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PubMed1995

Cosmids and transcribed sequences from chromosome 11q23.

To obtain cosmid markers and transcribed sequences from a specific chromosome region, a series of radiation-reduced hybrids (RHs) containing various regions of human chromosome 11 was prepared from microcell hybrid A9 (neo11) cells containing a normal human chromosome 11 tagged with pSV2neo at 11p11.2. Among 15 radiation hybrid clones isolated, RH(11)-9 which contains a q23 fragment in addition to the neo integration site, was used for the construction of a cosmid library. Cosmid clones having human DNA sequences were screened, and localized by Southern hybridization with the radiation hybrid panel. Fifty-nine cosmids were assigned to 11q23 and 6 cosmids to 11p11.2. Exon amplification proceeded with 23 of the 59 cosmids and 16 putative exons were cloned. Three of them were identical to those constituting a known gene which locates on q23 (ATDC), and the others were unknown. Thus, the RHs containing various subchromosomal fragments of chromosome 11 were useful for constructing region-specific DNA markers. The RH(11)-9 cells and putative exons also facilitate the positional cloning of genes in the 11q23 region.

Animals↗
PubMed1989

Instability of extrachromosomal cosmid DNA in SV40-transformed human (ataxia-telangiectasia) cells.

The ability of SV40-transformed human (ataxia-telangiectasia) fibroblasts to maintain Epstein-Barr virus (EBV)-based plasmids and cosmids extrachromosomally has been investigated. Transfection of a culture of cells with two different plasmids gave rise to cell clones which were able to maintain both plasmids extrachromosomally. When an EBV-based cosmid library was transfected into the cells and an individual cell clone was isolated, the extrachromosomal DNA derived from the cosmid contained numerous deletions and rearrangements. When individual cosmids were transfected into the culture, and several cell clones were isolated, the intracellular cosmid-derived DNA again showed the presence of multiple deletions and rearrangements. We conclude that although SV40-transformed cells are able to maintain more than one different EBV-based plasmid extrachromosomally, large EBV-derived molecules are extensively rearranged. SV40-transformed human fibroblasts cannot therefore be usefully used in attempting to clone genes from EBV-based cosmid libraries.

Ataxia Telangiectasia↗
PubMed1993

Shuttle cosmid vectors for the trypanosomatid parasite Leishmania.

We have developed two shuttle cosmid vectors for the trypanosomatid protozoan parasite Leishmania. Cosmids cLHYG and cLNEO contain hyg and neo markers, conferring resistance to hygromycin B and G418, respectively, replicate extrachromosomally after transfection into promastigotes, and bear a unique BamHI cloning site. To ensure the representation of telomeric sequences, which represent about 5% of the Leishmania genome, random insert DNAs were prepared by shearing followed by blunt-end ligation with BamHI adapters. Representative genomic libraries from Leishmania species representing the four major pathogenic complexes were prepared using cosmid cLHYG. The cosmid libraries were efficiently transfected into Leishmania, and individual cosmids were readily recovered by transformation back into Escherichia coli. The relatively small size of the Leishmania genome (50 Mb) combined with the capacity and transfection efficiency of these cosmid libraries (> 1000 Leishmania transfectants/plate) suggests the feasibility of functional genetic complementation in this parasite.

Animals↗
PubMed1990

Efficient procedure for preparing cosmid libraries from microgram quantities of genomic DNA fragments size fractionated by gel electrophoresis.

A modified procedure for preparing cosmid libraries from genomic DNA is described. Genomic DNA was partially digested with a restriction endonuclease, and DNA fragments of appropriate size fractionated by agarose gel electrophoresis. A cosmid library was prepared, prescreened, and used to isolate gene inserts with previously published procedures. In one series of experiments, a modified cosmid vector containing stuffer fragments was used to prepare cosmid libraries containing partial SphI digests of 25 to 35 kb. From 10(5) to 10(7) clones were obtained per microgram of size-fractionated genomic DNA. From 10 to 100 hybridization-positive clones of a single copy gene (COL2A1) were obtained from plates that were positive in the prescreening step. Restriction mapping of over 20 clones and nucleotide sequencing of over 20,000 bp in each of two clones indicated that the inserts were faithful copies of the gene. In another experiment, a standard cosmid vector was used to prepare a cosmid library containing partial BamHI fragments of 30 to 45 kb. Genomic libraries can be prepared with 5 to 20 micrograms of genomic DNA and a large number of clones containing 25 to 45 kb fragments of a single copy gene can be isolated in about three weeks.

