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PubMed1997

Interpreting a sequenced genome: toward a cosmid transgenic library of Caenorhabditis elegans.

We have generated a library of transgenic Caenorhabditis elegans strains that carry sequenced cosmids from the genome of the nematode. Each strain carries an extrachromosomal array containing a single cosmid, sequenced by the C. elegans Genome Sequencing Consortium, and a dominate Rol-6 marker. More than 500 transgenic strains representing 250 cosmids have been constructed. Collectively, these strains contain approximately 8 Mb of sequence data, or approximately 8% of the C. elegans genome. The transgenic strains are being used to rescue mutant phenotypes, resulting in a high-resolution map alignment of the genetic, physical, and DNA sequence maps of the nematode. We have chosen the region of chromosome III deleted by sDf127 and not covered by the duplication sDp8(III;I) as a starting point for a systematic correlation of mutant phenotypes with nucleotide sequence. In this defined region, we have identified 10 new essential genes whose mutant phenotypes range from developmental arrest at early larva, to maternal effect lethal. To date, 8 of these 10 essential genes have been rescued. In this region, these rescues represent approximately 10% of the genes predicted by GENEFINDER and considerably enhance the map alignment. Furthermore, this alignment facilitates future efforts to physically position and clone other genes in the region. [Updated information about the Transgenic Library is available via the Internet at http://darwin.mbb.sfu.ca/imbb/dbaillie/cos mid.html.]

Animals↗

PubMed1988

Cosmid cloning of five Zymomonas trp genes by complementation of Escherichia coli and Pseudomonas putida trp mutants.

A library of Zymomonas mobilis genomic DNA was constructed in the broad-host-range cosmid pLAFR1. The library was mobilized into a variety of Escherichia coli and Pseudomonas putida trp mutants by using the helper plasmid pRK2013. Five Z. mobilis trp genes were identified by the ability to complement the trp mutants. The trpF, trpB, and trpA genes were on one cosmid, while the trpD and trpC genes were on two separate cosmids. The organization of the Z. mobilis trp genes seems to be similar to the organization found in Rhizobium spp., Acinetobacter calcoaceticus, and Pseudomonas acidovorans. The trpF, trpB, and trpA genes appeared to be linked, but they were not closely associated with trpD or trpC genes.

Chromosome Mapping↗

PubMed1996

Defined large-scale alterations of the human cytomegalovirus genome constructed by cotransfection of overlapping cosmids.

We have constructed defined human cytomegalovirus (CMV) mutants by cotransfecting overlapping cosmid clones spanning the 230-kbp genome. Using this strategy, we have introduced a 13-kbp region of DNA from a virulent strain of CMV into a defined position within the avirulent CMV(Towne) genome. Although more than 80% of the genome of these recombinant viruses was derived from Towne DNA, their plaque morphology more closely resembled that of Toledo. To date, CMV is the largest virus and requires the greatest number of cosmids to be regenerated via overlapping cosmid cotransfection.

Cell Line↗

PubMed1993

Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. We have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Our available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5'-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Our evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed from several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeasts, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Our analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B.

Amino Acid Sequence↗

PubMed1994

Isolation and characterisation of a panel of cosmids which allows unequivocal identification of chromosome deletions involving the RB1 gene using fluorescence in situ hybridisation.

A series of cosmids covering the majority of the RB1 gene have been isolated from a flow sorted human chromosome 13 specific library. Using fluorescence in situ hybridisation these cosmids were all shown to hybridise to the 13q14 region but not to chromosomes known to carry subband deletions involving the RB1 gene. This panel of cosmids, therefore, can be used objectively for identification of RB1 gene deletions in tumour and normal cells.

Carboxylesterase↗

PubMed1990

Rapid subchromosomal localization of cosmids by nonradioactive in situ hybridization.

A rapid method for localizing large numbers of complete cosmids by nonradioactive in situ hybridization is described. The cosmids are nick translated in the presence of biotin-16-dUTP, incubated with an excess of sonicated human DNA, and used as a probe for in situ hybridization. Sites of hybridization are detected by successive treatments with FITC-labeled avidin and biotinylated anti-avidin antibody. Fifty-two cosmids were localized on chromosome 16 in 5 d relative to translocation breakpoints contained in two cell lines. Rapid identification of chromosome 16 was achieved by cohybridization with a chromosome 16-specific centromeric repeat probe.

