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PubMed1994

Direct characterization of bovine microsatellites from cosmids: polymorphism and synteny mapping.

A (TG)8 oligonucleotide probe was used to screen 186 cosmids from a commercial bovine cosmid library. Of the 56 positive discovered, 7 were sequenced in the region of the microsatellite and analysed for polymorphism. These microsatellites, IDVGA-2, -3, -7, -8, -9, -10, -11 showed the following number of alleles and polymorphism information content (PIC) values (7/0.616, 8/0.693, 6/0.641, 5/0.643, 2/0.239, 10/0.844, 6/0.720). The microsatellites were also assigned to synteny groups as follows: IDVGA-2/U17, IDVGA-3/U16, IDVGA-7/U7, IDVGA-8/U29, IDVGA-9/U3, IDVGA-10/U19, IDVGA-11/A (probably U18).

Animals↗
PubMed2001

Genomic characterization of Helicobacter hepaticus: ordered cosmid library and comparative sequence analysis.

Helicobacter hepaticus is an important pathogen in laboratory mice and induces the development of liver tumors and gastrointestinal disease in susceptible strains of mice. In this study, a miniset of 36 cosmid clones from a genomic library of H. hepaticus was ordered and grouped into four large contigs representing approximately 1 Mb of the H. hepaticus genome using PCR, DNA sequencing, Southern and dot-blot hybridization and pulsed-field gel electrophoresis. From the 200-300 terminal nucleotide sequences of 38 cosmid clones, 56 coding regions were predicted, of which 51 were found to have orthologs in the public databases and five appeared to be unique to H. hepaticus. Of these 51 genes, 36 have orthologs in Helicobacter pylori and 25 display the highest sequence similarity to H. pylori. However, chromosomal positions of these genes are not conserved between these two helicobacters. In addition, 10 H. hepaticus genes had the highest sequence similarity to orthologs in Campylobacter jejuni. The GC content in a randomly selected 21-kb H. hepaticus genomic sequence was 35.8%, which approximates the average between H. pylori (39%) and C. jejuni (30.6%). These results demonstrate that: (1) H. hepaticus is more closely related to H. pylori than C. jejuni; (2) significant genomic alterations exist between H. hepaticus and H. pylori, including gene organization, protein sequences and GC content, probably in part due to specific adaptation to distinct ecological niches.

Animals↗
PubMed1990

High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones.

Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.

Blotting, Southern↗
PubMed1996

Two pathogenicity islands in uropathogenic Escherichia coli J96: cosmid cloning and sample sequencing.

Many of the virulence genes of pathogenic strains of Escherichia coli are carried in large multigene chromosomal segments called pathogenicity islands (PAIs) that are absent from normal fecal and laboratory K-12 strains of this bacterium. We are studying PAIs in order to better understand factors that govern virulence and to assess how such DNA segments are gained or lost during evolution. The isolation and sample sequencing of a set of 11 cosmid clones that cover all of one and much of a second large PAI in the uropathogenic E. coli J96 are described. These PAIs were mapped to the 64- and 94-min regions of the E. coli K-12 chromosome, which differ from the locations of three PAIs identified in other pathogenic E. coli strains. Analysis of the junction sequences with E. coli K-12-like DNAs showed that the insert at 94 min is within the 3' end of a phenylalanine tRNA gene, pheR, and is flanked by a 135-bp imperfect direct repeat. Analysis of the one junction recovered from the insert at 64 min indicated that it lies near another tRNA gene, pheV. To identify possible genes unique to these PAIs, 100 independent subclones of the cosmids were made by PstI digestion and ligation into a pBS+ plasmid and used in one-pass sample DNA sequencing from primer binding sites at the cloning site in the vector DNA. Database searches of the J96 PAI-specific sequences identified numerous instances in which the cloned DNAs shared significant sequence similarities to adhesins, toxins, and other virulence determinants of diverse pathogens. Several likely insertion sequence elements (IS100, IS630, and IS911) and conjugative R1 plasmid and P4 phage genes were also found. We propose that such mobile genetic elements may have facilitated the spread of virulence determinants within PAIs among bacteria.

Base Sequence↗
PubMed1994

FISH detection of trisomy 21 in interphase by the simultaneous use of two differentially labelled cosmid contigs.

Techniques have been reported in which fluorescence in situ hybridisation (FISH) and cosmid probes are used to detect trisomy 21 (and other abnormalities involving chromosomes X, Y, 13, and 18) on uncultured amniocytes. However the detection rate of trisomy 21 is lower than for the other anomalies owing to a larger number of uninformative results and false negatives. We report the simultaneous use of two differentially labelled cosmid contigs to improve the detection rate of trisomy 21 on uncultured amniocyte samples thus allowing the prenatal diagnosis of Down's syndrome even if only few labelled nuclei are available.

