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PubMed1996

A 350-kb cosmid contig in 3p14.2 that crosses the t(3;8) hereditary renal cell carcinoma translocation breakpoint and 17 aphidicolin-induced FRA3B breakpoints.

The constitutive fragile site at human chromosomal band 3p14.2, FRA3B, has been described as the most active common fragile site in the human genome. FRA3B is cytologically indistinguishable from the chromosome 3 breakpoint observed in the hereditary renal cell carcinoma (hRCC) translocation t(3;8) (p14.2;q24.13). Previous work demonstrated that a 1330-kb YAC clone, YC850A6, spans both the t(3;8) translocation and FRA3B and also encompasses FRA3B-associated breakpoints induced in hamster-human hybrids. This YAC was used to construct a multi-hit cosmid library. Screening of this library resulted in a 350-kb cosmid contig that extends distally from the t(3;8) translocation breakpoint. Seventeen aphidicolin-induced 3p14. 2 breakpoints derived from hamster-human hybrids were mapped within this cosmid contig. These breakpoints were found to localize as two distinct clusters, separated by 200 kb, which lie on either side of a region of frequent breakage within FRA3B as defined by FISH analysis using cosmids from the contigs. The most proximal of the breakpoint clusters lies approximately 100 kb distal to the hRCC t(3;8) breakpoint. The distribution of these breakpoints, together with the region of frequent chromosomal breakage mapped by FISH analysis, further confirms the position of FRA3B and helps to define the extent over which its fragility is exerted. These data indicate that FRA3B comprises several hundred kilobases of DNA sequence within 3p14.2. The 350-kb contig and the cosmid library constructed from YAC YC850A6 will be essential for further characterization of the region surrounding FRA3B and in experiments to determine the molecular basis of the fragility of FRA3B.

Animals↗
PubMed1997

Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial Mediterranean fever locus (MEFV) on chromosome 16p 13.3.

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAC, and YAC clones, we have generated a contig of genomic clones spanning approximately 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAC, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the approximately 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene.

Base Sequence↗
PubMed1997

Cosmid contig and transcriptional map of three regions of human chromosome 21q22: identification of 37 novel transcripts by direct selection.

Human chromosome 21 is associated with many disorders, including Down syndrome (DS). In an effort to identify genes involved in brain development or function and therefore implicated in the mental retardation associated with DS, we chose YACs from three regions of chromosome 21: a region within the so-called "Down syndrome critical region," a region proximal to it, and one distal to it. We made cosmid libraries from these YACs and generated high-resolution physical maps by constructing cosmid contigs. These are the first cosmid contigs on chromosome 21 outside the critical region. The cosmids were used for direct selection of cDNAs to isolate chromosome 21 expressed sequences. We have isolated 45 nonredundant partial cDNAs and mapped these back to the cosmid contigs. We isolated 3 nonoverlapping portions of DSCR1 and a part of GIRK2 and identified 3 nonoverlapping partial cDNAs with similarity to the rat Dyrk gene, which turned out to be the human homologue (MNB) of the Drosophila minibrain gene. Twelve sequences had matches with either STS or EST entries in the databases, including a chromosome 21 EST, a chromosome 21 STS, and 6 unmapped expressed sequence entries. Only 1 sequence resulted in a match with a protein entry. The remaining 25 sequences revealed no similarity to any database entry. All of these partial cDNAs are expressed as determined by Northern blotting or by RT-PCR.

Animals↗
PubMed1999

Closing in on the BPES gene on 3q23: mapping of a de Novo reciprocal translocation t(3;4)(q23;p15.2) breakpoint within a 45-kb cosmid and mapping of three candidate genes, RBP1, RBP2, and beta'-COP, distal to the breakpoint.