Cell Line, Transformed↗

PubMed1994

Isolation and mapping of 328 new cosmid markers on human chromosome 8: construction of a high-resolution cytogenetic map of chromosome 8 with 416 markers.

We have determined the chromosomal localizations of newly isolated cosmids by fluorescent in situ hybridization (FISH) on prometaphase R-banded chromosomes and have constructed a high-resolution cytogenetic map for human chromosome 8 with 416 cosmid markers, including 328 new markers and 88 reported previously. Of the 416 markers, 229 were mapped to the long arm of chromosome 8, 181 to the short arm, and 6 to the centromere. Although the clones were scattered throughout the chromosome, they were concentrated in R-positive bands. Since the estimated physical length of chromosome 8 is 135 Mb, the overall average distance between loci is 320 kb, but the average separation of loci on R-positive bands is nearly 130-200 kb. This cytogenetic map will serve as a resource for efforts to characterize chromosomal and molecular aberrations involved in cancers, to clone genes associated with hereditary diseases, and to construct a detailed physical map of large electrophoretic fragments and/or contiguous cosmids and yeast artificial chromosomes.

Animals↗

PubMed1995

A human 12p-derived cosmid hybridizing to subsets of human and chimpanzee telomeres.

The chromosomal locations of a cosmid-linking clone, cLN12-43, isolated from band 12p13 in man and containing subtelomeric repeat sequences, were determined by FISH. The cosmid hybridized to the telomeres of 3q, 6p, 9p, 12p, 15q, 19p, 20p, and 20q at high frequency and showed variable hybridization to 1p, 6q, and 9q. Two interstitial loci were detected, one at bands 2q13-->q14 and the other at band 12q13. The former corresponds to the fusion point of chimpanzee chromosomes 12 and 13, resulting in the human chromosome 2, whereas the latter may represent a similar but more ancient fusion event. The cLN12-43 cosmid was also shown to hybridize to a set of chimpanzee telomeres.

Animals↗

PubMed1995

FISH mapping of 22 novel X chromosome cosmids and the isolation of a novel microsatellite on proximal Xp.

Twenty-two X-linked cosmid clones have been localised by fluorescence in situ hybridisation (FISH). Twelve map to the long arm of the X chromosome and 10 to the short arm. Seven of the latter cosmids and an additional one were mapped by two colour FISH relative to reference markers DXS7 and DXS426 in proximal Xp. Finer localisation of one of the cosmids, namely HX43 was achieved by isolation of a microsatellite followed by genetic mapping with respect to reference markers in the region.

Base Sequence↗

PubMed1996

Partial CviJI digestion as an alternative approach to generate cosmid sublibraries for large-scale sequencing projects.

We demonstrate an alternative method for the generation of random subclones for large-scale shotgun human DNA sequencing projects. Miniprep DNA from a human cosmid clone was partially digested with CviJI, size-fractionated by agarose gel electrophoresis and cloned into bacteriophage M13. A library consisting of 10(5) subclones of 1.1 kb average size was recovered from one gel fraction containing approximately 300 ng of partially digested DNA. DNA sequences from an initial 103 subclones demonstrate that 100 of the subclones cover 90% of the cosmid, which is close to the 92% expected if the subclones were generated at random. DNA sequences from three of the subclones did not match the cosmid sequences, establishing that miniprep DNA can be used for library construction with little concern for contamination with genomic E. coli DNA. The use of CviJI to generate random DNA fragments thus offers a simple alternative to other commonly used fragmentation methods, such as shearing or sonication, for the generation of random sublibraries for large-scale human shotgun DNA sequencing projects.

Bacteriophages↗

PubMed1994

Construction of a cosmid contig and of an EcoRI restriction map of yeast chromosome X.

We report here the construction of a complete physical map of the chromosome X of yeast Saccharomyces cerevisiae. Fragments resulting from partial Sau3AI digestion of DNA from a diploid strain derived from S288C were ligated to linearized pWE15, a cosmid vector with T3 and T7 promoters. Another library, made in the cosmid vector pOU61 cos, that lacks T3 and T7 promoters, was also used as a source of target clones. Chromosome-X-specific clones were sorted out by hybridization with radiolabelled pulse-field-gel-purified chromosome X as a probe. Then, 254 cosmids were ordered by walking from one to another by hybridization with end-specific T3 or T7 RNA transcripts as probes. The construction was put to the test by hybridization with a battery of chromosome X gene markers, that showed that the physical map and the genetic map were colinear. The validity of the contig was further strengthened by the results of chromosome nested fractionation with meganuclease I-SceI. An EcoRI restriction map of the contig enabled further verification and measurement of the total length of the contig, that was found to be approximately 700 kb in size. In addition to providing a base for the ongoing yeast genome sequencing project, the physical map can be used to map any sequence belonging to chromosome X.