Cloning, Molecular↗
PubMed1993

Generation of varicella-zoster virus (VZV) and viral mutants from cosmid DNAs: VZV thymidylate synthetase is not essential for replication in vitro.

Four overlapping cosmid clones were constructed that contain the complete genome of the attenuated Oka strain of VZV. Transfection of human melanoma cells with the four cosmids resulted in production of infectious VZV. A double-stranded oligonucleotide, encoding a stop codon in all three open reading frames, was inserted into one of the cosmids at the 5' end of the viral thymidylate synthetase gene. Transfection of melanoma cells with the mutant cosmid, along with the other three cosmids, resulted in VZV that does not express the viral thymidylate synthetase protein. The mutant virus grew at a rate similar to that of the parental Oka strain virus. Production of recombinant VZV using cosmid DNAs will be useful for studying the function of viral genes in VZV replication and establishment of latency. Furthermore, manipulation of the Oka strain of VZV might allow one to produce a vaccine virus that does not establish latency in the central nervous system or a virus that encodes foreign antigens for use as a polyvalent live virus vaccine.

Acyclovir↗
PubMed1987

Physical and genetic analysis of cosmids from the vicinity of the cystic fibrosis locus.

Cosmid libraries have been constructed from DNA of somatic cell hybrid cell lines, each containing a fragment of human chromosome seven and including sequences closely linked to cystic fibrosis (CF). Cosmids containing human DNA as insert were isolated from the library. Three cosmids, when used as probes to total genomic DNA, detected polymorphic loci, each of which was shown to be in strong linkage disequilibrium with CF. Restriction endonuclease digestion of cosmid clones and use of a new, rapid method of chromosome walking based on competitive hybridisation of cosmid inserts has allowed identification of several groups of overlapping cosmids ("contigs") from the vicinity of CF.

Animals↗
PubMed1990

Towards a physical map of the Drosophila melanogaster genome: mapping of cosmid clones within defined genomic divisions.

A physical map of the D. melanogaster genome is being constructed, in the form of overlapping cosmid clones that are assigned to specific polytene chromosome sites. A master library of ca. 20,000 cosmids is screened with probes that correspond to numbered chromosomal divisions (ca. 1% of the genome); these probes are prepared by microdissection and PCR-amplification of individual chromosomes. The 120 to 250 cosmids selected by each probe are fingerprinted by Hinfl digestion and gel electrophoresis, and overlaps are detected by computer analysis of the fingerprints, permitting us to assemble sets of contiguous clones (contigs). Selected cosmids, both from contigs and unattached, are then localized by in situ hybridization to polytene chromosomes. Crosshybridization analysis using end probes links some contigs, and hybridization to previously cloned genes relates the physical to the genetic map. This approach has been used to construct a physical map of the 3.8 megabase DNA in the three distal divisions of the x chromosome. The map is represented by 181 canonical cosmids, of which 108 clones in contigs and 32 unattached clones have been mapped individually by in situ hybridization to chromosomes. Our current database of in situ hybridization results also includes the beginning of a physical map for the rest of the genome: 162 cosmids have been assigned by in situ hybridization to 129 chromosomal subdivisions elsewhere in the genome, representing 5 to 6 megabases of additional mapped DNA.

Animals↗
PubMed1992

Isolation of region-specific cosmids from chromosome 5 by hybridization with microdissection clones.

A method is described for the isolation of chromosome region specific cosmids. The 5q35 region of the long arm of human chromosome 5 was microdissected, digested with MboI, ligated to oligonucleotide adaptors, amplified by the polymerase chain reaction and cloned into a plasmid vector. Inserts which did not contain highly repetitive sequences were used to screen a chromosome 5 cosmid library by direct hybridization. There were 33 positive cosmid clones identified with 4 microclones. Individual cosmid clones were biotinylated and used as probes for fluorescence in situ hybridization to metaphase chromosomes. Of the 33 cosmids that were mapped, 29 localized to q35 and 4 to q34, demonstrating the specificity of the microdissection library and the cosmids.

Base Sequence↗
PubMed1992

Assembly and analysis of cosmid contigs in the CEA-gene family region of human chromosome 19.

