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PubMed1993

A high-resolution cytogenetic map of human chromosome 5: localization of 206 new cosmid markers by direct R-banding fluorescence in situ hybridization.

We have constructed a high-resolution cytogenetic map of human chromosome 5 with 206 new cosmid clones by direct R-banding fluorescence in situ hybridization. The fluorescent signals of 206 clones were evenly distributed throughout chromosome 5, although they were sublocalized preferentially to R-positive bands. This high-resolution cytogenetic map with an average distance of nearly 1 Mb will serve as important resource for construction of a genetic linkage map, which is essential for positional cloning of responsible genes. Moreover, these mapping data provide many useful landmarks for the construction of contig maps of chromosome 5, as required in the Human Genome Project.

Animals↗
PubMed1994

Nucleotide sequence analysis of 77.7 kb of the human V beta T-cell receptor gene locus: direct primer-walking using cosmid template DNAs.

The nucleotide sequence of 77.7 kb from the human T-cell receptor beta-chain locus was determined directly from three overlapping cosmid clones using the primer-walking approach. Computer-aided analyses of this sequence reveal the presence of at least 11 genic regions that are closely related to the human T-cell receptor beta variable region (TCRBV) gene family. These include five germline sequences that have previously been determined, V beta 21.2, V beta 8.1, V beta 8.2, V beta 8.3, and V beta 16, and four whose sequences have partially been determined at the mRNA level, V beta 6, V beta 23, V beta 12.2, V beta 24. The two remaining V beta Tcr-related sequences have eluded discovery by cDNA and RT-PCR cloning and genomic blot hybridization methods. These two V beta Tcr-related genes lack > 75% nucleotide sequence identity with any other V beta Tcr gene member and therefore, by convention, are referred to as new subfamily members V beta 25 and V beta 26. This lack of shared identity with other subfamily members explains why they were not detected by hybridization. The promoter regions of these V beta Tcr genes contain the conserved Tcr decamer element located between 80 and 110 bp 5' of the translation start site, generally near a putative TATAA promoter element. Our sequence analysis also reveals that a 3.3-kb duplication unit was involved in the recombination event that produced the closely related V beta 8.1 and 8.2 gene subfamily members. This sequenced region of the V beta locus contains an average number of repetitive DNA elements (21 Alu, three L1, three MER, and three retrovirus-related elements.

Amino Acid Sequence↗
PubMed1994

Selection of chromosome 22-specific clones from human genomic BAC library using a chromosome-specific cosmid library pool.

A new approach to rapidly identify chromosome-specific subsets of clones from a total human genomic library is described. We report here the results of screening a human bacterial artificial chromosome (BAC) library using the total pool of clones from a chromosome 22-specific cosmid library as a composite probe. The human BAC library was gridded on filters at high density and hybridized with DNA from the pooled chromosome 22-specific Lawrist library under suppressive conditions. In a single hybridization, we picked 280 candidates from the BAC library representing over 30,000 clones (or 1.2x coverage of human genome). This subset contained more than 60% of the chromosome 22-specific BAC clones that were previously found to be present in the original BAC library. In principle, this approach can be applied to select a subset of clones from other global libraries with relatively large inserts using a pool from a regional library as a composite probe. It is important to note that the target and probe libraries must be based on vectors that share no homology with each other.

Chromosomes, Bacterial↗
PubMed1996

Cosmids map two incontinentia pigmenti type 1 (IP1) translocation breakpoints to a 180-kb region within a 1.2-Mb YAC contig.

