共查询到20条相似文献,搜索用时 31 毫秒
1.
De-Xing Hou Kazuo Ozawa Naohiro Tomita Yoshizane Maeda Tsutomu Hashiguchi Kazushige Yokoyama Eiichi Soeda 《Journal of human genetics》1993,38(4):371-380
Summary Chymotrypsinogen is a principal precursor of pancreatic proteolytic enzymes. We previously isolated a cDNA clone for human prechymotrypsinogen from a human pancreatic cDNA library. In the present study, we used this cDNA sequences to isolate genomic DNA clones. Three overlapping cosmid clones spanning approximately 65-kb genomic sequences were isolated from a human cosmid library. The genomic DNA clones were characterized by restriction enzyme mapping and by hybridizing them to subfragments of the cDNA. The sequence tagged sites for human chymotrypsinogen gene were created by designing two oligonucleotides. Furthermore, the isolated genomic clones were confirmed to be localized on chromosome 16q23 by fluorescencein situ hybridization and G-banding analysis. 相似文献
2.
Takaya Tohma Toshiya Tamura Tohru Ohta Hidenobu Soejima Takeo Kubota Yoshihiro Jinno Kazuhiro Tsukamoto Yusuke Nakamura Kenji Naritomi Norio Niikawa 《Journal of human genetics》1993,38(3):267-275
Summary A human chromosomal region, 15q11–q13, was microdissected, its DNA was amplified with the primer-linker PCR method, and the PCR products were cloned into a plasmid vector to construct a microclone library. Of 193 microclones analyzed with Southern blot hybridization on hybrid cell panels, 26 (13.5%) were either single-copy (unique) or low-repetitive fragments. By screening of a cosmid library of human genomic DNA using the 26 microclones as probes, 47 positive cosmids were obtained and underwent regional mapping with chromosome fluorescencein situ hybridization (FISH). Sixteen cosmids gave FISH signals at 15p-cen, 5 at 15q11–q13, 6 at 15q22–q26, 3 at other chromosomes, and 17 no signal. These 27 cosmids mapped to chromosome 15 are useful additions to the inventory of DNA markers of this chromosome including the much interested Prader-Willi/Angelman syndrome region. 相似文献
3.
Weber B. H. F. Stöhr H. Siedlaczck I. Longmire J. L. Deaven L. L. Duncan A. M. V. Riess O. 《Chromosome research》1994,2(3):201-207
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. 相似文献
4.
Motonobu Katoh Yuzuki Nakagawa Toshio Yawata Satoshi Kumano Eisuke Kobayashi Akihiro Kurimasa Hiroyuki Kugoh Mitsuo Oshimura 《Journal of human genetics》1995,40(4):307-317
Summary To obtain cosmid markers and transcribed sequences from a specific chromosome region, a series of radiation-reduced hybrids (RHs) containing various regions of human chromosome 11 was prepared from microcell hybrid A9 (neo11) cells containing a normal human chromosome 11 tagged with pSV2neo at 11p11.2. Among 15 radiation hybrid clones isolated, RH(11)-9 which contains a q23 fragment in addition to theneo integration site, was used for the construction of a cosmid library. Cosmid clones having human DNA sequences were screened, and localized by Southern hybridization with the radiation hybrid panel. Fifty-nine cosmids were assigned to 11q23 and 6 cosmids to 11p11.2. Exon amplification proceeded with 23 of the 59 cosmids and 16 putative exons were cloned. Three of them were identical to those constituting a known gene which locates on q23 (ATDC), and the others were unknown. Thus, the RHs containing various subchromosomal fragments of chromosome 11 were useful for constructing region-specific DNA markers. The RH-(11)-9 cells and putative exons also facilitate the positional cloning of genes in the 11q23 region. 相似文献
5.
Shoichi Fujita Eisei Shin Tsutomu Nakamura Hiroki Kurahashi Yasufumi Kaneda Kiyoji Tanaka Takesada Mori Shin-ichiro Takai Isamu Nishisho 《Journal of human genetics》1993,38(4):361-370
Summary Radiation-reduced hybrids for mapping of DNA markers in the pericentromeric region of chromosome 10 were developed. A Chinese hamster/human somatic cell hybrid (762-8A) carrying chromosomes 10 and Y as the only human material were exposed to 40,000 rads of irradiation and then rescued by fusion with non-irradiated recipient Chinese hamster cells (GM459). Southern hybridization analyses revealed that 10 of 128 HAT-resistant clones contained human chromosomal fragments corresponding to at least one marker locus betweenFNRB (10p-11.2) andRBP3 (10q11.2). These hybrids were then used to map microdissection clones previously isolated and roughly mapped to this chromosomal region by fluorescencein situ hybridization (FISH). Two of the six microclones studied could be mapped to the proximity of the D10-S102 locus. These radiation hybrids are useful for the construction of refined genetic maps of the pericentromeric region of chromosome 10. 相似文献
6.
