A yeast artificial chromosome contig encompassing the type 1 neurofibromatosis gene.

The yeast artificial chromosome (YAC) system (Burke et al., 1987, Science 236: 806-812) allows the direct cloning of large regions of the genome. A YAC contig map of approximately 700 kb encompassing the region surrounding the type 1 neurofibromatosis (NF1) locus on 17q11.2 has been constructed. A single YAC containing the entire NF1 locus has been constructed by homologous recombination in yeast. In the process of contig construction a novel method of YAC end rescue has been developed by YAC circularization in yeast and plasmid rescue in bacteria. YACs containing homology to the NF1 region but mapping to another chromosome have also been discovered. Sequences of portions of the homologous locus indicate that this other locus is a nonprocessed pseudogene.     

A yeast artificial chromosome contig encompassing the cystic fibrosis locus.

The gene responsible for cystic fibrosis (CF) has recently been identified. Coding sequence for the cystic fibrosis transmembrane conductance regulator (CFTR) spans at least 230 kb of the human genome. Although all 27 exons of the gene are represented in cosmid or bacteriophage clones, there are still several gaps in the physical map of this region. It should be possible to complete the map and to clone the entire CFTR gene in a single fragment of DNA using a yeast artificial chromosome (YAC) vector. Herein we describe the construction and physical mapping of a 1.5-Mb YAC contig which encompasses D7S8 (J3.11) and D7S23 (KM19), two genetic loci flanking the CF locus. One of the clones in the contig, 37AB12, contains a 310-kb YAC which includes the entire CFTR gene and flanking sequence in both the 5' and 3' directions.     

Isolation and characterization of a yeast artificial chromosome (YAC) contig around the human steroid sulfatase gene.

The region surrounding the steroid sulfatase (STS) locus on Xp22.3 is of particular interest since it represents a deletion hot spot, shares homology with the proximal long arm of the Y chromosome (Yq11.2), and contains genes for several well-described X-linked disorders. Here we describe yeast artificial chromosomes (YACs) covering 450 kb around the STS gene. Eight YAC clones were isolated from a human YAC library. Their STS exon content was determined and the overlap of the clones characterized. Two of the YAC clones were found to contain the entire STS gene. The most proximal and the most distal ends of the YAC contig were cloned but neither of them crossed the breakpoints in any of the previously described patients with entire STS gene deletions. This is consistent with deletions larger than 500 kb in all these patients. One of the YAC clones was found to contain sequences from the STS pseudogene on Yq11.2. Two anonymous DNA sequences, GMGXY19 and GMGXY3, previously mapped in the vicinity of the STS locus, were found within the YAC contig and their assignment with respect to the STS locus was thus possible. This contig is useful for the overlap cloning of the Xp22.3 region and for reverse genetic strategies for the isolation of disease genes in the region. Furthermore, it may provide insight into the molecular mechanisms of deletion and translocation events on Xp22.3 and in the evolution of sex chromosomes.     

A yeast artificial chromosome contig containing the complete Duchenne muscular dystrophy gene.

A contig of 36 overlapping yeast artificial chromosome (YAC) clones has been constructed for the complete Duchenne muscular dystrophy (DMD) gene in Xp21. The YACs were isolated from a human 48,XXXX YAC library using the DMD cDNA and brain promoter fragments as hybridization probes. The YAC clones were characterized for exon content using HindIII or EcoRI digests, hybridization of individual DMD cDNA probes, and polymerase chain reaction (PCR) amplification of specific exons near the 5' end of the gene. For comparison to the known long-range restriction map of the DMD gene, YAC clones were digested with SfiI and hybridized with DMD cDNA probes. The combined analysis of the exon content and the SfiI map allowed an approximately 3.2-Mb YAC contig to be constructed. The complete 2.4-Mb DMD gene could be represented in a minimum set of 7 overlapping YAC clones.     