BPES is a genetic disorder presenting with blepharophimosis, ptosis of the eyelids, epicanthus inversus, and telecanthus. BPES type I is associated with female infertility, whereas type II presents without additional symptoms. Hitherto, it remains unknown whether BPES type I results from a defect in a single gene or from a contiguous gene syndrome. Previous cytogenetic and linkage analyses have assigned a BPES locus to 3q23, in a 5-cM interval between D3S1615 and D3S1316. In this report, we describe the molecular and physical characterization of the 3q23 breakpoint in a BPES patient with a t(3;4)(q23;p15.2) translocation. Eight YACs located around and within the D3S1615-D3S1316 interval were mapped relative to the 3q23 breakpoint; 5 YACs spanning the 3q23 breakpoint were identified. Thirteen STSs and ESTs were localized on the YAC map. Subsequent hybridization of 2 YACs spanning the breakpoint to the Human RPCI1 PAC Library and the Human Chromosome 3 LLNL Cosmid Library resulted in the identification of 12 PACs and 50 cosmids respectively, allowing the construction of a detailed PAC and cosmid physical map. A refined position-telomeric to the breakpoint-of 3 candidate genes, cellular retinol-binding proteins 1 and 2 (RBP1, RBP2) and the coatomer beta' subunit (beta'-COP), was obtained on this physical map. Furthermore, a PAC and cosmid contig encompassing the breakpoint was constructed. PAC 169-C 10 and cosmid 11-L 10 crossing the breakpoint have sizes of 110 and 45 kb, respectively. The isolation of coding sequences in these clones and in the rest of the contig will greatly facilitate further efforts toward positional cloning of the gene(s) involved in BPES.

Blepharophimosis↗
PubMed1989

Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains.

A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB lambda receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.

Animals↗
PubMed1995

Physical and linkage mapping of the bovine genome with cosmids.

We have initiated a mapping strategy using cosmid clones to chromosomally anchor a high-resolution bovine genetic linkage map. Ten cosmids containing microsatellites were assigned to bovine chromosomes by fluorescence in situ suppression hybridization (FISH). Four cosmid clones, three of which contain an informative microsatellite, were assigned to autosomes 5, 13, 24, and 28. The assignment to autosome 13 anchors bovine syntenic group U11. Two additional cosmid clones, each containing informative microsatellites, are assigned to autosomes 9 and 29, anchoring bovine linkage groups U2 and U8, respectively. Four cosmid clones, three of which contain informative microsatellites, also provide the first assignment to autosome 25, anchoring bovine syntenic group U7 and orienting the corresponding linkage group relative to the centromere.

Alleles↗
PubMed1992

Mapping and characterization of 129 cosmids on human chromosome 11p.

We constructed cosmid libraries from human-hamster somatic cell hybrids that possess all or part of the short arm of chromosome 11 as their only human complement and isolated 129 human 11p clones. These cosmids map to 22 of 25 intervals distinguished by a hybrid panel for chromosome 11p. Forty-eight single-copy sequences were subcloned from 25 cosmids. Six of 17 (35%) single-copy sequences tested identify 11 new polymorphisms. Restriction endonuclease analysis identified CpG islands in 16 of 68 cosmids (23.5%). Analysis of the distribution of restriction endonuclease sites recognizing CpG dinucleotides showed that clusters of these sites, including those associated with the 5' region of an 11p13 Wilms' tumor gene, WT1, can span greater distances than generally recognized. The cosmids reported here should contribute to the construction of long-range physical maps and the isolation of additional genes on the short arm of chromosome 11.

Blotting, Southern↗
PubMed1992

Generation and characterization of a human chromosome 9 cosmid library.

A cosmid library has been constructed from the hamster-human hybrid cell line PK-87-9, which contains chromosome 9 as its sole known human component. Ten thousand colonies were produced, of which approximately 200, or 2%, contain human material. Fifty of these 200 were regionally mapped by an Alu-primed PCR product hybridization procedure. These cosmids were localized to all regions of chromosome 9, but were especially concentrated in the distal portion of 9q. The map location derived by the Alu-primed PCR product hybridization procedure was compared to the map location derived by fluorescent in situ hybridization. Assignment of chromosomal location by the two methods was correspondent in all but a few cases. The presumptive presence of HTF islands was investigated for 130 cosmids by digestion with the restriction enzyme NotI. Twenty percent of cosmids contained at least one NotI site. A number of simple sequence repeat polymorphisms identified from the cosmid set were characterized and will provide a link between the genetic and physical maps for this chromosome.

Animals↗
PubMed2003

Genomic EcoRI polymorphism and cosmid sequencing reveal an insertion/deletion and a new IGLV5 allele in the human immunoglobulin lambda variable locus (22q11.2/IGLV).