Incontinentia pigmenti (IP) is an X-linked dominant disorder of neuroectodermal development. Based on the observation of six unrelated females with clinical features of nonfamilial IP with constitutional de novo reciprocal X;autosome translocations, a putative incontinentia pigmenti type 1 locus (IP1; MIM No. 308300) was localized to region Xp11.21. Using available regional DNA markers, we constructed a yeast artificial chromosome (YAC) contig that contained 1.2 Mb of distal Xp11.21 and spanned two IP1 X-chromosomal breakpoints. This contig was used to generate a detailed molecular map of the region and identify three regional CpG islands. YAC-derived cosmids were used to clone and map the IP1 breakpoints to a 180-kb interval that was flanked by DNA markers DXS705 and DXS741. The physical map and genomic clones should facilitate the isolation and characterization of transcripts associated with the IP1 translocation breakpoints.

Base Sequence↗
PubMed1997

Construction of a 780-kb PAC, BAC, and cosmid contig encompassing the minimal critical deletion involved in B cell chronic lymphocytic leukemia at 13q14.3.

A putative tumor suppressor gene involved in B cell chronic lymphocytic leukemia (B-CLL) was mapped to human chromosome 13q14.3 close to the genetic markers D13S25 and D13S319. We constructed a 780-kb-long contig composed of cosmids, bacterial artificial chromosomes, and bacteriophage P1-derived artificial chromosomes that provides essential information and tools for the positional cloning of this gene. The conting contains both flanking markers as well as several additional genetic markers, three ESTs, and one potential CpG island. In addition, using one B-CLL patient, we characterized a small internal deleted region of 550 kb. Comparing this deletion with other recently published deletions narrows the minimally deleted area to less than 100 kb in our physical map. This deletion core region should contain all or part of the disrupted in B cell malignancies tumor suppressor gene.

Bacteriophages↗
PubMed1997

A 370-kb cosmid contig of the serpin gene cluster on human chromosome 14q32.1: molecular linkage of the genes encoding alpha 1-antichymotrypsin, protein C inhibitor, kallistatin, alpha 1-antitrypsin, and corticosteroid-binding globulin.

The human genes encoding alpha 1-antitrypsin (alpha 1AT, gene symbol PI), corticosteroid-binding globulin (CBG), alpha 1-antichymotrypsin (AACT), and protein C inhibitor (PCI) are related by descent, and they all map to human chromosome 14q32.1. This serine protease inhibitor (serpin) gene cluster also contains an antitrypsin-related sequence (ATR, gene symbol PIL), but the precise molecular organization of this region has not been defined. In this report we describe the generation and characterization of an approximately 370-kb cosmid contig that includes all five serpin genes. Moreover, a newly described serpin, kallistatin (KAL, gene symbol PI4), was also mapped within the region. Gene order within this interval is cen-CBG-ATR-alpha 1 AT-KAL-PCI-AACT-tel. The genes occupy approximately 320 kb of genomic DNA, and they are organized into two discrete subclusters of three genes each that are separated by approximately 170 kb. The distal subcluster includes KAL, PCI, and AACT; it occupies approximately 63 kb of DNA, and all three genes are transcribed in a proximal-to-distal orientation. Within the subcluster, there is approximately 12 kb of intergenic DNA between KAL and PCI and approximately 19 kb between PCI and AACT. The proximal subcluster includes alpha 1AT, ATR, and CBG; it occupies approximately 90 kb of genomic DNA, with approximately 12 kb of DNA between alpha 1AT and ATR and approximately 40 kb between ATR and CBG. These genes are all transcribed in a distal-to-proximal orientation. This represents the first detailed physical map of the serpin gene cluster on 14q32.1.

Carrier Proteins↗
PubMed1994

Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library.

We report the rescue of an arginine-requiring mutant (arg7-8) of Chlamydomonas reinhardtii by complementation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were obtained from 3 x 10(9) treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene (ARG7) encoding argininosuccinate lyase (ASL). The arginine-independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones. We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach.

Animals↗
PubMed1995

Isolation and characterization of a cosmid contig for the GCPS gene region.