Chromosomes, Fungal↗

PubMed1998

[Study of alpha-satellite DNA in cosmid libraries, specific for chromosomes 13, 21, and 22, using fluorescence in situ hybridization].

Fluorescent in situ hybridization (FISH) was employed in mapping the alpha-satellite DNA that was revealed in the cosmid libraries specific for human chromosomes 13, 21, and 22. In total, 131 clones were revealed. They contained various elements of centromeric alphoid DNA sequences of acrocentric chromosomes, including those located close to SINEs, LINEs, and classical satellite sequences. The heterochromatin of acrocentric chromosomes was shown to contain two different groups of alphoid sequences: (1) those immediately adjacent to the centromeric regions (alpha 13-1, alpha 21-1, and alpha 22-1 loci) and (2) those located in the short arm of acrocentric chromosomes (alpha 13-2, alpha 21-2, and alpha 22-2 loci). Alphoid DNA sequences from the alpha 13-2, alpha 21-2, and alpha 22-2 loci are apparently not involved in the formation of centromeres and are absent from mitotically stable marker chromosomes with a deleted short arm. Robertsonian translocations t(13q; 21q) and t(14q; 22q), and chromosome 21p-. The heterochromatic regions of chromosomes 13, 21, and 22 were also shown to contain relatively chromosome-specific repetitive sequences of various alphoid DNA families, whose numerous copies occur in other chromosomes. Pools of centromeric alphoid cosmids can be of use in further studies of the structural and functional properties of heterochromatic DNA and the identification of centromeric sequences. Moreover, these clones can be employed in high-resolution mapping and in sequencing the heterochromatic regions of the human genome. The detailed FISH analysis of numerous alphoid cosmid clones allowed the identification of several new, highly specific DNA probes of molecular cytogenetic studies--in particular, the interphase and metaphase analyses of chromosomes 2, 9, 11, 14, 15, 16, 18, 20, 21-13, 22-14, and X.

Chromosomes, Human, Pair 13↗

PubMed1989

Ig VH, DH, and JH germ-line gene segments linked by overlapping cosmid clones of rabbit DNA.

Overlapping cosmid clones of rabbit germ-line DNA containing VH, DH and JH gene segments were isolated. The map of this cluster of cosmid clones indicated that the rabbit VH and JH regions were separated by 63 kb. Hybridization of Southern blots of these cosmid clones with two different DH segment probes identified a total of six DH segments within the region between the VH and JH regions. The nucleotide sequences of the JH region and one of the DH segments have been determined. The DH segment has conserved heptamer and nonamer sequences separated by 12 and 11 bp at the 3' and 5' sides, respectively, of the coding region and hence, appears to be a functional gene. The nucleotide sequence of the JH region revealed four functional JH gene segments and one JH pseudogene. Inasmuch as the JH region had previously been linked by contiguous overlapping clones with C mu, C gamma, C epsilon, and one C alpha gene, this VH-DH-JH cluster and the clones containing the Ig H chain C region genes represent 190 kb of contiguous germ-line DNA of the Ig H chain locus.

Amino Acid Sequence↗

PubMed1989

Identification and characterization of 23 RFLP loci by screening random cosmid genomic clones.

As part of our search for polymorphic DNA probes, we have screened cosmids from a human genomic DNA library for their ability to reveal RFLPs. A total of 101 randomly isolated cosmid clones were tested in Southern hybridizations for polymorphic band patterns. Fifty-four of these clones revealed RFLPs with one or more of nine restriction enzymes. Twenty-three of these clones have been further characterized and assigned to 10 different chromosomes by linkage analysis or by hybridization to panels of human-hamster hybrid cell lines. Fifteen of the probes have heterozygosities greater than or equal to .5. The relative efficiency of RsaI and PstI restriction enzymes in detecting polymorphism was different from results obtained with libraries constructed in bacteriophage vectors. Screening randomly selected cosmid probes is an efficient method for detecting RFLPs.