Isolation and characterization of human random cDNA clones homologous to DNA from the X chromosome 总被引:9,自引:0,他引:9
I. Balazs M. Purrello D. M. Kurnit K. H. Grzeschik M. Siniscalco 《Somatic Cell and Molecular Genetics》1984,10(4):385-397
To search for human X-chromosome-specific probes useful for molecular mapping, we studied recombinant clones isolated from a human cDNA library. DNA preparations from 150 randomly selected clones were labeled and annealed to XY and 4XY human DNA, and to DNA from a human-mouse hybrid cell line that had retained only the human X-chromosome (A9/HRBC2). cDNA clones sharing homology with DNA from the X chromosome annealed to A9/HRBC2-DNA and hybridized more intensely to 4XY DNA than to XY DNA. Eleven such clones were identified. Of these, three hybridized only to X chromosomal DNA while the rest also annealed to DNA from one or more autosomes. Chromosomal assignment of the autosomal DNA fragments showed that, in addition to hybridization to X chromosomal DNA, four of the clones hybridized to DNA sequences from chromosome 2 and two clones to chromosome 7. Subregional mapping of the relevant X chromosomal DNA fragments indicated that one clone is homologous to DNA sequences located at Xp21-Xp22, whereas the others are located in the telomeric region of the long arm. The cDNA clones were used to search for restriction fragment length polymorphisms. Several restriction-site polymorphisms were detected. Some corresponded to variants of X chromosomal DNA sequences while others were from autosomes such as chromosomes 2 and 7. 相似文献
7.
Yoriko Watanabe Naomichi Matsumoto Tohru Ohta Takahiro Tsujita Norio Niikawa 《Journal of human genetics》1996,41(1):149-158
Summary We previously mapped the putative humanHYRC (the hyper-radiosensitivity of the scid mutation, complementing gene) to human chromosome 8q11.1 by fluorescencein situ hybridization (FISH) using Alu-based PCR products from a mouse-human scid radiation cell hybrid (RD15/5) as probes. From a cosmid library constructed from RD15/5, 57 cosmid clones containing human DNA inserts were isolated, 18 of which were mapped to 8q11. Based on the sequences of plasmid subclones of the 18 cosmids, five novel sequence-tagged-sites (STSs) were made. By a screening of the CEPH-YAC library with these STSs, five yeast artificial chromosome, (YAC) clones were isolated. All these YAC clones were confirmed not to be chimeric by FISH, but two of them showed deleted human insert DNAs. Using the other 3 non-deleted YACs, we constructed a physical map covering theHYRC region. We confirmed that the recently isolated gene (the DNA-PKcs gene) which is a strong candidate forHYRC is located within the present contig and spans less than 200 kb. This map will be useful for the analysis of the genomic structure of the DNA-PKcs gene and for isolation of other complementing genes in theHYRC region. 相似文献
8.
Isolation of genomic clones homologous to transcribed sequences from human X chromosome 总被引:9,自引:0,他引:9
Pauline H. Yen Birgit Marsh T. K. Mohandas Larry J. Shapiro 《Somatic Cell and Molecular Genetics》1984,10(6):561-571
Several X chromosome DNA clones homologous to transcribed sequences were isolated from a human X chromosome library. The clones were selected for their ability to hybridize either with32P -labeled human cDNA in the presence of an excess of unlabeled human repetitive DNA or with mouse fibroblast cDNA. The X chromosome specificity of these sequences was demonstrated by two criteria: A dosage effect was seen when the clones were hybridized to Southern blots of DNA from 1X and 5X cells, and they hybridized to DNA from mouse-human hybrid cells containing only the human X chromosome. The presence of transcribed sequences in these X clones was detected by hybridization with mouse cDNA or with human cDNA in the presence of unlabeled human repetitive sequences, by identifying restriction fragments which hybridize with cDNA but not with human repetitive DNA, and by hybridization with poly A+ RNA on Northern blots. These clones were mapped on the human X chromosomes using a panel of mouse-human somatic cell hybrids carrying various translocated human X chromosomes. 相似文献
9.