Complete nucleotide sequence of the region encompassing the first twenty-five exons of the human pro alpha 1(I) collagen gene (COL1A1)

Dysfunctions of the genes coding for the two chains of the human type-I procollagen result in genetic disorders that affect the integrity of bone, ligaments, tendons, and other connective tissues. While the primary amino acid (aa) sequence of one of the two type-I subunits, pro alpha 2(I), has been derived in its entirety from the analysis of overlapping cDNAs, the sequence of the first 247 aa residues of the helical domain of the other polypeptide, pro alpha 1(I), had yet to be determined. To this end, we have sequenced nearly 4 kb of the human pro alpha 1(I) collagen gene and identified twelve open reading frames whose conceptual amino acid translation exhibits 95% homology to the first 247 aa of rat alpha 1(I) chain. Furthermore, with these and other data, some of which previously unpublished, we have derived the complete sequence of the first 7618 bp of the gene. This region comprises the 25 exons encoding the N-terminal pre-propeptide and five of the eight cyanogen-bromide-derived peptides. This information therefore represents a most useful reference for the characterization of molecular defects in individuals affected by various connective tissue disorders.     

A YAC contig encompassing the recessive Stargardt disease gene (STGD) on chromosome 1p.

Stargardt disease (STGD) and fundus flavimaculatus are infrequent autosomal recessive conditions characterized by a juvenile macular dystrophy and variable degrees of peripheral retinal changes. Linkage analysis performed in 47 STGD/fundus flavimaculatus families demonstrated significant linkage to 13 polymorphic DNA markers on chromosome 1p. The maximum combined two-point lod score was 32.7 (maximum recombination fraction [phi max] = .006) with the polymorphic marker D1S188. Our data demonstrate that STGD and fundus flavimaculatus are the same disorder clinically and genetically and provide further evidence for genetic homogeneity of this phenotype. Analysis of recombination events on disease chromosomes placed the STGD gene within a 4-cM interval between markers D1S435 and D1S236. A physical map was constructed of a YAC contig flanking STGD, from markers D1S500 to D1S495, and includes the critical interval delineated by historical recombinants. This contig spans approximately 31 cM, with one gap (3-5 cM) that is outside the 4-cM critical region. Localization of STGD to a single YAC contig will facilitate its positional cloning.     

DNA rearrangements in the alpha 5(IV) collagen gene (COL4A5) of individuals with Alport syndrome: further refinement using pulsed-field gel electrophoresis.

Alport syndrome (AS), an X-linked kidney disorder, has been shown to be caused by mutations in the gene for the alpha 5-chain of type IV collagen (COL4A5), which maps to Xq22. On the basis of the results of conventional Southern blot analysis of AS patient DNAs, we employed pulsed-field gel electrophoresis to characterize further three gene rearrangements at the 3'-end of alpha 5(IV). We were able to construct long-range restriction maps for all three of these patients and deduce the extent and nature of each rearrangement. One of these mutations is a 450-kb simple deletion that includes 12 kb of the alpha 5(IV) gene. A second mutation has been shown to be a direct duplication of 35 kb of alpha 5(IV) genomic DNA, and a third mutation involves a complex insertion/deletion event resulting in an overall loss of 25 kb.     

Assignment of the human collagen alpha 1 (XIII) chain gene (COL13A1) to the q22 region of chromosome 10

Type XIII collagen is a recently described collagen that resembles in structure the short-chain collagens of types IX, X, and XII. Unlike any other collagen, the type XIII is found in several different forms generated through alternative splicing. A 2.0-kb genomic fragment from the human alpha 1 (XIII) collagen gene was isolated and shown by DNA sequencing to contain exon 12 as counted from the 3' end. This fragment was used as a probe to localize the gene. The gene (COL13A1) was assigned to chromosome 10 by hybridization of the probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes. Furthermore, the gene was mapped to the q22 region by in situ hybridization to metaphase chromosomes.     

Mapping of a human fibrillar collagen gene, pro alpha 1 (XI) (COL11A1), to the p21 region of chromosome 1

Type XI collagen is a minor and poorly characterized structural component of cartilage. Recently, cDNA and genomic clones coding for the pro alpha 1 chain of human Type XI collagen, formerly 1 alpha collagen, have been isolated and fully characterized. Here we have used one such probe to establish the chromosomal localization of the pro alpha 1 (XI) collagen gene (COL11A1) by hybridization to filter-bound DNA isolated from flow-sorted chromosomes and by in situ hybridization on metaphase chromosomes. This combination of approaches has enabled us to locate COL1A11 in the p21 region of chromosome 1. This represents the first mapping of a Type XI collagen gene and the first assignment of a collagen locus to chromosome 1. These studies also provide additional evidence for the nearly uniform dispersion of the human fibrillar collagen genes in the human genome.     