Alleles↗

PubMed1986

Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to chromosome 19.

We have discovered and characterized a compound polymorphic locus on chromosome 19, defined by an arbitrary genomic DNA segment cloned into a cosmid vector. Four different restriction fragment length polymorphisms with minor allele frequencies equal to or greater than 10% are revealed by Southern hybridization of subclones of cosmid 1-13 with TaqI, MspI, BamHI, and HindIII digests of human DNAs. Seventy-two percent of unrelated individuals are heterozygous at one or more loci, and seven of the 24 possible haplotypes occur with frequencies of 3%-38%. Using a somatic cell hybrid panel, we have mapped this locus to 19p13.2----19q13.3, whereas in situ hybridization suggests the probe is on 19p. Taken together, these results suggest localization to 19p13.2----19cen. The locus revealed by probes from cosmid 1-13 has been designated D19S11.

Animals↗

PubMed1995

Fish mapping of 250 cosmid and 26 YAC clones to chromosome 4 with special emphasis on the FSHD region at 4q35.

Facioscapulohumeral muscular dystrophy (FSHD) is located on chromosome 4q35, close to the telomere. FSHD patients carry deletions within a cluster of tandemly repeated DNA. Although expression of a functional FSHD gene will be altered in patients, the sequence itself may be unaffected by this deletion. Hence, the FSHD gene could lie outside of the deleted region. This study employs fluorescent in situ hybridization using chromosome 4-specific cosmid and YAC clones to rapidly saturate chromosome 4 with new markers. Some 250 cosmids and 26 YACs were regionally mapped, of which 5 YACs and 55 cosmids mapped to the distal portion of 4q. Only one of these clones (D4S1454) mapped telomerically to a translocation breakpoint specified by D4S187. Using two-color interphase mapping, the following marker order was obtained: Cen-D4S187-D4S1454-HSPCAL2-D4S163-D4S139-+ ++D4F35S1. Absence of additional markers mapping distal to D4F35S1 indicates that the linkage group containing the FSHD gene lies extremely close to the 4q telomere.

Cell Line↗

PubMed1994

Non-isotopic restriction mapping of cosmid DNA.

A protocol for the non-isotopic restriction mapping of cosmid DNA was developed. After digestion with lambda terminase and partial digestion with restriction enzymes, DNA fragments containing right or left cohesive cos termini were selectively captured by hybridization with biotinylated oligonucleotides, bound to magnetic beads coated with streptavidin and recovered by heating. Recovered DNA fragments containing cos ends were resolved by agarose gel electrophoresis, and fluorescence from the DNA fragments in the ethidium bromide-stained gel was detected with the fluorescent image analyzer, FMBIO. Restriction maps were directly determined from the ladder of the partial digestion products. Two micrograms of cosmid DNA for each partial digestion were sufficient for mapping the restriction enzyme sites. This procedure provides a prototype for other cos sequence-mediated or other specific sequence-mediated DNA isolation technologies and a convenient method for non-isotopic DNA analysis. The rapid physical analysis of cosmid DNA that we present here will contribute to large DNA structural analyses like the human genome project.

Base Sequence↗

PubMed1994

One-megabase yeast artificial chromosome and 400-kilobase cosmid-phage contigs containing the von Hippel-Lindau tumor suppressor and Ca(2+)-transporting adenosine triphosphatase isoform 2 genes.

We have isolated and ordered yeast artificial chromosomes (YACs) and cosmids surrounding the von Hippel-Lindau (VHL) tumor suppressor and plasma membrane Ca(2+)-transporting ATPase isoform 2 (PMCA-2) genes on chromosome 3p25-26. The YAC contig consists of six YACs and covers a region of 1 megabase. A cosmid-phage contig around VHL and PMCA-2 genes (400 kilobases) was established and integrated into the YAC map. Using these clones, we generated an EcoRI map of the 400-kilobase region. PMCA-2 and VHL complementary DNA were positioned entirely within the cosmid-phage contig as well as two polymorphic markers (D3S601 and D3S1317). This physical map of the cloned region will allow a detailed analysis of both the PMCA-2 and VHL genes. Some of the genomic clones may be useful for isolation of the full-length VHL complementary DNA.

Base Sequence↗