F. Yang B. S. Milne C. Schelling G. Dolf J. Schläpfer M. Switonski D. Ladon A. Pienkowska A. A. Bosma D. R. Sargan M. A. Ferguson-Smith 《Chromosome research》2000,8(2):93-100
We have developed a novel method for identifying dog chromosomes and unambiguously mapping specific clones onto canine chromosomes. This method uses a previously established red fox/dog comparative chromosome map to guide the FISH mapping of cloned canine DNA. Mixing metaphase preparations of the red fox and dog enabled a single hybridization to be performed on both species. We used this approach to map the chromosomal locations of twenty-six canine cosmids. Each cosmid contains highly polymorphic microsatellite markers currently used by the DogMap project to compile the canine linkage map. All but two cosmids were successfully assigned to subchromosomal regions on red fox and dog chromosomes. For eight cosmids previously mapped on dog chromosomes, we confirmed and refined the canine chromosomal assignments of seven cosmids and corrected an erroneous assignment regarding cosmid CanBern1. These results demonstrate that the red fox and dog comparative chromosome map can greatly improve the accuracy and efficiency of chromosomal assignments of canine genetic markers by FISH. 相似文献
10.
Isolation of cosmids and fetal brain cDNAs from the proximal long arm of human chromosome 22 总被引:1,自引:1,他引:1
Lamour Valerie; Levy Nicolas; Desmaze Chantal; Baude Veronique; Lecluse Yann; Delattre Olivier; Bernheim Alain; Thomas Gilles; Aurias Alain; Lipinski Marc 《Human molecular genetics》1993,2(5):535-540
The proximal portion of human chromosome 22q appears to carrygenes implicated in the pathogenesis of various developmentaldisorders, including the cat eye syndrome (CES) and the DiGeorgesyndrome (DGS). A cosmid library was prepared from a radiationhybrid selected for its content in chromosome 22 fragments.A large fraction of cosmids containing human DNA were foundto derive from the juxtacentromeric region of chromosome 22,as shown by fluorescence in situ hybridization (FISH) performedusing individual cosmids or cosmid pools as probes. Finer mappingwas obtained for individual cosmids by hybridization to a somaticcell hybrid mapping panel which splits the long arm of the chromosomeinto 14 bins numbered 1 to 14 from the centromere to the telomere.Of the 10 cosmids mapped, eight belonged to group 1, the othertwo to group 14, in agreement with FISH data. Rare endonucleasesites and fragments conserved between species were searchedin single cosmids, resulting in the selection of seven cosmidfragments which were used to screen a human fetal brain cDNAlibrary. Three cDNAs were identified, encoded from two chromosome22 genes which appeared to be novel, as determined from partialend sequence and comparison with the database entries. Finelocalization of the 30.9 cDNA indicated that the correspondinggene was located in a segment of proximal 22q overlapping withthe critical DGS region. 相似文献
11.
G. Rettenberger T. Leeb S. Meier-Ewert J. Bruch C. Klett B. Brenig H. Hameister 《Chromosome research》1996,4(2):147-150
We have mapped two genes from human chromosome 1, urate oxidase (UOX) and transforming growth factor beta 2 (TGFB2), by fluorescencein situ hybridization (FISH) in the pig genome. Porcine-specific polymerase chain reaction (PCR) primers for both genes were designed from the porcine cDNA sequence. With the help of these primers yeast artificial chromosome (YAC) clones forUOX andTGFB2 were isolated from a pig YAC library. These DNA probes were used for FISH analysis.TGFB2 was localized to SSC 10p16. With the YAC probe forUOX two porcine chromosome regions 6q26 and 6q32, revealed specific signals. These results, help to refine the comparative mapping data between human and pig.accepted for publication by H. Schmidt 相似文献
12.
M. R. Littlejohn J. Camakaris D. M. Woodcock 《Somatic Cell and Molecular Genetics》1995,21(6):385-398
We have isolated sites of de novo rearrangements from interspecific cell hybrids. Of 147,000 clones screened from a human/hamster
hybrid genomic library, 14 clones were found with homology to both human and hamster repetitive DNA sequences. Five of these
clones contained recombination events involving less than 13 kb of DNA, three with human DNA recombined into a section of
hamster DNA, and two with hamster DNA recombined into human DNA. None of the clones involving human L1 sequences were found
to be de novo transposition events, but simply short recombination or insertion events. Considering the apparent random nature
of these events, they are likely to involve unique as well as repetitive sequences and also involve integration into the homologous
as well as heterologous chromosome sets. These results suggest that the chromosome sets in somatic cell hybrids may be randomly
contaminated with small DNA segments derived from either set of chromosomes. 相似文献
13.
