, the genes located in proximity to D7S522 have remained elusive. Recently, we identified five independent BAC clones (approximately 100-200 kb) containing D7S522 and the human genes encoding caveolins 1 and 2. Here, we present the detailed organization of the caveolin locus and its relationship to D7S522, as deduced using a shot-gun sequencing approach. We derived two adjacent contigs for a total coverage of approximately 250 kb. Analysis of these contigs reveals that D7S522 is located approximately 67 kb upstream of the caveolin-2 gene and that the caveolin-2 gene is located approximately 19 kb upstream of the caveolin-1 gene, providing for the first time a detailed genetic map of this region. Further sequence analysis reveals many interesting features of the caveolin genes; these include the intron-exon boundaries and several previously unrecognized CA repeats that lie within or in close proximity to the caveolin genes. The first and second exons of both caveolin genes are embedded within CpG islands. These results suggest that regulation of caveolin gene expression may be controlled, in part, by methylation of these CpG regions. In support of this notion, we show here that the CGs in the 5' promoter region of the caveolin-1 gene are functionally methylated in two human breast cancer cell lines (MCF7 and T-47D) that fail to express the caveolin-1 protein. In contrast, the same CGs in cultured normal human mammary epithelial cells (NHMECs) are non-methylated and these cells express high levels of the caveolin-1 protein. Comparison of the human locus with the same locus in the pufferfish Fugu rubripes reveals that the overall organization of the caveolin-1/-2 locus is conserved from pufferfish to man. In conclusion, our current studies provide a systematic basis for diagnostically evaluating the potential deletion, mutation, or methylation of the caveolin genes in a variety of human tumors.     

Subregional mapping of 13 single-copy genes on the long arm of chromosome 12 by fluorescence in situ hybridization.

Subregional localization of 13 single-copy DNA sequences previously assigned to the long arm of chromosome 12 has been performed using the fluorescence in situ hybridization (FISH) technique. The following order is suggested for the 13 mapped genes: cen-->COL2A1-->(VDR-D12S15)-->(D12S17-D12S4++ +-D12S14-D12S6)-->D12S8-->(IAPP-MGF- D12S7-D12S12)-->IGF1-->qter. Eight of the mapped genes clustered at two regions, one at 12q13 (D12S17-D12S4-D12S14-D12S6) and the other at 12q22 (IAPP-MGF-D12S7-D12S12). Our results show that single-copy DNA sequences as small as 500 bp can be successfully mapped by FISH.     

Partial physical map of human chromosome 21 from fibroblast and lymphocyte DNA

A partial physical map of the human chromosome 21 including 26 genes and anonymous sequences was established by pulsed-field gel electrophoresis analysis of restriction fragments obtained from lymphocyte and fibroblast DNAs. The sizes of the restriction fragments obtained by total digestion with eight different enzymes were compared in these two tissues. Differences resulting from the variations in the methylation state of the restriction sites were frequently observed. These differences and partial digestions were used to estimate the order and the distances between genes and sequences. Six linkage groups were defined: D21S13-D21S16, D21S1-D21S11, D21S65-D21S17, (D21S55,ERG)-ETS2, BCEI-D21S19-D21S42-D21S113-CBS-CRYA1, and COL6A2-S100B. For six intergenic distances the resolution of previous maps was significantly increased.     

Genomic organization and chromosomal localization of the human casein gene family

