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PubMed1994

P1 and cosmid clones define the organization of 280 kb of the mouse H-2 complex containing the Cps-1 and Hsp70 loci.

A 280 kilobase (kb) contig was isolated from mouse genomic P1 and cosmid libraries, using as probes human cDNA and genomic DNA fragments that map in the interval between the second component of complement and tumor necrosis factor genes of the HLA complex. The clone contig demonstrates synteny of eleven mouse genes that are homologous to genes initially mapped within the human major histocompatibility complex. These include the mouse homologs of BAT2 (HLA-B-associated transcript 2) through BAT9 and also three HSP70-related genes. Five P1 clones form a contig of 240 kb that spans from BAT9 through BAT3. Twelve cosmid clones are arranged in three contigs that confirm most of the structure of the P1 contig and link the mouse BAT3 homolog to the BAT2 homolog approximately 15 kb farther telomeric. Polymorphic DNA markers within the cloned region were used to map the cleft palate susceptibility-1 (Cps-1) locus to the interval between Hsp70.1 and BAT6 (valyl-tRNA synthetase). This refines the location of the Cps-1 locus to a 45 kb region contained in the H2-124 P1 insert.

Adenosine Triphosphatases↗
PubMed1990

Direct carrier detection by in situ suppression hybridization with cosmid clones of the Duchenne/Becker muscular dystrophy locus.

A basic problem in genetic counseling of families with Duchenne/Becker muscular dystrophy (DMD/BMD) concerns the carrier status of female relatives of an affected male. In about 60% of these patients, deletions of one or more exons of the dystrophin gene can be identified. These deletions preferentially include exon 45, which can be detected by multiplex polymerase chain reaction (PCR) and Southern blot analysis of genomic cosmid clones that map to this critical region. As a new approach for definitive carrier detection, we have performed chromosomal in situ suppression (CISS) hybridization with these cosmid clones in female relatives of four unrelated patients. In normal females, most metaphases showed signals on both X chromosomes, whereas only one X chromosome was labeled in carriers. Our results demonstrate that CISS hybridization can define the carrier status in female relatives of DMD patients exhibiting a deletion in the dystrophin gene.

Blotting, Southern↗
PubMed1990

Isolation and regional localisation of DNA sequences from a human chromosome 11-specific cosmid library.

A cosmid library has been prepared in the lorist-B vector from a mouse/human somatic cell hybrid containing region 11q23-11pter as the only human component. This chromosome region is stably maintained in the hybrid as a result of translocation onto one copy of mouse chromosome 13. Individual cosmids containing human DNA were isolated by their ability to hybridise with total human DNA, digested with either HindIII or EcoRI, and 33 individual unique sequences were identified. These fragments were then isolated and subcloned into the bluescribe plasmid vector. Regional localisation of these unique sequences was achieved using a panel of somatic cell hybrids containing different overlapping deletions of chromosome 11. The majority of the 33 mapped sequences derived from the long arm of chromosome 11. Two clones were located within the 11p13-p14 region, which is associated with a predisposition to Wilms' tumour. These probes supplement those already mapped to this chromosome and will assist in the generation of a detailed chromosome 11 linkage map.

Animals↗
PubMed1994

Multicolor fluorescence in situ hybridization on metaphase chromosomes and interphase Halo-preparations using cosmid and YAC clones for the simultaneous high resolution mapping of deletions in the dystrophin gene.

We report on multicolor fluorescence in situ hybridization protocols for the simultaneous visualization of deletion-prone regions for carrier detection of Duchenne/Becker (DMD/BMD) muscular dystrophy. Cosmid and yeast artificial chromosome (YAC) clones specific for preferentially deleted subregions of the dystrophin gene were labeled differentially and detected with three different fluorochromes using digital imaging microscopy. This approach allows for an assessment of the carrier status of female relatives even in families where no index patient is available. Cosmid and YAC clones, and different probe-generation protocols are compared with respect to their feasibility for carrier detection. The use of histone-depleted interphase nuclei (Halo-preparations) for deletion mapping is demonstrated and shown to have a resolution power of 5 kb.

Cell Line↗
PubMed1992

Organization and nature of fortimicin A (astromicin) biosynthetic genes studied using a cosmid library of Micromonospora olivasterospora DNA.

