Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Cosmids”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources
PubMed1997

FISH mapping of three bovine cosmids to cattle, goat, sheep and buffalo X chromosomes.

Contrary to extensive banding similarities of the autosomes, there are variations in the morphology as well as banding patterns of the X chromosomes of various species of bovidae family. We used three bovine X chromosome specific cosmid clones for fluorescence in situ hybridization mapping on cattle, goat, sheep, and river buffalo chromosomes to identify homologous regions in the genomes of the four species. The three cosmids (cIOBT 314, 945, and 1489) mapped to well spaced locations on buffalo X, and both arms of cattle X chromosome. However, of the three probes, cosmids cIOBT 314 and 1489 showed no hybridization signal on any of the goat-sheep chromosomes. Incidentally, the two cosmids show correspondence to those regions of the buffalo X chromosome which are proposed to be lost during the structural reshuffle of the latter, leading to sheep/goat X chromosomes. The lack of hybridization signal of the two cosmids in sheep and goat, therefore, indicates that these DNA sequences are most likely absent in the two genomes and probably correspond to the segments lost during evolution of their X chromosomes.

Animals↗
PubMed1989

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector.

The Bacteroides conjugal tetracycline resistance (Tcr) elements appear not to be plasmids. In many cases, resistance to erythromycin (Emr) is cotransferred with Tcr. Using a newly constructed shuttle cosmid, pNJR1, we cloned 44 to 50 kilobase pairs of a conjugal Tcr Emr element on overlapping cosmid clones. Cosmid libraries were made in Escherichia coli with DNA from the original clinical Bacteroides thetaiotaomicron DOT strain containing Tcr Emr-DOT or from a Bacteroides uniformis Tcr Emr-DOT transconjugant strain. The cosmid clones were mobilized from E. coli into B. uniformis in groups of 10 to 20 per filter mating, with selection for Tcr or Emr transconjugants. The Tcr and Emr genes were cloned both separately and together on 30-kilobase-pair fragments. Several of the Tcr clones also contained transfer genes that permitted self-transfer of the cosmid from B. uniformis donors to E. coli or B. uniformis recipients. Neither the Tcr nor the Emr gene conferred resistance on E. coli, and the transfer-proficient clones did not self-transfer out of E. coli. Southern blot analysis was used to compare DNA from independently isolated Bacteroides strains carrying conjugal Tcr or Tcr Emr elements and their respective B. uniformis transconjugants. Results of these analyses indicate that there are large regions of homology, including regions outside the Tcr and Emr genes, but that the elements are not identical. Some Tcr clones contained a region which hybridized to chromosomal DNA from the wild-type B. uniformis recipient strain that did not carry the Tcr Emr-DOT element. This region of homology appeared not to be a junction fragment. It was not required in a Bacteroides recipient for successful transfer of the Tcr Emr element. Although we are not sure we have cloned a junction fragment between the Tcr Emr-DOT element and the B. uniformis chromosome, the preliminary function and restriction map appears to be linear.

Bacteroides↗
PubMed2000

Generation of mutant murine cytomegalovirus strains from overlapping cosmid and plasmid clones.

We have developed a cosmid and plasmid system to generate mutant strains of murine cytomegalovirus (MCMV). The system is based on a series of seven overlapping cosmid clones that regenerate MCMV when cotransfected into mouse cells. The unaltered cosmids produce MCMV that is indistinguishable from wild-type MCMV based on restriction enzyme digest patterns of virus DNA and growth rates both in vitro and in vivo. Analysis of viral DNA from plaque-purified recombinant isolates taken from in vitro and in vivo stocks indicated that regeneration did not introduce novel mutations in the recombinant viral genomes. Isolation of specific genes and subsequent generation of specific mutant MCMVs was accomplished by replacement of cosmids with overlapping plasmid subclones. A new vector, PmeSUB, featuring a multiple cloning site and a stringent origin of replication, was constructed to make large subclones for use with smaller subclones containing the gene of interest. The utility of this system was demonstrated by the generation of two different mutant MCMVs from different combinations of overlapping plasmid subclones of one cosmid. The advantages of this system are that (i) target genes are maintained as small clones making them amenable to standard in vitro mutagenesis manipulations and that (ii) no reporter or selection genes are necessary to identify mutants.

Animals↗
PubMed1986

Eucaryotic chromosome transfer: production of a murine-specific cosmid library from a neor-linked fragment of murine chromosome 17.