Five yeast artificial chromosome (YAC) clones containing the human casein gene family were isolated and characterized to study the control mechanisms for the expression of these genes. Partial restriction analysis in conjunction with the chromosomal fragmentation method and fluorescence in situ hybridization (FISH) analysis were performed to construct a detailed physical map of the casein gene family and to determine the chromosomal localization of these genes. The isolated YAC clones 748F3, 750D11, 882G11, 886B3 and 960D2 were 1.2 Mb, 860 kb, 800 kb 1.5 Mb and 1.5 Mb in size, respectively. The clones 748F3, 882G11, 886B3 and 960D2 contained the entire casein gene family, while the κ-casein gene was absent in 750D11. The human αS1-, β- and κ-casein genes were found to be closely linked and arranged in the order αS1-β-κ. The distance between αS1 and β, and between αS1 and κ was approximately 10 and 300 kb, respectively. The β-casein gene was oriented in the opposite direction to the αS1- and κ-casein genes. The casein gene family was localized to chromosome 4q21.1 by FISH analysis. Received: 7 July 1996 / Revised: 29 October 1996     

Characterization of cloned ribosomal DNA from Drosophila hydei.

The structure of ribosomal genes from the fly Drosophila hydei has been analyzed. EcoRI fragments, cloned in a plasmid vector, were mapped by restriction enzyme analysis. The lengths of the regions coding for 18S and 28S rRNA were defined by R-loop formation. From these data a physical map of the rRNA genes was constructed. There are two major types of rDNA units in D. hydei, one having a size of 11 kb and the other a size of 17 kb. The 17 kb unit results from an intervening sequence (ivs) of 6.0 kb, interrupting the beta-28S rRNA coding region. Some homology between th D. hydei ivs and D. melanogaster type 1 ivs has been described previously (1). However, the restriction sites within these ivs show considerable divergence. Whereas D. hydei rDNA D. melanogaster rDNA, the nontranscribed spacer has little, if any, sequence homology. Despite difference in sequence, D. hydei and D. melanogaster spacers show structural similarities in that both contain repeated sequence elements of similar size and location.     

Genomic and phylogenetic analysis of the S100A7 (Psoriasin) gene duplications within the region of the S100 gene cluster on human chromosome 1q21

Abstract The human S100 gene family encodes the EF-hand superfamily of calcium-binding proteins, with at least 14 family members clustered relatively closely together on chromosome 1q21. We have analyzed the most recently available genomic sequence of the human S100 gene cluster for evidence of tandem gene duplications during primate evolutionary history. The sequences obtained from both GenBank and GoldenPath were analyzed in detail using various comparative sequence analysis tools. We found that of the S100A genes clustered relatively closely together within a genomic region of 260 kb, only the S100A7 (psoriasin) gene region showed evidence of recent duplications. The S100A7 gene duplicated region is composed of three distinct genomic regions, 33, 11, and 31 kb, respectively, that together harbor at least five identifiable S100A7-like genes. Regions 1 and 3 are in opposite orientation to each other, but each region carries two S100A7-like genes separated by an 11-kb intergenic region (region 2) that has only one S100A7-like gene, providing limited sequence resemblance to regions 1 and 3. The duplicated genomic regions 1 and 3 share a number of different retroelements including five Alu subfamily members that serve as molecular clocks. The shared (paralogous) Alu S insertions suggest that regions 1 and 3 were probably duplicated during or after the phase of AluS amplification some 30–40 mya. We used PCR to amplify an indel within intron 1 of the S100A7a and S100A7c genes that gave the same two expected product sizes using 40 human DNA samples and 1 chimpanzee sample, therefore confirming the presence of the region 1 and 3 duplication in these species. Comparative genomic analysis of the other S100 gene members shows no similarity between intergenic regions, suggesting that they diverged long before the emergence of the primates. This view was supported by the phylogenetic analysis of different human S100 proteins including the human S100A7 protein members. The S100A7 protein, also known as psoriasin, has important functions as a mediator and regulator in skin differentiation and disease (psoriasis), in breast cancer, and as a chemotactic factor for inflammatory cells. This is the first report of five copies of the S100A7 gene in the human genome, which may impact on our understanding of the possible dose effects of these genes in inflammation and normal skin development and pathogenesis.     

Physical and genetic map of the Serpulina hyodysenteriae B78T chromosome.