A yeast artificial chromosome contig spanning the mouse immunoglobulin kappa light chain locus

 A single contig spanning the entire mouse immunoglobulin kappa light chain (Igk) locus on chromosome 6 has been established using yeast and bacterial artificial chromosome clones. Detailed mapping of the Igk locus indicates that a member of the Igk-V2 gene family, located about 3.5 megabases upstream of the Igk-J-C complex, is the most distal functional Igk-V gene. Sequence analyses of Igk-V genes and anonymous DNA segments provide indications for internal duplications at the 5′ end of the Igk-V locus and identify the likely origin of Igk-V orphon gene clusters located elsewhere in the mouse genome. Received: 17 July 1996 / Revised: 2 September 1996     

Construction of a bovine yeast artificial chromosome (YAC) library

We have constructed a bovine yeast artificial chromosome (YAC) library to provide a common resource for bovine genome research. We used leukocytes of a Japanese black bull ( Bos taurus ) as the DNA source, AB1380 for the yeast host, and pYAC4 for the vector. The library consists of 24 576 clones arranged in 256 96-well microtiter plates. An average insert size estimated from the analysis of 251 randomly selected clones was 480 kb. The rate of chimeric YACs evaluated by fluorescence in situ hybridization (FISH) analysis of 44 randomly selected clones was 36·4%. To estimate the number of genome equivalents, PCR-based screening was performed with 48 primer pairs and isolated 3·2 clones on average. In order to provide broad access for the scientific community, this library has been incorporated into the Reference Library system which provides high density filters for colony hybridization screening and a common database of the library.     

The human alpha 2(XI) collagen gene (COL11A2) maps to the centromeric border of the major histocompatibility complex on chromosome 6

Type XI collagen, a minor structural component of cartilage fibrils, is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI). Using a cloned fragment of the human alpha 2(XI) collagen gene (COL11A2) as a molecular probe for in situ hybridization and somatic cell hybrid mapping, we have localized the gene to the short arm of chromosome 6, region 21.3. By exploiting the rich source of probes provided by the major histocompatibility complex (MHC) genes, which also map to this chromosomal band, we have constructed macrorestriction maps of the region by pulsed-field gel electrophoresis and have localized the alpha 2(XI) collagen gene to the centromeric extreme of the MHC. Finally, we have demonstrated, by the isolation of overlapping cosmid clones, that the gene is 45 kb centromeric to the HLA-DPB2 locus and oriented with the 3' end toward the MHC. The COL11A2 locus thus demarcates the proximal boundary of the MHC. This finding may have implications for the understanding of certain MHC-linked diseases.     

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.     

Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach.

A sequence tagged site (STS)-based approach has been used to construct a 2.6-Mb contig in yeast artificial chromosomes (YACs) spanning the human dystrophin gene. Twenty-seven STSs were used to identify and overlap 34 YAC clones. A DNA fingerprint of each clone produced by direct Alu-PCR amplification of YAC colonies and the isolation of YAC insert ends by vectorette PCR were used to detect overlaps in intron 1 (280 kb) where no DNA sequence data were available, thereby achieving closure of the map. This study has evaluated methods for mapping large regions of the X chromosome and provides a valuable resource of the dystrophin gene in cloned form for detailed analysis of gene structure and function in the future.     

A three-megabase yeast artificial chromosome contig spanning the C57BL mouse Igh locus

The mouse Ig H chain (Igh) complex locus is composed of >100 gene segments encoding the variable, diversity, joining, and constant portions of the Ab H chain protein. To advance the characterization of this locus and to identify all the V(H) genes, we have isolated the entire region from C57BL/6 and C57BL/10 as a yeast artificial chromosome contig. The mouse Igh locus extends approximately three megabases and contains at least 134 V(H) genes classified in 15 partially interspersed families. Two non-Igh pseudogenes (Odc-rs8 and Rpl32-rs14) were localized in the distal part of the locus. This physical yeast artificial chromosome map will provide important structure and guidance for the sequencing of this large, complex, and highly repetitive locus.