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PubMed1993

Isolation and mapping of human chromosome 21 cosmids using a probe for RTVL-H retrovirus-like elements.

We have explored the usefulness of a cloned low-repetitive DNA element, termed RTVL-H, for the region-specific isolation of DNA cosmids from chromosome 21. Hybridization of the RTVL-H element as a probe to genomic Southern blots of DNAs from a chromosome 21-specific somatic cell hybrid panel demonstrated the presence of at least seven RTVL-H-related sequences in defined regions dispersed over the chromosome. Some of the RTVL-H sequences detected by using the chromosome 21-specific somatic cell hybrid panel were subsequently isolated from a chromosome 21-specific cosmid library and mapped using somatic cell hybrids. In general, close correspondence was found between mapping results obtained using the consensus RTVL-H probe on the chromosome 21-specific somatic cell hybrid panel and the more precise mapping obtained using cosmids. The results demonstrate that sequences such as the RTVL-H element can be used for isolating region-specific sequences.

Animals↗
PubMed1993

Isolation and FISH mapping of 80 cosmid clones on the short arm of human chromosome 3.

We have isolated and mapped by fluorescence in situ hybridization 80 new cosmids on the short arm of chromosome 3. These markers were isolated from a radiation-reduced hybrid, DM1, made from a cell line that was monochromosomal for human chromosome 3. Selected cosmids were used in double-label cohybridization experiments in which polymerase chain reaction products, generated by an Alu oligonucleotide primer from genomic DNA, were used for chromosome banding. Fifty-six of the cosmid probes map between 3p14.3 and 3p22 while 24 other probes cluster around bands 3p23-3p25. Three probes that appeared to map close to the chromosome 3 region bearing a t(3,8)p14.2; q24.1 translocation associated with renal cell carcinoma were analyzed by interphase mapping techniques and hybridized to metaphase spreads from the translocation cell line. These 80 probes will be useful in the elucidation of genetic alterations associated with diseases such as small cell lung carcinoma, renal cell carcinoma, and von Hippel-Lindau disease.

Animals↗
PubMed1993

A fluorescence in situ hybridization map of human chromosome 21 consisting of 30 genetic and physical markers on the chromosome: localization of 137 additional YAC and cosmid clones with respect to this map.

A fluorescence in situ hybridization (FISH) map of human chromosome 21 was compiled using yeast artificial chromosome (YAC) DNA probes that encode 28 markers physically and/or genetically mapped on the chromosome. Probes that recognize the centromere and rDNA repeat sequences in the p arm were also placed as reference markers on the FISH map. For each probe, the location of the fluorescence hybridization signal was measured on metaphase chromosomes with respect to fractional chromosome length (FL) from p-ter. The location of the markers was established with a standard error of +/- 1.9 Mb using from 9 to 63 FL measurements for each probe. The relative order and separation of the markers as determined by FISH are shown to correspond well to those of other maps of the chromosome. Fifty-one additional YAC and 86 cosmid clones were also localized by FISH with respect to the 30 markers on the chromosome. The cosmids, chosen at random from a flow-sorter chromosome 21 cosmid library, show some biases in chromosome distribution.

Chromosome Mapping↗
PubMed1993

High-resolution cytogenetic mapping of 342 new cosmid markers including 43 RFLP markers on human chromosome 17 by fluorescence in situ hybridization.

We have constructed a high-resolution cytogenetic map of human chromosome 17 with 342 cosmid markers, each newly isolated from a cosmid library constructed from a human-mouse hybrid cell line containing a single human chromosome 17. Direct mapping on R- and/or G-banded (pro)metaphase chromosomes by fluorescence in situ hybridization localized these markers throughout the chromosome, although density was highest in the R-band-dominant regions of 17p13, 17p11.2, 17q11.2-q12, 17q21.3, 17q23, and 17q25. By screening some of the cosmid clones, we identified 71 polymorphic systems with 43 markers; 11 of these are VNTRs. As our high-resolution cytogenetic map contains a large number of markers, it can provide useful landmarks for a contig map of chromosome 17. Furthermore, the map will contribute to positional cloning of aberrant genes responsible for inherited diseases such as Miller-Dieker syndrome (MDS), Smith-Magenis syndrome (SMS), and familial early-onset breast cancer, as well as putative tumor suppressor genes on this chromosome.

Animals↗
PubMed1992

Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type 1 (MEN1) locus.

Multiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves unmasking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chromosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced somatic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12-13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction fragment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene.