We recently developed a procedure for the molecular analysis of specific mammalian chromosomal fragments. This procedure allows for the transfer of contiguous chromosomal fragments, varying in size from a fraction to several centimorgans in length, from the donor cell of one species into a recipient cell of a different species. Specifically, we inserted a neor gene, encoded by a recombinant retrovirus, into the murine major histocompatibility complex (MHC). Metaphase chromosome transfers with this neor-tagged chromosome into recipient hamster, primate, and canine fibroblasts produced a panel of primary neor transferents, each containing a portion of, or all of, the murine MHC. A cosmid library was made from one such transferent, CHMD(D)B1. Cosmid clones were divided, using species-specific repeat probes, into those containing murine (donor) DNA sequences and those containing sequences derived from the recipient cell. The murine-specific cosmids were clustered into overlapping DNA segments by restriction enzyme digest analysis of the cosmid DNAs coupled with Southern blot analysis with, as probes, murine-specific repeat sequences and nick-translated murine genomic DNA. These cosmid clusters were analyzed for their position within or outside of the MHC, using recombinant mouse strains, and for the presence within them of known murine MHC genes.

Animals↗
PubMed1996

Characterization of a flow-sorted human chromosome 10 cosmid library by FISH.

Fluoresence in situ hybridization (FISH) was used to localize cosmids to regions of human chromosome 10. A total of 301 cosmids were selected randomly from a flow-sorted human chromosome 10 cosmid library constructed from human x hamster cell line 762-8A and arrayed in microtiter storage dishes. Over 70% (211/301) of the cosmids mapped to unique regions of chromosome 10. About 7% (22/301) produced multiple hybridization signals indicative of chimeric clones or sequences repeated at low copy number. Three cosmids (3/301, or 1%) hybridized to the centromeric regions of chromosome 10 and one or more other human chromosomes. About 19% (59/301) consisted mostly or entirely of hamster DNA inserts, and about 2% (6/301) appeared to be nonrecombinants.

Animals↗
PubMed1996

The telomeric and centromeric ends of HLA class I: MCD maps of YAC derived cosmids and sequence analysis of 740 kb of genomic DNA.

We previously isolated and characterized a set of overlapping yeast artificial chromosome (YAC) clones spanning 2.4 Mb, including the entire MHC class I region, as a first step towards a detailed genetic analysis. We report here the genomic sequence of the two ends of HLA class I. The centromeric portion of HLA class I, extending from TNF to HLA-C was determined using two different sources of genomic DNA. As a first source, we sequenced cosmids (provided by T. Spies) derived from a total human DNA library which were mapped with conventional restriction digestion and fingerprinting. A second more generalizable approach was used to obtain cosmids for the remainder of the region. The new technology of Multiple-Complete-Digest (MCD) mapping, developed in Maynard Olson's laboratory, was used to map cosmids derived from YACs. This technique involves screening deep cosmid libraries derived from selected YACs and subjecting the cosmids to complete digestion with restriction enzymes, followed by computational assembly into completed maps. This method was also used to obtain material for sequence from three overlapping YACs covering a contiguous region from HLA-G to a point 330 kb telomeric. Among the plethora of new genetic information is a detailed picture of the organization of new multigene families contained within the telomeric end and of members of families spread throughout HLA class I. A clear relationship between the telomeric and centromeric ends of HLA class I has been defined, suggesting that large portions of these regions derived from a common ancestor. Our results demonstrate genomic sequencing to be one of the most effective and efficient means of identifying new genes, yielding information about genomic structure, regulation, and offering new insights into the meaning of physical relationships among functionally interacting genes.

Centromere↗
PubMed1999

Analysis of two cosmid clones from chromosome 4 of Drosophila melanogaster reveals two new genes amid an unusual arrangement of repeated sequences.

Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, we are assembling a physical map of this chromosome. Here we report the sequence of two cosmids representing approximately 5% of the polytenized region. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis, which are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus (ci) gene and two new genes: that a gene with a predicted amino acid sequence similar to ribosomal protein S3a which is consistent with the Minute(4)101 locus thought to be in the region, and a novel member of the protein family that includes plexin and met-hepatocyte growth factor receptor. The other cosmid contains only the two short 5'-most exons from the zinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the beta-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome.

Amino Acid Sequence↗
PubMed1992

High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones.

Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier to efforts to construct a contiguous cloned library of a genome. We have encountered such sequences during our efforts to isolate cosmid and bacteriophage lambda clones carrying members of the human type 2 cystatin gene family. Several cosmid clones constructed in the pWE 15 vector did not survive purification, and using standard techniques, we were unable to obtain significant amounts of cosmid DNA from those clones we could purify. Similarly, several lambda bacteriophage clones constructed in the lambda DASH II vector could not be purified, and those lambda clones we were able to isolate gave low titers in liquid lysates. In this paper, we describe generally applicable methods for preparing high yields of recombinant DNA from such recalcitrant cosmid and lambda clones constructed in these vectors.