The cloning of five DNA segments carrying at least seven genes (fms1, fms3, fms4, fms5, fms7, fms11, and fms12) that participate in fortimicin A (astromicin) biosynthesis was described previously. These DNA fragments were used to screen a cosmid library of genomic DNA in order to examine if these biosynthetic genes are clustered in Micromonospora olivasterospora. One cosmid clone (pGLM990) was obtained, which hybridized to all the probes. Complementation analysis, using mutants blocked at various steps and chimeric plasmids subcloned from pGLM990, showed that three additional genes (fms8, fms10, and fms13) are present in pGLM990. A gene conferring self-resistance to the antibiotic, which was independently cloned in Streptomyces lividans, using the plasmid vector pIJ702 was also found to be linked to the cluster of biosynthetic genes. Thus, at least ten biosynthetic genes and a self-defense gene are clustered in a chromosomal region of about 27 kb in M. olivasterospora. Interestingly, the fms8 gene which participates in the dehydroxylation step of fortimicin A biosynthesis was found to have homology with a neomycin resistance gene nmrA from the neomycin-producing Micromonospora sp. MK50. Studies using a cell-free extract of the fms8 mutant and its parent strain showed that the enzyme encoded by fms8 phosphorylates a biosynthetic precursor, fortimicin KK1, in the presence of ATP. Thus the dehydroxylation reaction is suggested to occur via the phosphorylation of the target hydroxyl group. DNA regions homologous to fms genes were found in Micromonospora sp. SF-2098 and Dactylosporangium matsuzakiense, both producers of fortimicin group antibiotics.

Actinomycetales↗
PubMed1986

Isolation of a random cosmid clone, cX5, which defines a new polymorphic locus DXS148 near the locus for Duchenne muscular dystrophy.

We have isolated a random cosmid cX5 (DXS148), which maps into a small Xp21 deletion associated with Duchenne muscular dystrophy (DMD), chronic granulomatous disease (CGD), retinitis pigmentosa (RP) and McLeod syndrome, cX5 maps proximally outside several other deletions associated with DMD, glycerol kinase deficiency (GK) and adrenal hypoplasia (AHC). The following order of loci is proposed: centromere-OTC-cX5 (DXS148)-754 (DXS84)-PERT87 (DXS164)/DMD-telomere. A subclone cX5.7, isolated from this cosmid, identifies an MspI RFLP, with a minor allele frequency of 35%. This probe forms an important adjunct to the existing RFLPs for family studies in Duchenne muscular dystrophy.

Chromosome Deletion↗
PubMed1994

Characterization and expression of a P-450-like mycinamicin biosynthesis gene using a novel Micromonospora-Escherichia coli shuttle cosmid vector.

A 29 kb shuttle cosmid vector, pTYS507, was constructed from a cryptic Micromonospora griseorubida plasmid and the Escherichia coli cosmid pJB8. Subcloning of mycinamicin II biosynthesis genes in pTYS507 led to the identification of a DNA region that could complement a mutant of M. griseorubida that lacked both hydroxylase and epoxidase activities. Nucleotide sequence and mutational analysis suggested that a single P-450-like protein catalyzes both reactions.

Amino Acid Sequence↗
PubMed1995

The cosmid CSSM25 assigns syntenic group U2 to bovine chromosome 9 and is localized to ovine chromosome 8.

The cosmid-derived microsatellite CSSM 25 has previously been shown to map to bovine syntenic group U2 by linkage and hybrid somatic cell analysis. We have mapped the cosmid by fluorescent in situ hybridization to bovine Chromosome (Chr) 9q17-21 and ovine Chr 8q17-21 and hence assign U2 to Chr 9 in cattle. Bovine Chr 9 and ovine Chr 8 show strong banding pattern homology, and the localization of CSSM 25 to the same region confirms the strong conservation of gene locations on these chromosomes.

Animals↗
PubMed1993

A cosmid library specific for human chromosome regions 6p21.3 and 6q27.