The human immunoglobulin lambda locus ( IGL) is mapped at Chromosome 22q11.2, spanning about 1 Mb of DNA, and directs the synthesis of lambda-type immunoglobulin light chains. The positions of about 73-74 germline V-lambda genes, depending on the haplotypes, are known, with 29-33 of them being functional IGLV genes. These genes were divided into 11 subgroups ( IGLV1 to IGLV11) distributed into three gene clusters ( VA, VB, and VC). We constructed a high-resolution restriction map of a 37-kb cosmid clone (cosmid 8.3) harboring genes of the IGLV1, IGLV7, and IGLV5 families and the non-coding sequences IGLV(I)-42 and IGLV(VII)-41-1, located at cluster VB of the IGL locus. These IGLV genes were associated with unique EcoRI fragments detectable in Southern blots of genomic DNA. Population RFLP has revealed new IGLV alleles and haplotypes. We used the restriction map of cosmid 8.3 and the IMGT database as a reference for RFLP studies. EcoRI Southern blot hybridizations with subgroup-specific probes of the functional and open reading frame sequences present in cosmid 8.3 revealed different frequencies of IGLV gene fragments, as well as deletions of IGLV1-50 and IGLV5-39 genes and RFLP involving IGLV5-45 and IGLV5-48 genes. All members of the IGLV7 subgroup were monomorphic. Sequencing of the genes present in cosmid 8.3 revealed a new allelic variant of the IGLV5 subgroup. These data contribute to a better understanding of the contribution of the germline IGLV genes to the human genetic background and polymorphism.

Alleles↗
PubMed1990

The use of a drug resistance cartridge for in vitro insertion and deletion mutagenesis of a cosmid clone.

A drug-resistant cartridge was employed in the construction of families of insertion mutants of a cosmid clone. The cartridge contains a cml gene and has identical restriction enzyme sites, EcoRI, BamHI, SalI, and PstI, on both ends. The families of mutants were made by ligation of the cartridge to the cosmid, which was linearized or partially digested, followed by in vitro packaging and transduction. From these families we selected cosmid derivatives which either have a unique BamHI site at a predetermined site in the cosmid or have deletions covering different portions of the original clone. The extent of a large gene cluster cloned into the original cosmid was identified by confirming the gene function in some of the deletion mutants. The possibility for further and various uses of this cartridge is discussed.

Chromosome Deletion↗
PubMed1994

An approach to functional complementation by introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani using a cosmid shuttle vector.

To extend the range of genetic tools available for the functional analysis of trypanosomatid genes we have constructed a cosmid shuttle vector (pcosTL) which facilitates the introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani. The vector contains several features to simplify library construction and insert mapping and transformed cells can be selected on the basis of G418 resistance. To evaluate the vector and to determine the fidelity of replication we first constructed cosmid libraries and isolated clones containing the T. cruzi major cysteine protease genes (a tandemly repeated array) and the L. donovani trypanothione reductase gene (a single copy gene). T. cruzi and L. donovani cells transfected with their respective cosmids were characterised by the presence of multiple copies of cosmid DNA and by a considerable over-expression of the corresponding enzyme activity. Rearrangements or deletions of insert sequences were not detected. These findings and the observation that cosmid DNA can be rescued unaltered from transformed parasites suggest that the pcosTL vector will be ideally suited for studies involving functional complementation.

Animals↗
PubMed1985

Analysis of cosmids using linearization by phage lambda terminase.

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Animals↗
PubMed1989

Simplified cosmid vectors for gene transfer to cultured mammalian cells: isolation of the gene for elongation factor 2 from the mouse.

We constructed a series of cosmid vectors that carry two tandemly arranged lambda cos and mammalian selective markers. We achieved cloning efficiencies of 1-3 x 10(7) and greater than 10(6) colony-forming units per microgram of insert, using a cloned 42-kb BamHI fragment and Sau3AI fragments of 40-50 kb from mouse genomic DNA, respectively. The modified Ca.phosphate coprecipitation method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607-616] considerably improved the efficiency of gene transfer of cosmids into cultured mammalian cells: when genes encoding thymidine kinase from herpes simplex virus type 1 and aminoglycoside 3'-phosphoribosyltransferase from Tn5 were selected, the efficiencies of gene transfer into mouse L cells were about 10(-6). The mouse genome contains one copy of the functional gene for elongation factor 2 (EF2) per haploid genome and multiple copies of the EF2-related gene. We isolated a cosmid that carried functional full-length mouse EF2 from a cosmid library of L-cell genomic DNA, by colony hybridization and subsequent gene transfer of candidate cosmids into human 143B cells.

Animals↗
PubMed1994

Rapid isolation of cosmid insert DNA by triple-helix-mediated affinity capture.