A combined physical and genetic map of the Serpulina hyodysenteriae B78T genome was constructed by using pulsed-field gel electrophoresis and DNA blot hybridizations. The S. hyodysenteriae genome is a single circular chromosome about 3.2 Mb in size. The physical map of the chromosome was constructed with the restriction enzymes BssHII, EclXI, NotI, SalI, and SmaI. The physical map was used to constructed a linkage map for genes encoding rRNA, flagellum subunit proteins, DNA gyrase, NADH oxidase, and three distinct hemolysins. Several flaB2-related loci, encoding core flagellum subunit proteins, were detected and are dispersed around the chromosome. The rRNA gene organization in S. hyodysenteriae is unusual. S. hyodysenteriae has one gene each for 5S (rrf), 16S (rrs), and 23S (rrl) rRNAs. The rrf and rrl genes are closely linked (within 5 kb), while the rrs gene is about 860 kb from the other two rRNA genes. Using a probe for the S. hyodysenteriae gyrA gene, we identified a possible location for the chromosomal replication origin. The size and genetic organization of the S. hyodysenteriae chromosome are different from those of previously characterized spirochetes.     

Identification and molecular characterization of two novel mutations in COL1A2 in two Chinese families with osteogenesis imperfecta

Osteogenesis imperfecta (OI, also known as brittle bone disease) is caused mostly by mutations in two type Ⅰ collagen genes, COL1A1 and COL1A2 encoding the pro-α1 (Ⅰ) and pro-α2 (Ⅰ) chains of type Ⅰ collagen, respectively. Two Chinese families with autosomal dominant OI were identified and characterized. Linkage analysis revealed linkage of both families to COL1A2 on chromosome 7q21.3-q22.1. Mutational analysis was carried out using direct DNA sequence analysis. Two novel missense mutations, c.3350A＞G and c.3305G＞C, were identified in exon 49 of COL1A2 in the two families, respectively. The c.3305G＞C mutation resulted in substitution of a glycine residue (G) by an alanine residue (A) at codon 1102 (p.G1102A), which was found to be mutated into serine (S), argine (R), aspartic acid (D), or valine (V) in other families. The c.3350A＞G variant may be a de novo mutation resulting in p.Y1117C. Both mutations co-segregated with OI in respective families, and were not found in 100 normal controls. The G1102 and Y1117 residues were evolutionarily highly conserved from zebrafish to humans. Mutational analysis did not identify any mutation in the COX-2 gene (a modifier gene of OI). This study identifies two novel mutations p.G1102A and p.Y1117C that cause OI, significantly expands the spectrum of COL1A2 mutations causing OI, and has a significant implication in prenatal diagnosis of OI.     

Refined linkage map of chromosome 7 in the region of the cystic fibrosis gene

The genetic map in the region of human chromosome 7 that harbors the gene for cystic fibrosis (CF) has been refined by multilocus linkage studies in an expanded database including a large set of normal families. Six loci known to be linked to CF were examined: MET, an oncogene; COL1A2, collagen; TCRB, T-cell-receptor beta polypeptide; and three arbitrary loci—D7S8, D7S13, and D7S16—defined by probes pJ3.11, pB79a, and p7C22, respectively. The gene order with greatest statistical support is COL1A2-D7S13-D7S16-MET-D7S8-TCRB. Linkage analysis in families segregating for CF suggested that the most likely location of the CF gene on this map is between MET and D7S8.     

A 2.5-Mb physical map within 3p21.1 spans the breakpoint associated with Greig cephalopolysyndactyly syndrome.