Blotting, Southern↗
PubMed1995

Chromosomal localization and molecular characterization of 53 cosmid-derived bovine microsatellites.

Gene mapping in cattle has progressed rapidly in recent years largely owing to the introduction of powerful genetic markers, such as the microsatellites, and through advances in physical mapping techniques such as synteny mapping and fluorescence in situ hybridization (FISH). Microsatellite markers are often not physically mapped because they are generally isolated from small insert plasmid libraries, which makes their chromosomal localization inefficient. In this report we describe the FISH mapping of a large group of cosmid-derived bovine microsatellite markers, as our contribution to the European mapping initiative, BovMap. One objective of BovMap is to develop a set of anchored loci for the cattle genome map. Two cosmid libraries were screened with probes corresponding to the (AC)n microsatellite motif. Positive clones were mapped by FISH, and then a subset was further analyzed by sequencing the region flanking the microsatellite repeat. In total, 58 clones were hybridized with chromosomes and identified loci on 22 of the 31 different bovine chromosomes. Three clones contained satellite DNA. Two or more markers were placed on 12 chromosomes. Sequencing of the microsatellites and flanking regions was performed directly from 43 cosmids, as previously reported (Ferretti et al. Anim. Genet. 25, 209-214, 1994). Primers were developed for 39 markers and used to describe the polymorphism associated with the corresponding loci.

Animals↗
PubMed1993

A mathematically designed STS primer without any mismatches for direct sequencing of cosmid DNA clones.

We report here a new method for the direct sequencing of large DNA inserts of cosmid clones from human chromosomes using STS primers of 14 nucleotides without any mismatches, which are designed from results of a mathematical analysis. It is clear that STS primer of 14 nucleotides is optimum for direct sequencing of cosmid recombinant DNA clones. We also provide examples of direct sequencing of cosmid clones of human chromosome 21 using these STS primers.

Animals↗
PubMed1997

Characterization, genetic and physical mapping analysis of 36 horse plasmid and cosmid-derived microsatellites.

Thirty-six new horse microsatellites (11 from plasmid libraries and 25 from a cosmid library) were isolated and characterized on a panel of four horse breeds. Thirty were found to be polymorphic with heterozygosity levels ranging between 0.20 and 0.87. Twenty-two of the cosmids were physically mapped to R-banded single horse Chromosomes (Chrs) 1, 3, 4, 9, 11, 12, 13, 15, 18, 19, 21, 22, 23 and three to pericentromeric regions. Furthermore, linkage analysis between a selection of 42 DNA markers, including those presented in this study, and 16 conventional markers of the horse hemotype was performed on six paternal half-sib horse families. Five linkage groups were detected, of which four were assigned to Chr 10, 11, 15, and 18. This work increased by one-third the number of published polymorphic DNA markers suitable for horse mapping and approximately doubled the number of known linkage groups. Our cosmids labeled 14 out of the 31 horse autosomes. Moreover, the physical anchoring of part of these markers will orient linkage and synteny groups on the chromosomes and will contribute to their assignment.

Animals↗
PubMed1997

Alignment of the genetic and physical maps in the dpy-5 bli-4 (I) region of C. elegans by the serial cosmid rescue of lethal mutations.

Lethal mutations in the 0.5 map unit region between dpy-5 and bli-4 on chromosome I in Caenorhabditis elegans were serially rescued using cosmid-containing transgenic strains. All the lethal mutations analyzed came from a set of 495 EMS-induced, sDp2-rescued lethals described previously. Germline transformation with cosmid DNA was used to create 25 transgenic strains bearing heritable extrachromosomal arrays. These arrays were used as small duplications for the fine-scale mapping of essential genes, via the rescue of lethal mutations. Lethal mutations in 13 essential genes have been phenotypically rescued, allowing the alignment of the genetic and physical maps in this region. Extrachromosomal arrays were found to be transmitted 2- to 7-fold less frequently in oocytes than in hermaphrodite sperm for 12 of the 16 arrays that were examined. Three of these strains showed a subsequent 4- to 13-fold increase in array stability in oocytes. This phenomenon may be influenced by cosmid sequences. Early mitotic loss of the arrays was observed in all 17 transgenic strains examined, suggesting that loss of the array can occur at any time during development when cell divisions are occurring. As a result of this work, 13 of the essential loci positioned between dpy-5 and bli-4 are anchored to the physical map, thereby providing links between the physical and genetic maps on average every 85 kb.

Animals↗
PubMed1987

The human V kappa locus. Characterization of extended immunoglobulin gene regions by cosmid cloning.