Normal mouse fibroblasts at early passage levels were used as a starting material to construct mouse-hamster microcell hybrids (MCH). Theneo
r
gene, carried on the pSV2neo and pZIP-NeoSV(X)1 plasmids, was introduced into the mouse fibroblasts by gene transfection and retroviral infection, respectively, prior to microcell hybridization into the E36 Chinese hamster cell line. In total about 180 MCH clones were isolated and their amount of mouse DNA was estimated by dot-blot analysis. About 50% of the transfection based hybrids (T-hybrids) showed signals indicating one mouse chromosome, less than 10% more than one mouse chromosome, and the remaining clones contained only subchromosomal amounts of mouse DNA. In the infection-based hybrid series (I-hybrids) more than 95% showed only subchromosomal mouse DNA content. Chromosomal integration analysis verified the presence ofneo
r
insertions in all 42 hybrid clones analyzed. C-banding analysis verified 14 of 15 hybrids scored as monochromosomals on dot blots. Chromosome fragmentation in T-type MCH was found to be (1) nonrandom, preferentially occurring in MCH derived from certain transfectants, (2) late in clonal establishment, and (3) essentially not related to prolonged cultivation in vitro. Once established, most T-type MCH clones including mono- and subchromosomal hybrids were essentially stable during prolonged cultivation. In contrast MCH initially containing several mouse chromosomes tend to lose the nonselectable ones during prolonged cultivation. In total we estimate the number of independent monochromosomal MCH derived in this study to more than 30. 相似文献
14.
Molecular cloning of MER-2, a human chromosome-11-encoded red blood cell antigen,using linkage of cotransfected markers 总被引:1,自引:0,他引:1
We report the molecular cloning of a human gene MER-2located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO ×human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2expression. 相似文献
15.
Kevin L. Bentley Wan-Iiang Li Fredrik O. VannBerg Julie Y. Choi Jingwei Yu Fa-Ten Kao Gualberto Ruaño 《Somatic Cell and Molecular Genetics》1997,23(5):353-365
A region-specific microdissection library originating from human chromosome 17q21, was constructed using the MboI linker-adaptor
microcloning techique. DNA sequencing of 241 microclones resulted in the identification of 74 novel coding sequences, paralogs
of known genes, and known, but previously unmapped, genes or expressed sequence tags that were “virtually” mapped to chromosome
17q21. By pooling the microclones as multiplexed hybridizaion probes, and by virtue of their origin on 17q21, we were able
to identify approximately 150 P1 clones from the human Reference Library Data Base P1 Library that potentially map to chromosome
17q21. Verification of the 17q21 location of 16 P1 clones was accomplished by PCR analysis, with STS primer pairs to known
17q21 genes or by FISH. Our results demonstrate the substantial advantage of combining the sequence analysis of microclones
with multiplex hybridization strategies for gene discovery and mapping specific gene rich regions of the genome. 相似文献
16.
Construction of cosmid contigs and high-resolution restriction mapping of the Huntington disease region of human chromosome 4 总被引:1,自引:0,他引:1
Zuo Jlan; Robblns Carolyn; Bahariloo Slamak; Cox David R.; Myers Richard M. 《Human molecular genetics》1993,2(7):889-899
The gene responsible for Huntington disease (HD) has been localizedto a 2.2 million base pair (Mbp) region between the loci D4S10and D4S98 on the short arm of human chromosome 4. As part ofa strategy originally designed to clone the gene based on itschromosomal location, we and others previously identified overlappingyeast artificial chromosome (YAC) clones covering most of thisregion. While these YAC clones were useful for initially obtaininglong-range clone continuity, a number of features of the YACsindicated that smaller clones are generally more useful in thesubsequent steps of the positional cloning strategy. In thispaper, we use these YAC clones to generate sets of overlappingcosmid clones covering most of the HD region. We Isolated alarge number of cosmids by screening a chromosome 4-specificcosmld library with labeled DNA from a minimal overlapping setof YAC clones. These cosmid clones were further analyzed byrestriction mapping and hybridization experiments, leading tothe assembly of 185 cosmids Into eleven contigs covering morethan 1.65 Mbp and to a fine-structure restriction map of theregion. Nine of these contigs cover 90 percent of the 1.7 Mbpsubregion between loci D4S125 and D4S98 where the HD gene isnow known to lie. The detailed restriction map and the cosmidclones should facilitate the identification and localizationof cDNAs and polymorphic markers, and they provide reagentsfor large scale DNA sequencing of this region of the human genome.Our results suggest that this strategy should be generally usefulfor converting YAC clones into cosmid contigs and generatinghigh-resolution restriction maps of genomioc regions of interest. 相似文献
17.