The zinc finger gene GLI3 has been shown to be involved in the embryonal development of the limbs and skull. Mutations in GLI3 lead to the development of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutations extra toes (Xt) and anterior digit deformity (add). The GCPS locus on human chromosome 7p13 has recently been isolated in a yeast artificial chromosome (YAC) contig. Here, we describe the establishment of a cosmid contig that was derived from two of the YAC clones, that spans 550 kb of human DNA, and that includes the GLI3 gene. In this contig, three GCPS translocation breakpoints have been mapped to distinct EcoRI fragments in the 3' half of the gene. In addition, exon-carrying fragments have been identified and the size of the GLI3 gene could be determined as at least 280 kb. The gene is flanked by a CpG island that lies on the 5' side and that is in close proximity to the first exon detected by the cloned GLI3 cDNA. Further upstream, five segments were found that have been conserved between man and mouse. In the mouse, this region has been characterized as the transgene integration site resulting in the add phenotype. Both the CpG island and the conserved regions are probable candidates for a search for GLI3 promoter and control elements.

Abnormalities, Multiple↗
PubMed1986

A polymorphic locus on the long arm of chromosome 20 defined by two probes from a single cosmid.

Two probes from the random human cosmid c1-37 detect restriction fragment length polymorphisms in humans. The loci revealed by these probes are in linkage equilibrium and constitute a compound polymorphic locus with a polymorphism information content of 0.54. A somatic cell hybrid panel has been used to map the probes to chromosome 20; in situ hybridization studies confirm this localization and indicate that the locus is on 20q13. This is the first polymorphic locus to be assigned to the long arm of chromosome 20.

Animals↗
PubMed1988

Cosmids carrying Aspergillus terreus DNA can integrate into Saccharomyces cerevisiae chromosome XII via recombination between yeast and foreign DNAs.

A genome clonotheque consisting of 25- to 40-kb Sau3AI fragments of Aspergillus terreus DNA was constructed in the episomal cosmid vector pES33 containing the yeast ARG4 gene. From the 475 transformants of cir0 yeast strain ESH-0, 23 stable Arg+ transformants were independently selected. Genetic and Southern analysis of these stable transformants showed that 39% arose as a result of recombination between cloned A. terreus DNA sequences and yeast chromosome XII. The recombination events most likely occurred in the regions of homology within the rDNA clusters of A. terreus and Saccharomyces cerevisiae.

Aspergillus↗
PubMed1992

Localization of 24 cosmid clones on the human Y chromosome.

Twenty-four novel cosmid clones were cloned and mapped on the human Y chromosome using a panel consisting of DNA from seven individuals each having a different segment of the Y chromosome. Eight were assigned to the short arm, 15 to the long arm and 1 to the both short and long arms.

Animals↗
PubMed1996

High-resolution fluorescence in situ hybridization of RBM- and TSPY-related cosmids on released Y chromatin in humans and pygmy chimpanzees.

Applying two-colour fluorescence in situ hybridization (FISH) we simultaneously hybridized RBM- and TSPY-related cosmids to Y chromosomes in prophase and to released Y chromatin in interphase nuclei of man and pygmy chimpanzee. Whereas, even on prophasic Y chromosomes, no resolution of the overlapping RBM and TSPY signal clusters could be achieved, the RBM and TSPY signals are completely separated from each other in our maximum released Y chromatin stretches in interphase nuclei. These results unequivocally lend support to the view that the RBM and TSPY families have an interspersed organization on the Y chromosomes of man and higher apes. Thus, the distribution of RBM and TSPY signals might well go back to a common organization of these genes next to each other on an ancient Y chromosome.

Animals↗
PubMed1996

Genetic mapping studies of 40 loci and 23 cosmids in chromosome 11p13-11q13, and exclusion of mu-calpain as the multiple endocrine neoplasia type 1 gene.