The carcinoembryonic antigen (CEA)-like genes are members of a large gene family which is part of the immunoglobulin superfamily. The CEA family is divided into two major subgroups, the CEA-subgroup and the pregnancy-specific glycoprotein (PSG)-subgroup. In the course of an effort to develop a set of overlapping cosmids spanning human chromosome 19, we identified 245 cosmids in a human chromosome 19 cosmid library (6-7X redundant) by hybridization with an IgC-like domain fragment of the CEA gene. A fluorescence-based restriction enzyme digest fingerprinting strategy was used to assemble 212 probe-positive cosmids, along with 115 additional cosmids from a collection of approximately 8,000 randomly selected cosmids, into five contigs. Two of the contigs contain CEA-subgroup genes while the remaining three contigs contain PSG-subgroup genes. These five contigs range in size from 100 kb to over 300 kb and span an estimated 1 Mb. The CEA-like gene family was determined by fluorescence in situ hybridization to map in the q13.1-q13.2 region of human chromosome 19. Analysis of the two CEA-subgroup contigs provided verification of the contig assembly strategy and insight into the organization of 9 CEA-subgroup genes.

Carcinoembryonic Antigen↗
PubMed1994

Ordered cosmid library and high-resolution physical-genetic map of Helicobacter pylori strain NCTC11638.

Helicobacter pylori is a Gram-negative bacterium that infects the human gastric mucosa, causes gastritis and contributes to the development of peptic ulcers and gastric cancer. To facilitate molecular genetic analysis of this pathogen, we constructed a approximately 20-fold redundant cosmid library and physical/genetic map of strain NCTC11638. Genomic DNA fragments were cloned into the cosmid vector Lorist6, and clones were ordered by hybridization with several types of probes: (i) ends of cloned DNAs; (ii) chromosomal Notl digest fragments; (iii) cosmids containing Notl sites; and (iv) specific genes. Seven hundred and fifty-one cosmids were mapped to one of three contigs covering > 90% of the chromosome, and are represented by a 68-cosmid miniset. The order of cosmids was confirmed and extents of overlap among them were estimated by restriction analysis. All currently known H. pylori genes were mapped, including those for a cytotoxin (vacA), cytotoxin-associated protein (cagA), urease and regulatory functions (ureAb, ureD and ureH), catalase (katA), major and minor flagellins (flaA and flaB), heat-shock (stress) and chaperone proteins (dnaK, htA, hspB (groEL)), prokaryotic ferritin (pfr), an adhesin subunit (hpaA), a surface protein (26 kDa), and 16S and 23S ribosomal RNAs (two genes each). The orientations of eight genes or clusters were determined, and two repetitive sequences were also found. The gene order and rRNA gene copy number determined here differed from that reported for an unrelated strain, which suggests considerable flexibility in H. pylori genome organization.

Base Sequence↗
PubMed1995

Cosmid contigs spanning 9q34 including the candidate region for TSC1.

The tuberous sclerosis disease gene TSC1 has been mapped to 9q34. However, its precise localisation has proved problematic because of conflicting recombination data. Therefore, we have attempted to clone the entire target area into cosmid contigs prior to gene isolation studies. We have used Alu-PCR from irradiation hybrids to produce complex probes from the target region which have identified 1,400 cosmids from a chromosome-specific library. These, along with cosmids obtained by other methods, have been assembled into contigs by a fingerprinting technique. We estimate that we have obtained most of the region in cosmid contigs. These cosmids are a resource for the isolation of expressed genes within the TSC1 interval. In addition, the cosmid contig assembly has demonstrated a number of previously unknown physical connections between genes and markers in 9q34.

Animals↗
PubMed1984

Direct isolation of the functional human thymidine kinase gene with a cosmid shuttle vector.