Amniotic Fluid↗
PubMed2000

Diagnostic analysis of the Rubinstein-Taybi syndrome: five cosmids should be used for microdeletion detection and low number of protein truncating mutations.

Rubinstein-Taybi syndrome (RTS) is a malformation syndrome characterised by facial abnormalities, broad thumbs, broad big toes, and mental retardation. In a subset of RTS patients, microdeletions, translocations, and inversions involving chromosome band 16p13.3 can be detected. We have previously shown that disruption of the human CREB binding protein (CREBBP or CBP) gene, either by these gross chromosomal rearrangements or by point mutations, leads to RTS. CBP is a large nuclear protein involved in transcription regulation, chromatin remodelling, and the integration of several different signal transduction pathways. Here we report diagnostic analysis of CBP in 194 RTS patients, divided into several subsets. In one case the mother is also suspect of having RTS. Analyses of the entire CBP gene by the protein truncation test showed 4/37 truncating mutations. Two point mutations, one 11 bp deletion, and one mutation affecting the splicing of the second exon were detected by subsequent sequencing. Screening the CBP gene for larger deletions, by using different cosmid probes in FISH, showed 14/171 microdeletions. Using five cosmid probes that contain the entire gene, we found 8/89 microdeletions of which 4/8 were 5' or interstitial. This last subset of microdeletions would not have been detected using the commonly used 3' probe RT1, showing the necessity of using all five probes.

Amino Acid Sequence↗
PubMed1988

Gene transfer of a putative mammalian K+ channel gene from genomic and cosmid DNA.

We have successfully transfected mouse genomic DNA containing a mutant putative K+ channel gene into murine LMTK- cells. Incorporation of the mutant gene allowed primary transformants to express the mutant cell phenotype of growth at a subthreshold K+ concentration and, furthermore, caused these primary transformants to display the same characteristics of growth and unidirectional K+ flux as the mutant cell line, LTK-1, from which the genomic DNA was derived. A cosmid genomic library was formed from LTK-1 mutant DNA and shown by transfection experiments to contain at least one unique cosmid clone containing a functional copy of the mutant putative K+ channel gene.

Acyclovir↗
PubMed1994

Characterization of a human chromosome 4 flow-sorted cosmid library.

A cosmid library has been constructed from a hamster x human hybrid cell line and gridded into 270 microtiter plates containing a total of 25,920 single colonies. Approximately 84% of the recombinants contain human material, with an average length of 29 kb. This library represents a nearly three-fold coverage of human chromosome 4. We investigated this library for presumptive genes, using a set of oligonucleotides detecting SpI and splice-site consensus sequences. The presence of simple repeat motifs was investigated in the cosmids using the oligonucleotides (GGATTT)3, (GGAT)4, (CAC)5, (GCC)5, (AGC)5, (GATA)4, (GACA)4, and (CA)8 as hybridization probes.

Animals↗
PubMed1995

A high-resolution cytogenetic map of human chromosome 2: localization of 434 cosmid markers by direct R-banding fluorescence in situ hybridization.

We have constructed a high-resolution cytogenetic map of human chromosome 2 with 434 newly isolated cosmid markers by means of direct R-banding fluorescence in situ hybridization. Two markers were mapped to the centromeric region, 173 to the short arm, and 259 to the long arm. The clones were evenly distributed along the entire chromosome, although a tendency toward clustering in R-positive bands was observed. The mapped cosmids provide useful landmarks for construction of a contig map and for positional cloning of cancer-associated genes, as well as genes responsible for hereditary diseases.

Animals↗
PubMed1995

Mapping of an ordered set of 14 cosmids to human chromosome 12p by two-color in situ hybridization.

To map human chromosome 12p aberrations by fluorescence in situ hybridization (FISH), cosmids were isolated or obtained for 14 known 12p loci (D12S119, D12S158, D12S178, D12S370, D12S380E, A2M, CACNL1A1, FGF6, GAPD, KRAS2, PRB1, PZP, TPI1, and VWF). Using two-color FISH with three labeled probes to interphase nuclei, and to prometaphase chromosomes where possible, the order of these loci was sequentially determined to be pter-D12S158-D12S380E-CACNL1A1-FGF6- D12S370-VWF-GAPD-TPI1-A2M-PZP-PRB1-D12S 178-D12S119-KRAS2-cen. Two cell lines were analyzed with this set of cosmids. The EBV-transformed cell line TA carries a der(12) with a deletion of bands 12p13.1-->p11.2.D12S178, D12S119, and KRAS2 were absent in the der(12), whereas the other loci were present. The second cell line, GM01203A, exhibits a balanced t(4;12)(4q25; 12p13.3) with a breakpoint between FGF6 and D12S370.