Forty loci (16 polymorphic and 24 non-polymorphic) together with 23 cosmids isolated from a chromosome 11-specific library were used to construct a detailed genetic map of 11p13-11q13. The map was constructed by using a panel of 13 somatic cell hybrids that sub-divided this region into 19 intervals, a meiotic mapping panel of 33 multiple endocrine neoplasia type 1 (MEN1) families (134 affected and 269 unaffected members) and a mitotic mapping panel that was used to identify loss of heterozygosity in 38 MEN1-associated tumours. The results defined the most likely order of the 16 loci as being: 11pter-D11S871-(D11S288, D11S149)-11cen-CNTF-PGA-ROM1-D11S480-PYGM- SEA-D11S913-D11S970-D11S97- D11S146-INT2-D11S971-D11S533-11qter. The meiotic mapping studies indicated that the most likely location of the MEN1 gene was in the interval flanked by PYGM and D11S97, and the results of mitotic mapping suggested a possible location of the MEN1 gene telomeric to SEA. Mapping studies of the gene encoding mu-calpain (CAPN1) located CAPN1 to 11q13 and in the vicinity of the MEN1 locus. However, mutational analysis studies did not detect any germ-line CAPN1 DNA sequence abnormalities in 47 unrelated MEN1 patients and the results therefore exclude CAPN1 as the MEN1 gene. The detailed genetic map that has been constructed of the 11p13-11q13 region should facilitate the construction of a physical map and the identification of candidate genes for disease loci mapped to this region.

Animals↗
PubMed1996

Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes.

Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV) probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization (FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining, we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between HCMV and peripheral blood leukocytes at the single-cell level.

Cloning, Molecular↗
PubMed1997

Cosmid-derived markers anchoring the bovine genetic map to the physical map.

The mapping strategy for the bovine genome described in this paper uses large insert clones as a tool for physical mapping and as a source of highly polymorphic microsatellites for genetic typing, and was one objective of the BovMap Project funded by the European Union (UE). Eight-three cosmid and phage clones were characterized and used to physically anchor the linkage groups defining all the bovine autosomes and the X Chromosome (Chr). By combining physical and genetic mapping, clones described in this paper have led to the identification of the linkage groups corresponding to Chr 9, 12, 16, and 25. In addition, anchored loci from this study were used to orient the linkage groups corresponding to Chr 3, 7, 8, 9, 13, 16, 18, 19, and 28 as identified in previously published maps. Comparison of the estimated size of the physical and linkage maps suggests that the genetic length of the bovine genome may be around 4000 cM.

Animals↗
PubMed1993

A 13 kb resolution cosmid map of the 14 Mb fission yeast genome by nonrandom sequence-tagged site mapping.

We present the application of a nonrandom sequence-tagged site (STS) content detection method in mapping an entire genome, that of fission yeast. The novelty of our strategy is in the use of STS probes made from both ends of cosmid clones, selected on the basis of "sample without replacement" (only library clones that show no previous positive hybridization are selected and made into probes). We developed powerful techniques, based on consistency analysis, for error detection and contig assembly. In addition, we probed our library with genetically mapped markers and Notl or Sfil linking clones, thereby anchoring contigs onto chromosomes. Our map contains more than 1000 sites, including genes (most were previously unmapped), occurrences of known repetitive elements, and Notl-Sfil restriction sites.

Chromosome Mapping↗
PubMed1985

Novel high- and low-copy stable cosmids for use in Agrobacterium and Rhizobium.

Presented are a set of cosmids based on the unit copy Agrobacterium plasmid, pTAR, and the high-copy-number mutant plasmid, pUCD500, of pTiC58. The addition of a par function derived from pTAR to the vectors allowed them to be stably maintained throughout the cell population in the absence of selective pressure. These vectors, designed for Agrobacterium and Rhizobium, also work in Escherichia coli. The vectors can be cotransferred to Rhizobiaceae from E. coli with the helper plasmid, pRK2013. The pTiC58 origin containing vectors, pUCD1000 and pUCD1001 were found to be incompatible with a 250-kb plasmid harbored by R. meliloti RM102Z1. RM102Z1(pUCD1000) was still capable of nodulating roots in alfalfa.

Chromosome Mapping↗