We have developed a new recombinant DNA cloning system to isolate directly the functional unit of the human thymidine kinase (TK) gene. The system utilizes a cosmid vector that can shuttle cloned DNA sequences between bacteria and mammalian cells. A complete human cosmid library was constructed and DNA from the total library was transfected to mouse L cells deficient in TK (LTK-) by calcium phosphate precipitation. The transfected cells were then selected with hypoxanthine/aminopterin/thymidine (HAT) medium, and one HAT-resistant cell clone was isolated. This cell line became resistant to HAT selection by acquiring the TK gene derived from the human cosmid library. As the cosmid vector contains the cohesive ends of the bacteriophage, we could directly retrieve the human DNA sequences from the transformed mouse L cells. Total DNA from the transformed TK+ L cells was packaged in vitro with lysogenic bacterial extracts and used to infect Escherichia coli. One of the two recombinant cosmids isolated contained a 43.8-kilobase human DNA insert and was capable of converting TK- L cells to the TK+ phenotype in both acute and stable transformation assays. Thus, we have isolated the functional human TK gene in this recombinant cosmid. The gene was further localized on a 14.5-kilobase BamHI DNA fragment, and it transcribed a mature mRNA of about 1,500 nucleotides. This method of gene isolation has several special features: (i) an intact structural gene can be cloned directly based on its function without knowledge of its amino acid or nucleotide sequence; (ii) the functional gene sequences can be recovered faster and more efficiently than with the usual DNA transfection method; and (iii) in conjunction with cell-sorting techniques, this method can be used to clone genes encoding cell surface markers.

Animals↗
PubMed1995

Refinement of the high-resolution physical and genetic map of Rhodobacter capsulatus and genome surveys using blots of the cosmid encyclopedia.

Cosmids from a library containing Rhodobacter capsulatus DNA fragments were previously ordered in two contigs: one corresponding to the chromosome and one to a 134 kb plasmid. This map contained 40 regions connected only by colony hybridization. To confirm the linkage and correct the map, the actual sizes of the overlaps were determined by blot-hybridization with Rhodobacter chromosomal DNA and by mapping of additional cosmids. Several revisions of the earlier map include single cosmid shifts and inversions. One additional gap in a cosmid contig was also found, raising the possibility that the chromosome is not a contiguous circle. About 2500 additional EcoRI,BamHI and HindIII restriction sites were added to the 560 EcoRV sites previously mapped onto the Rhodobacter chromosome, increasing the resolution of the physical map to the size of individual genes. Twenty-five new markers were located on the genetic map. The 48 markers now mapped represent nearly 300 genes and ORFs cloned from different species of Rhodobacter. The orientation of transcription of the four rrn operons was established using 16S rRNA- and 23S rRNA-specific probes and digestion with the rare-cutting enzyme, CeuI. Gel blots of 192 cosmids of the miniset of R.capsulatus digested with EcoRV were prepared. Such a hybridization template represents the whole genome cut into 560 DNA fragments varying in size from 0.4 to 25 kb. This template was used for high-resolution mapping of single genes, analysis of total genomic DNAs from related Rhodobacter strains and differentially expressed RNAs.

Chromosome Mapping↗
PubMed1993

Isolation of region-specific cosmids by hybridization with microdissection clones from human chromosome 10q11.1-q21.1.

A region-specific plasmid library composed of 20,000 recombinants was constructed by microdissection of human chromosome 10 (10q11.2-q21.1) and subsequent amplification with the primer-linker method of polymerase chain reaction (PCR). Hybridization with total human DNA showed that 32 of 217 microclones studied contained highly repetitive sequences. Further analysis of the remaining 185 microclones proved that 43 microclones, each having an insert longer than 200 bp, contained unique sequences of human chromosome 10 origin. Twenty-five microclones randomly selected from the 43 were used directly as probes to isolate corresponding cosmid clones, resulting in 32 cosmids corresponding to 14 microclones. Of the 25 cosmids that could be mapped by fluorescence in situ hybridization, 24 proved to originate from the microdissected or adjacent region (10p11.2-q22.3) and 1 from a rather distal region (10q24.3-q25.1). In addition, 15 of the 32 cosmids revealed restriction fragment length polymorphisms, including 1 with a variable number of tandem repeats marker. The microdissection library and the obtained cosmids are valuable resources for constructing high-resolution physical and linkage maps of the pericentromeric region of chromosome 10, where the gene predisposing to multiple endocrine neoplasia type 2A (MEN2A) has been mapped.

Base Sequence↗
PubMed1994

Identification of YAC and cosmid clones encompassing the ZFX-POLA region using irradiation hybrid cell lines.

