scholarly journals Isolation of Human Transcripts Expressed in Hamster Cells from YACs by cDNA Representational Difference Analysis

1999 ◽  
Vol 9 (2) ◽  
pp. 182-188
Author(s):  
Jessie Gu ◽  
Xin-Yuan Guan ◽  
Melissa A. Ashlock
Keyword(s):  
Cell Line ◽  
Mammalian Cells ◽  
Positional Cloning ◽  
Yeast Artificial Chromosome ◽  
Expressed Sequence Tag ◽  
Gene Isolation ◽  
Representational Difference Analysis ◽  
Difference Analysis ◽  
Artificial Chromosomes ◽  
Link Type

Gene isolation methods used during positional cloning rely on physical contigs consisting of bacterial artificial chromosomes, P1, or cosmid clones. However, in most instances, the initial framework for physical mapping consists of contigs of yeast artificial chromosome (YACs), large vectors that are suboptimal substrates for gene isolation. Here we report a strategy to identify gene sequences contained within a YAC by using cDNA representational difference analysis (RDA) to directly isolate transcripts expressed from the YAC in mammalian cells. The RDA tester cDNAs were generated from a previously reported hamster cell line derived by stable transfer of a 590-kb YAC (911D5) that expressed NPC1, the human gene responsible for Niemann-Pick type C (NP-C). The driver cDNAs were generated from a control hamster cell line that did not contain the YAC that expressed NPC1. Among the gene fragments obtained by RDA,NPC1 was the most abundant product. In addition, two non-NPC1 fragments were isolated that were mapped to and expressed from 911D5. One of these RDA gene fragments (7-R) spans more than one exon and has 98% sequence identity with a human cDNA clone reported previously as an expressed sequence tag (EST), but not mapped to a chromosomal region. The other fragment (2-R) that had no significant sequence similarities with known mammalian genes or ESTs, was further localized to the region of overlap between YACs911D5 and 844E3. The latter YAC is part of a contig across the NP-C candidate region, but does not contain NPC1. This two-part approach in which stable YAC transfer is followed by cDNA RDA should be a useful adjunct strategy to expedite the cloning of human genes when a YAC contig is available across a candidate interval.[The sequence data described in this paper have been submitted to GenBank under accession nos. AF117641 and AF117642.]

Arabidopsis YAC restriction mapping

Genome ◽  
1998 ◽  
Vol 41 (6) ◽  
pp. 806-817
Author(s):  
Shaun M Morroll ◽  
Zoe A Wilson
Keyword(s):  
Positional Cloning ◽  
Floral Development ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Genomic Region ◽  
Restriction Mapping ◽  
Artificial Chromosomes ◽  
Number Of Genes ◽  
Yeast Artificial Chromosomes ◽  
Large Genomic Region

The approach of partial restriction mapping and vector hybridisation has been used to restriction map and align six yeast artificial chromosomes (YACs) corresponding to the top arm (~27.9 centiMorgans, cM) of Arabidopsis chromosome 5 and confirm the chimeric nature of a further four clones which map to this region. The restriction endonucleases Sma1 and Sfi1 which recognise rare-medium frequency sites in the Arabidopsis genome were used. This work has restriction mapped a 315 kb region that includes a number of genes implicated in floral development, namely PISTILLATA and TOUSLED, and a number of uncharacterised genes involved in male gametogenesis (e.g., Ms1 and Ms37). The information generated can be used to transcriptionally map genes to this contig and will provide data for the isolation of several uncharacterised floral development genes which lie in this region. This approach has demonstrated how large tracts of YAC DNA can be mapped and aligned to show the presence/absence of chimeric YAC clones and provide detailed restriction knowledge for a large genomic region to help facilitate the positional cloning of genes.Key words: yeast artificial chromosome, YAC, Arabidopsis thaliana, partial restriction mapping, floral development.

scholarly journals A Sequence-Ready BAC Contig of the GABAA Receptor Gene Cluster Gabrg1–Gabra2–Gabrb1 on Mouse Chromosome 5