A simple and rapid method for the isolation of cosmid insert DNA was developed based on triple-helix-mediated affinity capture (TAC). A modified cosmid was constructed from the SuperCos 1 cosmid vector by flanking the cloning site with two homopurine-homopyrimidine triple-helix-forming sequences. The cosmid DNA is digested with NotI restriction enzyme to release the insert DNA. The NotI-digested cosmid DNA is then combined with a biotinylated homopyrimidine oligonucleotide in an acidic buffer solution to form a triple-helix complex. The triple-helix complex is captured with streptavidin-coated magnetic beads. Insert DNA is eluted by adding a pH 9 buffered solution to the captured complex. The purified insert DNA is recovered with a yield of up to 95% and a purity of at least 95%. The isolated insert DNA was directly digested with CviJI restriction endonuclease to generate random fragments for shotgun sequencing.

Base Sequence↗
PubMed2005

A cosmid-based system for inserting mutations and foreign genes into the simian varicella virus genome.

Simian varicella is a natural varicella-like disease of nonhuman primates. The etiologic agent, simian varicella virus (SVV), is genetically related to varicella-zoster virus (VZV) and SVV infection of nonhuman primates is a useful model to investigate VZV pathogenesis and latency. In this study, we report development of a cosmid-based genetic system to generate SVV mutant viruses. SVV subgenomic DNA fragments (32-38kb) that span the viral genome were cloned into cosmid vectors. Co-transfection of Vero cells with four overlapping cosmid clones representing the entire SVV genome resulted in recombination and generation of infectious virus. SVV mutants were produced by manipulation of one cosmid and substitution into the genetic system. This genetic approach was used to insert a site-specific mutation within the SVV open reading frame 14 which encodes the nonessential glycoprotein C gene. In a subsequent experiment, the green fluorescent protein (GFP) gene was inserted into the SVV genome within ORF 14. These SVV mutants replicate as efficiently as wild-type SVV in cell culture. This cosmid-based genetic system will be useful to investigate the effect of viral mutations on SVV pathogenesis and latency and also to develop and evaluate recombinant varicella vaccines that express foreign antigens.

Animals↗
PubMed2002

Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library.

The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.

Chromosomes, Artificial, Bacterial↗
PubMed1990

RecA-independent high-frequency deletion of recombinant cosmid DNA in Escherichia coli.

Segments of DNA were deleted from recombinant cosmid DNAs during propagation in Escherichia coli hosts in liquid culture. DNAs of more than 1000 cosmids propagated in various E. coli hosts were analysed by agarose gel electrophoresis (AGE). The effects of vectors, insert DNAs and host genetic characters on the formation of deletions were examined. The probability of deletion and the pattern of deletion bands observed by AGE differed from clone to clone, and after extensive culture the deletion band patterns remained almost constant during further culture. Most recombinant clones eventually showed deletion during prolonged liquid culture. Mutations in the recA gene of E. coli hosts, including a deletion mutation, did not prevent deletion. Most deletions occurred in the insert portions of cosmid DNAs. Nucleotide sequence analysis of six deletion junctions in test cosmid cMB15 demonstrated that deletions occurred between two short complete direct repeats of about 4-10 bp, irrespective of whether the cosmid was propagated in a recA host or a rec+ host. Some deletions occurred at the same sites either in a recA host or a rec+ host. These results suggest that the deletion events are mainly mediated by a recA-independent recombination system(s) of E. coli host cells.

Base Sequence↗
PubMed1994

Integrating the porcine physical and linkage map using cosmid-derived markers.

An essential part in the development of informative linkage maps is to include genetic markers that have been anchored by physical mapping. Here a set of 18 porcine cosmid-derived genetic markers are reported that have been mapped by linkage analysis, and that also have been physically localized by fluorescence in situ hybridization (FISH). Three different strategies were used to establish polymorphic markers from the cosmid clones. Firstly, dinucleotide microsatellite loci were derived by sequencing cosmid subclones containing (CA)n repeats. Secondly, variable SINE 3' poly(A) tracts (SINEVA) were identified by direct SINE-PCR amplification of cosmid clones. Thirdly, the cosmids were used in Southern blot hybridization to detect restriction fragment length polymorphisms (RFLPs). Compared with the most recent consensus compilation of the porcine gene map, the present assignment of markers to chromosomes 2p, 3, 4, 10, 12q, and 16 represents the first loci mapped to these chromosomes, for which linkage as well as in situ data are now available.

Animals↗