As part of the ongoing work in our laboratory on the structural organization of the human V kappa locus we screened cosmid libraries with V kappa gene probes and obtained numerous V kappa gene-containing cosmid clones. Several genomic regions of the V kappa locus were reconstructed from overlapping cosmid inserts and were extended by one step of chromosomal walking. The regions that are called Wa, Wb, Oa, Ob and Ob' comprise about 370 kb (10(3) bases) of DNA and contain 24 V kappa genes and pseudogenes. The V kappa genes belong to the three dominant subgroups (V kappa I, V kappa II, V kappa III) and are arranged to form mixed clusters with members of the different subgroups being intermingled with each other. The distances between the genes range from 1 to 15 kb. Three genes of the Wa and Wb regions that were sequenced turned out to be pseudogenes. Terminal parts of the regions Wa and Ob that do not contain V kappa genes of one of the known subgroups may represent extended spacer regions within the V kappa locus. Wa and Wb are duplicated regions located at different positions of the locus. Region Wb was found to comprise inversely repeated sections of at least 14 kb each that contain V kappa genes oriented in opposite polarity. This finding is consistent with inversion-deletion models of V-J joining; it also shows that the V kappa locus contains not only unique and duplicated but also triplicated parts. The data on the W and O regions are discussed together with those on the L regions and on other regions established in our laboratory. Although the picture of the human V kappa locus with, to date, about 70 different non-allelic V kappa genes is still incomplete, some general features with respect to the organization of the genes and the limited duplication of genomic regions have emerged.

Base Sequence↗
PubMed1987

A low copy number cosmid.

A low copy number cosmid was constructed by subcloning the pair of cos sites and the kanamycin resistance gene of pcos2EMBL into pGB2. The resulting cosmid, pPR691, has the pSC101 replicon and specifies resistance to kanamycin, spectinomycin, and streptomycin. pPR691 also carries restriction sites suitable for cloning partial Sau3A digests using the strategy of Bates and Swift (P. F. Bates and R. A. Swift, 1983, Gene 26, 137-146). A library of Salmonella typhimurium chromosomal DNA was made using this cosmid and the rfb gene cluster (map position 42) was isolated from this library.

Cloning, Molecular↗
PubMed1986

A cosmid vector for systematic chromosome walking.

We describe the construction of a cosmid, LoristB, that contains SP6 and T7 phage-encoded RNA polymerase promoter sequences that are oriented towards and immediately adjacent to HindIII and BamHI cloning sites. We describe techniques for rapidly generating RNA probes from these promoters that must be complementary to the extreme left or right ends of the cloned DNA and can be used for library screening. Probe preparation requires neither prior knowledge of restriction sites nor fragment isolation. We also make extensive use of cos mapping restriction-mapping protocols that we have devised for our cosmid vectors for generation and alignment of steps in a cosmid walk.

Base Sequence↗
PubMed1986

Cosmid vectors for high efficiency DNA-mediated transformation and gene amplification in mammalian cells: studies with the human growth hormone gene.

We have constructed two new recombinant cosmid vectors that can be used for direct expression and amplification of genomic DNA in mammalian cells. The vectors allow cloning of DNA fragments up to 40 kb in size. Each carries two dominant selectable markers: the bacterial neo gene and the mouse DHFR gene. In the first vector, pCV001, the neo and DHFR genes are regulated by the SV40 early promoter, and in the second, pAVCV007, by the avian sarcoma virus LTR promoter. The neo gene served as a dominant marker for the selection of transformants in all mammalian cell types, and we demonstrate here that the LTR promoter significantly improved the efficiency of DNA-mediated transformation of a human cell line. We isolated the human growth hormone genes from genomic libraries prepared in these cosmid vectors and used these recombinant cosmids for direct transfections of cultured cells. Selection of transformants in increasing concentrations of methotrexate led to the outgrowth of resistant cell populations carrying amplified copies of the DHFR marker. A 40-1000-fold coamplification of the hGH genes was observed in the different transfected cell lines, along with a corresponding increase in transcription and translation activity of the hGH gene. Gene amplification could be achieved in both DHFR deficient or normal cell lines. High level expression of a cloned gene mediated by gene amplification should facilitate characterization of DNA sequences, as well as isolation of specific gene products for biochemical, functional, and pharmacological studies.

Animals↗
PubMed1987

A new shuttle cosmid vector, pKC505, for streptomycetes: its use in the cloning of three different spiramycin-resistance genes from a Streptomyces ambofaciens library.