Bacteriophage lambda↗
PubMed1989

Identification of two cosmids derived from within chromosomal band 3p21.1 that contain clusters of rare restriction sites and evolutionarily conserved sequences.

We have isolated large numbers of human recombinants from a cosmid library constructed from an interspecific (hamster/human) somatic cell hybrid whose only human component is an intact chromosome 3. Unique sequence probes were isolated from these recombinants and were used to localize them along the length of chromosome 3 by hybridization to a somatic cell hybrid deletion panel. We identified two cosmids, cA84 (D3S92) and cA199 (D3S93), derived from within chromosomal band 3p21.1. Both cosmids contained multiple rare restriction sites that were tightly clustered within the cosmids. We have therefore identified, in a region consistently deleted in a variety of lung cancers, two cosmids that may contain genes that are candidates for involvement in lung cancer.

Animals↗
PubMed1993

Isolation of cDNA clones mapping around DXS178: a search for human X-linked agammaglobulinaemia gene using yeast artificial chromosomes, cosmids and direct cDNA selection.

X-linked agammaglobulinaemia (XLA) was previously mapped using genetic linkage analysis to Xq22. No recombinants have been found so far between the XLA locus and DXS178 in over 30 informative meioses. Two overlapping yeast artificial chromosomes (YACs), which cover a region of approximately 1000 kb around DXS178 and form part of a larger YAC contig, were hybridised to an ordered cosmid library constructed from a human fibroblast cell line with karyotype 49,XXXXX. Positive cosmids were gridded in high density arrays onto nylon filters and rescreened with a series of YACs, YAC end probes and additional markers from the region to assign cosmids into intervals. A 640 kb-YAC hybridising to p212 (DXS178) was used for direct selection of PCR-amplified cDNA from two cDNA libraries. To identify the cosmids containing transcribed sequences the enriched cDNA from each library was radiolabelled and hybridised back to positive clones which mapped to the region of interest. These clones were then hybridised to the subcloned enriched cDNA sublibraries. Candidate cDNA clones were isolated, grouped within the region of interest and their position was confirmed by mapping back to the cosmids.

Agammaglobulinemia↗
PubMedDate not supplied

Double color in situ hybridization of alpha-satellite chromosome 13, 21 specific cosmid clones for a rapid screening of their specificity.

Double color in situ hybridization was used to determine the specificity of cloned alpha-satellite cosmid DNA sequences belonging to satellite DNA sequences shared by chromosomes 13 and 21. It was determined that cosmid clone cos56D8 is more specific to chromosome 13, cosmid clone cos37E is more specific to chromosome 21 and cosmid clones cosA5130 is specific to both chromosomes 13 and 21. These new cosmid probes could bw useful in the molecular-cytogenetic analysis of trisomic cells at Patau or Down syndromes as well as in analysis of marker chromosomes originated from the chromosomes 13 and 21.

Cells, Cultured↗
PubMed1984

Cosmid clones derived from both euchromatic and heterochromatic regions of the human Y chromosome.

Clones containing sequences derived from the human Y chromosome have been isolated from cosmid libraries of a human-mouse hybrid cell line. These libraries were constructed in the new expression vectors Homer V and Homer VI. The collection of cosmids isolated is enriched for unique sequence DNA and only a few of the cosmids contain the tandemly repeated sequences which constitute a major portion of the Y chromosome. Three cosmids have been studied in detail. One cosmid shows extensive homology over at least 20 kb with the long arm of the X chromosome; this homology is outside the predicted homology region required for sex chromosome pairing. The other two clones contain unique sequences specific to the Y chromosome and both map to the heterochromatic region of the Y chromosome long arm.

Animals↗
PubMed1985

A highly polymorphic locus in human DNA revealed by cosmid-derived probes.

Human gene mapping would be greatly facilitated if marker loci with sufficient heterozygosity were generally available. As a source of such markers, we have used cosmids from a human genomic library. We have developed a rapid method for screening random cosmids to identify those that are homologous to genomic regions especially rich in restriction fragment length polymorphisms. This method allows whole cosmids to be used as probes against Southern transfers of genomic DNA; regions of cosmid probes homologous to repeated genomic sequences are rendered unable to hybridize with Southern transfers by prehybridization of the probes with a vast excess of nonradioactive genomic DNA. From one cosmid identified by this procedure, we have isolated three single-copy probes that collectively identify seven polymorphic loci. Of 56 unrelated individuals, 52 were heterozygous at one or more of these marker loci.

Chromosome Mapping↗
PubMed1985

Efficient cloning of single-copy genes using specialized cosmid vectors: isolation of mutant dihydrofolate reductase genes.