1999 ◽  
Vol 9 (8) ◽  
pp. 732-738 ◽  
Author(s):  
Andreas Lengeling ◽  
Tim Wiltshire ◽  
Chris Otmani ◽  
Maja Bućan
Keyword(s):  
Gene Cluster ◽  
Mouse Chromosome ◽  
Radiation Hybrid ◽  
Expressed Sequence Tag ◽  
Gene Clusters ◽  
Receptor Subunit ◽  
Chromosome 5 ◽  
Bac Contig ◽  
Artificial Chromosomes ◽  
Link Type

The type-A receptors for the neurotransmitter GABA (γ-aminobutyric acid) are ligand-gated chloride channels that mediate postsynaptic inhibition. The functional diversity of these receptors comes from the use of a large repertoire of subunits encoded by separate genes, as well as from differences in subunit composition of individual receptors. In mammals, a majority of GABAAreceptor subunit genes are located in gene clusters that may be important for their regulated expression and function. We have established a high-resolution physical map of the cluster of genes encoding GABAA receptor subunits α2 (Gabra2), β1 (Gabrb1), and γ1 (Gabrg1) on mouse chromosome 5. Rat cDNA probes and specific sequence probes for all three GABAA receptor subunit genes have been used to initiate the construction of a sequence-ready contig of bacterial artificial chromosomes (BACs) encompassing this cluster. In the process of contig construction clones from 129/Sv and C57BL/6J BAC libraries were isolated. The assembled 1.3-Mb contig, consisting of 45 BACs, gives five- to sixfold coverage over the gene cluster and provides an average resolution of one marker every 32 kb. A number of BAC insert ends were sequenced, generating 30 new sequence tag sites (STS) in addition to 6 Gabr gene-based and 3 expressed sequence tag (EST)-based markers. STSs from, and surrounding, theGabrg1–Gabra2–Gabrb1 gene cluster were mapped in the T31 mouse radiation hybrid panel. The integration of the BAC contig with a map of loci ordered by radiation hybrid mapping suggested the most likely genomic orientation of this cluster on mouse chromosome 5: cen–D5Mit151–Gabrg1–Gabra2–Gabrb1–D5Mit58–tel. This established contig will serve as a template for genomic sequencing and for functional analysis of the GABAA gene cluster on mouse chromosome 5 and the corresponding region on human chromosome 4.The sequence data described in this paper have been submitted to the GenBank/GSS data libraries under accession nos.AF156490 and AQ589406–AQ589436.

Murine Albino-Deletion Complex: High-Resolution Microsatellite Map and Genetically Anchored YAC Framework Map

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 787-799
Author(s):  
Brad A Rikke ◽  
Dabney K Johnson ◽  
Thomas E Johnson
Keyword(s):  
Positional Cloning ◽  
Interspecific Backcross ◽  
Genetic Distances ◽  
Sequence Length ◽  
Recombination Rates ◽  
Oak Ridge ◽  
Artificial Chromosomes ◽  
Microsatellite Map ◽  
Specific Locus ◽  
Yeast Artificial Chromosomes

The murine albino-deletion complex developed as part of the Oak Ridge specific-locus test covers 6–11 cM of chromosome 7. This complex has proven to be a valuable resource for localizing traits to a small target region suitable for positional cloning. In this study, we mapped the endpoints of deletions in this complex using all of the available Mit simple-sequence length polymorphism (SSLP) markers. Concurrently, this mapping has determined the map order of nearly all of the SSLP markers, most of which were previously unresolved. The SSLP-based deletion map was confirmed and genetic distances were determined using the European Collaborative Interspecific Backcross panel of nearly a thousand mice. The average SSLP marker resolution is 0.3–0.4 cM, comparable to the cloning capacity of yeast artificial chromosomes (YACs). The SSLP markers were then used to construct a genetically anchored YAC framework map that further confirms the deletion map. We find that the largest deleted region distal to Tyr is about two to three times larger than the largest proximal deleted region, and the original C3H/101 regions flanking the deletions (moved to an St2A cch/cch background) are smaller than anticipated, which we suggest may result from increased recombination rates immediately flanking the deleted regions.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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