We have explored the potential of irradiation-fusion gene transfer (IFGT) hybrids as a source of well-defined human chromosome fragments from which probes can be derived. Extensive characterization of the IFGT hybrid 4J4 with a full panel of markers from Chromosome (Chr) 6 showed that the human DNA content derives largely from 6p21.3 and 6q27. A cosmid library has been constructed from 4J4 DNA, and 370 recombinants containing human DNA have been isolated and overlapping clones ordered into 20 contigs. Regional localization of representative clones from each contig, determined by fluorescent in situ hybridization (FISH), places 13 contigs in 6q27 and 6 in 6p21.3. Preliminary screening of cDNA libraries with selected cosmids has identified two expressed sequences. Since there are a number of medically important genes in both these regions of human Chr 6 with several disease loci linked to the HLA-A region in 6p21.3 and various tumor suppressor genes to 6q27, this library will provide a valuable resource to aid the isolation of candidate genes for these diseases. In addition, unique markers for detailed physical and genetic mapping of these regions of human Chr 6 can be easily obtained.

Base Sequence↗
PubMed1986

Characterization of inl+ transformants of Neurospora crassa obtained with a recombinant cosmid-pool.

We constructed a Neurospora crassa gene library in a cosmid vector and used the cosmid-pool DNA to transform an inl, rg Neurospora crassa strain to inositol prototrophy. The inl+ colonies obtained in this experiment proved to be integrative type transformants. Genetic analysis revealed that the integration event occurred at or near the inl locus. In one of the transformants the inl+ trait exhibited mitotic and meiotic instability. In hybridization experiments free plasmids were detected in the F1 progeny of the transformants. We were able to recover eleven different plasmids from the F1 progeny of the transformants. None of these plasmids proved to carry a functional copy of the inl+ gene as judged by its transforming ability. Possible explanations for the observed phenomena are discussed.

Cosmids↗
PubMed1985

An E. coli-yeast shuttle cosmid with positive selection for inserted fragments.

We describe the construction of a cosmid cloning vector, pMT555, which allows positive selection for the presence of an inserted DNA fragment. The vector contains sequences which enable its replication and selection in either E. coli or Saccharomyces cerevisiae. We demonstrate that pMT555 may be used for the efficient construction of total genomic banks from small quantities of donor DNA. The positive selection permits the stable maintenance of the cosmid in E. coli and the faithful replication of inserted sequences.

Cosmids↗
PubMed1992

Molecular analysis of human chromosome 16 cosmid clones containing NotI sites.

To test the feasibility of using cloned NotI sites as markers for physical mapping, we have screened for cosmid clones spanning the NotI sites on human Chromosome (Chr) 16. Fluorescence in situ hybridization analysis of these clones confirms the previously reported cluster of NotI sites on 16p13.3. Methylation status of the cloned NotI sites on genomic DNA was established by hybridization of the cosmids to Southern blots containing EcoRI and EcoRI/NotI digest of genomic DNA. These results indicated that four of six clones included in our study can be used as linking clones for physical mapping. Two clones have NotI sites which are not cleavable in the cell lines tested. In one clone, the NotI site exists as an isolated rare-cutting restriction enzyme site, whereas in the other clone the NotI site appears to be island-related.

Blotting, Southern↗
PubMed1995

Comparative mapping of YRRM- and TSPY-related cosmids in man and hominoid apes.

Using chromosomal in situ hybridization it has been demonstrated that specific members of the YRRM and the TSPY families are multicopy and Y chromosome specific in hominoids. After hybridization with the YRRM-related cosmid A5F and the TSPY-related cosmids cos36 and cY91, a reverse and complementary pattern of main and secondary signals is detected on the Y chromosomes of the human, the pygmy chimpanzee and the gorilla, while the location of signals coincides on the Y chromosomes of the chimpanzee, both orang-utan subspecies and the white hand gibbon. This complementary distribution of YRRM and TSPY sequences on the hominoid Y chromosomes possibly originates from a similar sequence motif that is shared by and evolutionarily conserved between certain members of both gene families and/or repeated elements flanking those genes. Otherwise this complementary distribution could go back to a common organization of these genes next to each other on an ancient Y chromosome which was disrupted by chromosomal rearrangements and amplification of one or other of the genes at each of the locations.

Animals↗
PubMed1993

A high-resolution cytogenetic map of human chromosome 12: localization of 195 new cosmid markers by direct R-banding fluorescence in situ hybridization.

We have constructed a high-resolution cytogenetic map of human chromosome 12 with 195 newly isolated cosmids by direct R-banding fluorescence in situ hybridization. The fluorescent signals of 195 clones were evenly distributed throughout chromosome 12, but sublocalized preferentially to R-positive bands. This high-resolution cytogenetic map with an average map distance of 0.73 Mb on bands can, in conjunction with a genetic linkage map, facilitate the analysis of chromosomal and molecular aberrations in genetic diseases and cancers. Moreover, the cytogenetic mapping data provide starting points for establishing contig maps with cosmid clones and yeast artificial chromosomes.