Chromosome 1 deletions in human neuroblastomas: generation and fine mapping of microclones from the distal 1p region 总被引:7,自引:0,他引:7
Human neuroblastomas show a high incidence of deletions in the distal region of the short arm of chromosome 1. In pursuit of a molecular analysis of these deletions, we have generated a microclone bank from microdissected 1p35-pter chromosomal fragments. To allow a rapid localization of the microclones, we have also generated a panel of (human X mouse) hybrid cell lines through microcell-mediated chromosome transfer. The hybrid cells contained different portions of the human chromosome 1 on a murine background. A total of 20 randomly chosen single or low-copy microclones were localized by Southern analysis on DNA of the hybrid panel: All probes were derived from chromosome 1. Sixteen mapped in region 1p36.I-pter, two in 1p22-p36.1, and another two in 1 cen-qter. The mapping of ten of these microclones was further refined by in situ hybridization. Cells of the neuroblastoma line GI-ME-N carry two types of chromosome 1, one cytogenetically normal and another with a translocation reported to be in 1p36.2, i.e., a t(1;?)(p36.2;?) marker. Using cell hybridization, we separated the two chromosome 1 types of GI-ME-N into different hybrid cell clones. Southern hybridization of three microclones from distal 1p to DNA of the hybrid cell clones revealed that the breakpoint in the translocated chromosome 1 was located in 1p36.1. 相似文献
18.
Fast-twitch and slow-twitch/cardiac Ca2+ ATPase genes map to human chromosomes 16 and 12 总被引:3,自引:0,他引:3
David H. MacLennan Christopher J. Brandl Shashikant Champaneria Paul C. Holland Vicki E. Powers Huntington F. Willard 《Somatic Cell and Molecular Genetics》1987,13(4):341-346
The fast-twitch and slow-twitch/cardiac Ca
2+
ATPase genes have been assigned to human chromosomes 16 and 12, respectively, using rodent-human somatic cell hybrids and filter hybridization analysis of cell hybrid DNA. A rabbit cDNA for the fast-twitch ATPase hybridizes to a prominent single fragment in human genomic DNA digested with the restriction enzyme BamHI. By correlating the presence of this fragment in somatic cell hybrid DNA with the human chromosome content of the hybrids, the fast-twitch ATPase gene can be assigned to human chromosome 16. A slow-twitch/cardiac ATPase cDNA clone was isolated from a human muscle cDNA library and used to detect human fragments in EcoRI-digested somatic cell hybrid DNA. By correlating the presence of these fragments with the human chromosome content of the hybrids, the slow-twitch/cardiac ATPase gene can be assigned to human chromosome 12. Thus, the two ATPase genes, which are probably related to each other by an ancient duplication event, are not syntenic in the human genome. 相似文献
19.
20.
Construction and characterization of radiation hybrids for chromosome 9, and their use in mapping cosmid probes on the chromosome 总被引:1,自引:0,他引:1
Cynthia L. Jackson Deborah E. Britt Sharon L. Graw Audrey Potts Kathleen Santoro Alan J. Buckler David E. Housman Hon Fong L. Mark 《Somatic Cell and Molecular Genetics》1992,18(3):285-301
Radiation hybrids were produced from a monochromosomal microcell hybrid (PK87-9) which contains only human chromosome 9 with an inserted marker on 9p. Doses of radiation ranging from 1000 to 8000 rads were used to produce a series of hybrids with different size fragments of human chromosome 9. The inserted dominant selectable marker was used to select for hybrids that preferentially maintain fragments of 9p. A panel of 53 radiation hybrids were characterized for 17 chromosome 9 markers. In addition, 17 hybrids were analyzed by fluorescent in situ hybridization (FISH). Hybrids were produced with breaks on both 9p and 9q, many of which appear to contain a single fragment of human chromosome 9. These hybrid cell lines were used to regionally localize 31 cosmids isolated from a chromosome 9 cosmid library. Six cosmids were mapped to intervals on 9p, six cosmids mapped to the centromeric region of the chromosome, and 19 mapped to 9q. 相似文献