The human Xp21.3-p22.1 region is poorly mapped relative to other X chromosome regions. To target cosmid and YAC clones specifically from Xp21.3-p22.1 for rapid contig construction, a hybridization-based screening approach using irradiation hybrids has been used. Alu-PCR products generated from hybrid lines containing small overlapping fragments from Xp21-p22 were hybridized to an X chromosome cosmid library, and cosmids predicted by their hybridization pattern to map to the region of interest were analyzed by fluorescence in situ hybridization (FISH). Hybridization of the cosmids in pools to gridded YAC libraries identified 15 YACs, which were verified and tested for chimerism by FISH. Cosmid content analysis of the YACs defined two contigs, one with 12 YACs covering about 1.5 Mb and one with 3 YACs. Five YACs from the 12-YAC cluster had been previously recognized by DNA polymerase alpha (POLA). ZFX identified a single YAC; hence, the physical linkage of ZFX and POLA was demonstrated within the contig. Four YACs had been isolated previously with ZFX and these extend the contig to 2 Mb. Restriction mapping of several YACs demonstrates that ZFX and POLA are about 700 kb apart, a distance similar to that reported in the mouse between Zfx and Pola. The order of these two loci and two additional loci identified by homologous mouse linking clones was found to be conserved between human and mouse: tel-ZFX-DXCrc57-DXCrc140-POLA-cen. We have shown that YAC contigs can be rapidly constructed from targeted regions without the need for time-consuming YAC end rescue and chromosomal walking.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Assignment of 55 novel cosmids to seven subregions of chromosome 13 using fluorescence in situ hybridization.

Fifty-five individual cosmids isolated from a chromosome 13-specific library have been regionally assigned using fluorescence in situ hybridization. These cosmids represent novel DNA markers that can be added to the limited number of DNA probes already available for this chromosome. Individual cosmids mapped along the length of the chromosome with almost equal distribution except that there appears to be an excess of probes from the distal 13q33-q34 region. Only two cosmids did not map back to chromosome 13, indicating that at least 96% of cosmids in this library are from this chromosome. The library is generally available as gridded colonies on nylon membranes suitable for hybridization screening.

Chromosome Walking↗
PubMed1996

Isolation of a cosmid sublibrary for a region of chromosome 12 frequently amplified in human cancers using a complex chromosome microdissection probe.

Chromosome-specific cosmid libraries are an extremely useful resource for positional cloning projects. Once a particular region of interest has been identified, it would be of value to have an approach for isolating chromosome band-specific cosmids that could be assembled into a sublibrary for rapid screening. We constructed a region-specific sublibrary of 700 cosmids by screening a chromosome 12-specific cosmid library with a complex probe generated by degenerate oligonucleotide-primed PCR of a microdissected homogeneously staining region containing sequences amplified from chromosome 12q13-q15. Based on fluorescence in situ hybridization, approximately 60% of the cosmids in the sublibrary were derived from the microdissected region. To demonstrate further the utility of this sublibrary, a 150-kb contig containing the SAS and CDK4 genes was constructed, as well as several additional contigs between CDK4 and MDM2. This study demonstrates the possibility of utilizing probes generated by microdissection for assembling band-specific sublibraries that are amenable to rapid screening with multiple markers.

Base Sequence↗
PubMed1996

Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries.

Three human chromosome 2-specific clone libraries were constructed and characterized. Chromosome 2-specific cosmid and fosmid clone libraries were constructed using flow-sorted DNA from the monochromosomal hybrid cell line GM10826. The cosmid and fosmid libraries consist of 38,496 and 26,400 arrayed clones, respectively, with an average size of 40 kb. Colony hybridization of a representative number of clones with both human and hamster genomic DNA probes demonstrates that between 58 and 66% of the clones in the flow-sorted libraries contain human inserts. Approximately 5% of the cosmid and fosmid clones are nonrecombinants. A chromosome 2-specific PAC library was also produced from the hybrid cell line GM10826. DNA from the hybrid cell line was cloned, and the human chromosome 2-specific clones were identified by colony hybridization. Approximately 5800 chromosome 2-specific PAC clones with an average insert size of approximately 85 kb were arrayed. Based on the size of the clones, the cosmid, fosmid, and PAC libraries are approximately 3.6x, approximately 2.5x, and approximately 1.9x, respectively in chromosomal coverage. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. The average number of clones identified for each STS in the library indicates the cosmid, fosmid, and PAC libraries to be approximately 3.2x, approximately 2.1x, and approximately 1.5x, respectively, in chromosome coverage. All except one of the 82 STSs were represented in the portions of the libraries screened.

Animals↗