A method for the efficient cloning of single-copy genes from restriction digests of mammalian DNA is described. The method is illustrated by the cloning of several mutant genes as well as the wild-type gene for Chinese hamster dihydrofolate reductase (DHFR; 7,8-dihydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). This gene is isolated within a 41-kilobase Bgl I fragment by using cosmid (plasmids containing a cohesive-end site) vectors that have been constructed especially for this purpose. Two cosmids are used: one contains a short region from the 5' flanking region of the dhfr gene, and the other contains a short region from the 3' flanking region. These two regions contain the Bgl I sites that bound the dhfr gene. Bgl I leaves staggered ends that are different depending on the DNA sequence within the enzyme binding site. When these cosmids are cut with Bgl I and hybridized with total Bgl I-cut genomic DNA, they preferentially associate with the fragment bearing the dhfr gene, since it has the same Bgl I ends. An approximately 500-fold enrichment for the dhfr gene in cosmid libraries from Chinese hamster ovary cells was achieved by using this method coupled with a single-step size fractionation. As a result, only several hundred cosmid colonies need to be screened in order to clone a dhfr gene from a particular mutant Chinese hamster ovary cell. This method should facilitate the repetitive cloning of any gene or gene fragment.

Animals↗
PubMed1982

An analysis of cosmid clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glycoproteins are clustered in the genome.

Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC). We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli. Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used. We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes. In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome. Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses. We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment.

Animals↗
PubMed1982

The construction of cosmid libraries which can be used to transform eukaryotic cells.

Cosmid vectors have been developed which carry selective markers for growth in bacteria (beta lactamase gene) and animal cells (the Herpes Simplex virus thymidine kinase gene, the transposon Tn-5 aminoglycosyl 3' phosphotransferase gene and the E. coli guanine phosphoribosyltransferase gene). The design of the cosmids allows the exchange of the eukaryotic markers in recombinant cosmids. Human and mouse cosmid libraries containing DNA inserts of about 40kb have been generated by an improved method. Several clones from the human beta-globin locus were isolated. These cosmids transform mouse L cells at high efficiency in both circular and linear form. The newly introduced genes are expressed accurately in L cells.

Animals↗
PubMed1981

Cloning of multiple copies of immunoglobulin variable kappa genes in cosmid vectors.

The possibility of cloning large segments of DNA in cosmid vectors offers distinct advantages, in particular for the study of multigene families. Large size fragments of mouse embryo DNA were successfully cloned in the cosmid pHC 79. Twelve recombinants hybridizing specifically to an immunoglobulin kappa chain variable region probe were identified. In 9 of these recombinants, the size of the insert ranges from 30 to 43 kilobases. Factors affecting the cloning efficiency of a complex mammalian genome in cosmids were studied. The stability of these recombinant cosmids and the preparation of recombinant cosmid DNA are also discussed.

Animals↗
PubMed1985

Cosmid cloning of Rickettsia prowazekii antigens in Escherichia coli K-12.

Rickettsia prowazekii DNA was partially digested with Sau3A or HindIII, ligated with the cosmid vector pHC79, packaged in vitro, and transduced into Escherichia coli HB101. Cosmid cloning of Sau3A-digested rickettsial DNA yielded 1,288 ampicillin-resistant colonies; 798 cosmid clones resulted with HindIII-digested rickettsial DNA. Chimeric cosmid DNA was extracted from the latter gene bank, digested to completion with HindIII, and compared by agarose gel electrophoresis with a HindIII digest of rickettsial genomic DNA. The two digestion profiles were quite similar in their overall banding patterns, indicating that the clone bank was significantly representative of the rickettsial genome. When both clone banks were screened for expression of rickettsial antigens by enzyme-linked immunosorbent assay with goat anti-R. prowazekii serum, ca. 20% of the clones reacted positively. Two clones were randomly selected for more detailed analysis. Each contained a large chimeric plasmid (40.2 and 38.1 kilobases) which apparently yielded smaller deletion derivatives (13.6 and 12.6 kilobases) when transformed into an E. coli minicell strain. Each recombinant plasmid directed the synthesis of new protein species not observed in control minicells. One of the clones produced a 51,000-dalton protein in minicells, which comigrated with a protein reactive with anti-R. prowazekii serum. This protein was not present in negative controls. When antibodies to this protein were incubated with a Western blot of rickettsial total protein, they bound to a 52,000-dalton polypeptide. Hence, the cloned rickettsial gene product in E. coli corresponds to a protein of similar size in R. prowazekii. This study demonstrates the feasibility of cosmid cloning of rickettsial antigens in E. coli.

Antigens, Bacterial↗