A new shuttle cosmid vector, pKC505, was constructed for the cloning of Streptomyces DNA. This vector, which can be conjugally transferred between different streptomycetes, was used to construct a genomic library from a spiramycin-producing S. ambofaciens strain. By transformation of the spiramycin-sensitive S. griseofuscus with the library, three phenotypically different spiramycin-resistance genes were isolated. S. ambofaciens DNA in these clones was colinear with the chromosome, and the cloned DNA was stable in E. coli, S. griseofuscus and S. fradiae. These cosmids could be isolated easily from S. griseofuscus, an improvement over the previous shuttle cosmid vector, pKC462a [Stanzak et al., Bio/Technology 4 (1986) 229-232], which was somewhat difficult to isolate from S. lividans.

Chromosome Mapping↗
PubMed1989

High efficiency vectors for cosmid microcloning and genomic analysis.

We describe the construction and use of cosmid vectors designed for microcloning, gene isolation and genomic mapping starting from submicrogram amounts of eukaryotic DNA. These vectors contain (1) multiple cos sites to allow for simple and efficient cloning using non size-selected DNA; (2) bacteriophage T3 and T7 promoter sequences flanking the cloning site to allow for the synthesis of end-specific probes for chromosome walking; (3) a selectable gene for immediate gene transfer of cosmid DNA into mammalian cells; (4) recognition sequences for specific oligodeoxyribonucleotides to allow rapid restriction mapping; (5) unique NotI, SacII or SfiI sites flanking the cloning site to allow for removal of the cloned DNA insert from the vector. These cosmid vectors allow the construction of high quality genomic libraries in situations where the quantity of purified DNA is extremely limited, such as when using DNA prepared from purified mammalian chromosomes isolated by fluorescence-activated cell sorting.

Bacteriophages↗
PubMed1994

An improved cosmid vector for the cloning of equine herpesvirus DNA.

We have modified the commercial cosmid vector, triple helix vector (THV), such that I-Sce-I restriction endonuclease sites flank the cloning site. I-Sce-I is a rare-cutting endonuclease which recognizes an 18-bp sequence. It does not restrict the genome of either of the equine herpesvirus 1 or 4 (EHV-1 and EHV-4) strains we have cosmid cloned. Thus, cosmid-cloned EHV fragments can be excised intact from the vector by I-Sce-I digestion, facilitating production of large overlapping EHV fragments for use in transfections to produce recombinant virus.

Base Sequence↗
PubMed1991

A cosmid clone for the 5HT1A receptor (HTR1A) reveals a TaqI RFLP that shows tight linkage to dna loci D5S6, D5S39, and D5S76.

A human neuroreceptor clone (G21), which was isolated by cross-hybridization with the human clone for the beta 2-adrenergic receptor, has recently been shown to encode the gene for the 5HT1A receptor (HTR1A) subtype. In situ hybridization to human metaphase chromosomes mapped the G21 sequence to chromosome 5 at bands 5q11.2-q13. The clone G21 recognizes a SacI RFLP with low heterozygosity (0.13). To increase the informativeness of the HTR1A locus we have isolated two new cosmid clones containing the receptor gene. No polymorphic microsatellites were present in the cosmids. However, one cosmid revealed a new TaqI RFLP that showed tight linkage to new highly polymorphic microsatellites for the loci D5S76, D5S39, and D5S6 in seven British and Icelandic reference pedigrees (maximum LOD of 13.2 with D5S76).

Chromosome Mapping↗
PubMed1991

Laser microdissection of the fragile X region: identification of cosmid clones and of conserved sequences in this region.

Laser microdissection has been used to dissect material from the X-chromosome region involved in fragile-X-linked mental retardation. After dissection, single chromosome slices corresponding to this fragile site were subjected to DNA amplification using either a vector ligation method (to provide known anchor sequences) or primer oligonucleotides corresponding to the ubiquitous Alu sequences. Amplified material was then cloned or, alternately, used to screen a gridded cosmid library. Eight cosmid clones identified in this way were regionally mapped using a panel of hybrid cell lines and shown to originate from a narrow interval centered on the fragile X site. Two clones are included in the approximately 6-cM interval defined by probes RNI (DXS369, 5 cM proximal) and VK21 (DXS 296, 1-2 cM distal) and which includes the fragile site, and at least one clone contains sequences conserved across species suggestive of a gene. This method combines the focused approach of microdissection and the convenience of obtaining cosmid (rather than small-insert) clones; it may be useful for studies of other defined chromosomal regions.

Animals↗