Animals↗
PubMed1994

Cosmid cloning and restriction endonuclease mapping of the herpesvirus of turkeys (HVT) genome.

The genomic libraries of the herpesvirus of turkeys (HVT) presented to date do not include the entire genome. To construct a complete genomic library, HVT DNA was partially digested with Sau3A and inserted into the double cos site vector pcos2EMBL. The PstI maps derived from the partial Sau3A library demonstrate that the left region of the genome compared to the right area is overrepresented. Similar to the libraries of the HVT genome established earlier, a defined portion of the middle genomic region, however, is not contained in the partial Sau3A library. Cloning of HVT DNA in pcos2EMBL, employing BamHI for the partial digestion step, enabled to find the recombinant cosmid cBL267 which carries the BamHI-C and -D fragment as DNA insertion. As shown by Southern blot hybridization, the BamHI-C fragment ranges in a size to close the gap in the partial Sau3A library and thus guarantees the completeness of the genomic library of HVT which consists of seven overlapping cosmid clones (cBL1, cBL328, cBL11, cBL267, cBL27, cBL33, and cBL34).

Animals↗
PubMed1986

Cleavage maps of human cytomegalovirus genome (strain Towne) determined by the use of cosmid cloning system.

Virion DNA of human cytomegalovirus strain Towne was partially digested with endonuclease Hind III and fragments larger than 29 kbp were ligated to cosmid pHC79. The whole viral DNA sequence has been cloned in large overlapping segments carried by 32 recombinant cosmid clones (a pIT series). A whole set of Hind III fragments has also been cloned (a pHI series). By using these, we have constructed corrected cleavage maps of strain Towne DNA for Hind III, Bam H I, EcoR I and Xba I.

Chromosome Mapping↗
PubMed1994

Characterization of a cosmid library from flow-sorted chromosomes 11.

A cosmid library specific for human chromosome 11 has been constructed from flow-sorted chromosomes. The flow-purified chromosomes were prepared from the hamster/human hybrid line J1 which contains chromosome 11 as the only human chromosome. Individual clones were sampled in 187 microtitre plates, resulting in a total of 17,952 colonies. Hybridization analysis revealed that 83.7% of these clones were of human and 10.4% of hamster origin. The average insert size was estimated at 33.6 kb, and only 2.4% of insert fragments appear to be rearranged. This should result in 494,487 kb of cloned human DNA representing 3.5 chromosome 11 equivalents. We have prepared high-density nylon membranes of the arrayed library containing 1,536 single colonies per filter. We have demonstrated the usefulness of the library in the molecular genetic analysis of human chromosome 11 by testing for the presence of possibly polymorphic simple repeat motifs, by identifying cosmids that contain inserts from the telomeric ends of chromosome 11 and by assessing the potential of the library for rapid chromosome walking.

Animals↗
PubMed1996

Mapping of a new MAP kinase activated protein kinase gene (3PK) to human chromosome band 3p21.2 and ordering of 3PK and two cosmid markers in the 3p22-p21 tumour-suppressor region by two-colour fluorescence in situ hybridization.

A Noti-linking clone NL1-210 (D3S1656) that contains the human MAP kinase activated protein kinase (3PK) gene was localized to 3p21.2 on DAPI-banded and propidium iodide (R-bands)-stained chromosomes by fluorescence in situ hybridization (FISH). For more precise localization of 3PK, two cosmid probes were used as a frame. In order to establish this frame, two Noti-linking clones, NL2-008 (D3S1648) and NL3-003 (D3S3872) were used to screen the cosmid library for locus extension. They mapped to 3p21 and were found to belong to two separate contigs of Noti-jumping and linking clones. Using FISH on DAPI-banded metaphase chromosomes, we have determined the precise localization of cosNL2-008 and cosNL3-003 to 3p21.2-p21.1 and 3p22-p21.3 respectively. The 3PK gene was localized to the 3p21.2 region within this frame by two-colour FISH. The orientation of the probes are tel-D3S3872-3PK-D3S1648-cen.

Chromosome Mapping↗