Numerous investigations suggest that one or more genes residing in the p14 to p21 region of human chromosome 3 are critical to the development of neoplastic diseases such as renal cell carcinoma and small-cell lung cancer (SCLC). This region is additionally involved in several interchromosomal translocations, one of which is associated with the developmental disorder Greig cephalopolysyndactyly syndrome. A series of five loci that map in close proximity to the Greig syndrome breakpoint [t(3;7)(p21.1;p13)] at 3p21.1 have been physically linked by pulsed-field gel analysis over a 2.5-Mb region. The probes include ACY1, cA84 (D3S92), cA199 (D3S93), pHF12-32 (D3S2), and MW-Not153 (D3S332). The Greig 3;7 translocation breakpoint was discovered between clones cA199 and MW-Not153, separated by 825 kb. Further analysis revealed comigration of a rearranged fragment detected by MW-Not153 and a chromosome 7 probe previously shown to be in close proximity to the breakpoint (CRI-R944). This latter probe also detects a rearrangement in a second Greig-associated translocation, (6;7)(q27;p13). The physical map resulting from this analysis orders the markers along the chromosome and identifies several locations for CpG islands, likely associated with genes. Although probe pEFD145.1 (D3S32) has been genetically linked to D3S2 (2 cM), physical linkage to the other five loci could not be demonstrated. One of the linked loci, D3S2, has been widely utilized in the analysis of chromosome 3p loss in several malignant diseases. Since expression of ACY1, a housekeeping gene, is specifically reduced in many cases of SCLC, knowledge of its precise chromosomal position and identification of neighboring putative gene loci should facilitate investigation into the mechanism of this reduction.     

The gene CYP3 encoding P450pcn1 (nifedipine oxidase) is tightly linked to the gene COL1A2 encoding collagen type 1 alpha on 7q21-q22.1.

CYP3, the gene which encodes the hepatic cytochrome P450pcn1, the isozyme responsible for the metabolic oxidation of the calcium channel-blocking drug nifedipine, has recently been mapped to human chromosome 7 using somatic cell hybrids. Using multilocus linkage analysis in CEPH families, we examined the linkage of a cDNA probe (hPCN1) for CYP3 to the oncogene MET, the pro-alpha 2(1) collagen gene COL1A2, and the T-cell receptor beta-chain gene TCRB, together with three arbitrary loci D7S8, D7S13, and D7S16, defined by the anonymous DNA probes pJ3.11, pB79a, and p7C22, respectively. From 70 CEPH parents screened with a StyI RFLP for hPCN1, four informative families were found each with both parental and maternal grandparents and 6-11 children per family. Tight linkage emerged between CYP3 and COL1A2, with a maximum combined lod score of 5.72 at theta = 0, suggesting the most likely subchromosomal localization of CYP3 is 7q21.3-q22.1.     

The procollagen type III, alpha 1 (COL3A1) gene first intron expresses poly-A+ RNA corresponding to multiple ESTs and putative miRNAs

The mouse COL3A1 first intron is 9684 bp. RNA's of approximately 1.6 and 3.0 kb were detected by Northern hybridization analysis of poly‐A RNA from fetal mice and total RNA from suckling and adult mouse intestine using ³²P‐labeled, anti‐sense RNA synthesized from a mouse COL3A1 first intron, 5 prime region, 5.4 kb Xba I fragment (1655–7030 bp), recombinant plasmid (pPI5.4x). Expression of the 1.6 and 3.0 kb RNA's was significantly reduced in adult mouse intestine, indicating that these RNAs are developmentally regulated. “BLAST” analysis indicated that the mouse first intron 5 prime sequence has 94–100% identity to 13 mouse ESTs. These mouse first intron EST's lie within the 5.4 Xba I fragment of the mouse COL3A1 first intron. Two of the mouse first intron EST's have significant identity to known miRNA, mature sequences, mmu‐miR‐466f‐3P, mmu‐miR‐1187, and mmu‐miR‐574‐5P as well as others. Predicted targets for mmu‐miR‐466f‐3P include COL1A1, COL19A1, COL11A2, COL4A1, and COL4A5 indicating that COL3A1 intronic miRNAs may regulate the expression of other collagen genes in development. J. Cell. Biochem. 112: 541–547, 2011. © 2010 Wiley‐Liss, Inc.