Animals↗
PubMed1995

Cosmid contigs from the tuberous sclerosis candidate region on chromosome 9q34.

Tuberous sclerosis (TSC) is a heterogeneous multisystem disorder with loci on 9q34 (TSC1) and 16p13.3 (TSC2). The TSC2 gene has recently been isolated, while the TSC1 gene has been mapped to a 5-cM region between the markers D9S149 and D9S114. In our effort to localise and clone TSC1, we have obtained three adjacent cosmid contigs that cover the core of the candidate region. The three contigs comprise approximately 600 kb and include 80 cosmids, 2 P1 clones, 1 YAC, 5 anonymous markers and 4 sequence-tagged sites. The ABO blood group locus, the Surfeit gene cluster, the dopamine beta-hydroxylase gene (DBH) and VAV2, a homologue of the vav oncogene, have all been mapped within the contigs. Exon trapping and mutation screening experiments, aimed at identifying the TSC1 gene, are currently in progress.

Bacteriophage P1↗
PubMed1996

Transduction of a cosmid with the R4 phage cos sequence by heterogeneous actinophages, SPA10 and SPA38.

A cosmid, pR4C1, composed of the actinophage R4 cos sequence and Streptomyces plasmid pIJ365, was encapsidated in R4 phage particles in vivo. [T. Morino et al., Mol. Gen. Genet., 198, 228-233 (1985)] In this report a cosmid derivative, pR4C4, is shown to be also encapsidated by heterogeneous actinophages, SPA10 and SPA38, and transferred to Streptomyces lividans. Use of this transduction and conservation of the DNA packaging mechanism are discussed.

Antigens, Bacterial↗
PubMed1996

Preparation of pure plasmid or cosmid DNA using single-strand affinity matrix and gel-filtration spin columns.

A rapid method has been developed for ultrapure plasmid or cosmid DNA isolation from ten-mL to several hundred-mL cultures of Escherichia coli (midi to maxi prep). A cleared lysate is prepared by alkaline lysis, followed by a quick alcohol precipitation step. Denatured bacterial DNA and RNA having at least 20 nucleotides of single-stranded regions are removed from the supercoiled plasmid by binding strongly to the single-strand affinity matrix (SSAMTM). Plasmid DNA is then effectively purified on a gel-filtration spin column to remove SSAM, proteins, small RNA and salts. This method produces consistent yields of high-quality plasmids that are suitable for use in many molecular biology applications. In addition, recombinant cosmids of approximately 46 kb can be purified intact, free of chromosomal DNA.

Chromatography, Affinity↗
PubMed1997

Isolation and characterisation of cosmids to intervals within a 4.5Mb region at 6p21.3.

The gene responsible for hereditary haemochromatosis (HH) has recently been identified. One mutation in this gene, termed HFE, has been found in all Australian HH patients. We previously identified a predominant HH ancestral haplotype covering 4.5Mb at 6p21.3, and showed that patients with two copies of this haplotype express a more severe form of the disorder. One key question to now be resolved is why haplotype related variation in phenotypic expression of HH is present if all patients tested have the same HFE mutation. A cosmid resource covering the 4.5Mb HH ancestral haplo type region was obtained. These cosmids provide the material for the completion of a transcript map of this region, and will assist the identification of candidate modifiers of HFE expression.

Chromosomes, Artificial, Yeast↗
PubMed1999

[Construction of a pufferfish gridded cosmid library].

OBJECTIVE: Pufferfish is a newly-established model organism in human genome research, which has been demonstrated its unique advantages in comparative genomics. Construction of a genomic library of pufferfish is the prerequisite to any studies of its genome. METHODS: Genomic DNA from Fugu rubripes, a species distribute only in Japan and China seas, was used to construct a library in a novel cosmid vector, named sCOGH2, which contains an exon-trapping cassette. RESULTS: This library is composed of 57,600 clones gridded in 60,096-well cell culture plates, one clone each well. The average size of the inserts is around 35 kb. It provides 99.2% probability to find any unique DNA fragment of pufferfish in this library. The clones of this library can survive after more than 10 times freeze-thaw, also demonstrating high stability in propagation. The clones shone highly positive signals when hybridized with the pufferfish genomic DNA. CONCLUSIONS: The library accords with qualitative demands of a cosmid library.

Animals↗
PubMed1992

A rapid method for cosmid cloning.

We present a method for genomic library construction using cosmid vectors. With a combination of backfilling with Klenow enzyme and a cosmid vector with two cos sites, a DNA bank in excess of 500,000 clones can be made from 10 micrograms of genomic DNA. The method is more rapid than conventional protocols because size fractionation of target DNA is not necessary. A further advantage is that libraries can be made from relatively small amounts of genomic DNA.

Cloning, Molecular↗