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Article: Isolation of human transcripts expressed in hamster cells from YACs by cDNA representational difference analysis

TitleIsolation of human transcripts expressed in hamster cells from YACs by cDNA representational difference analysis
Authors
Issue Date1999
PublisherCold Spring Harbor Laboratory Press, Publications Department. The Journal's web site is located at http://www.genome.org
Citation
Genome Research, 1999, v. 9 n. 2, p. 182-188 How to Cite?
AbstractGene 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 YACs 911D5 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.
Persistent Identifierhttp://hdl.handle.net/10722/150746
ISSN
2015 Impact Factor: 11.351
2015 SCImago Journal Rankings: 14.352
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorGu, Jen_US
dc.contributor.authorGuan, XYen_US
dc.contributor.authorAshlock, MAen_US
dc.date.accessioned2012-06-26T06:09:44Z-
dc.date.available2012-06-26T06:09:44Z-
dc.date.issued1999en_US
dc.identifier.citationGenome Research, 1999, v. 9 n. 2, p. 182-188en_US
dc.identifier.issn1088-9051en_US
dc.identifier.urihttp://hdl.handle.net/10722/150746-
dc.description.abstractGene 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 YACs 911D5 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.en_US
dc.languageengen_US
dc.publisherCold Spring Harbor Laboratory Press, Publications Department. The Journal's web site is located at http://www.genome.orgen_US
dc.relation.ispartofGenome Researchen_US
dc.subject.meshAnimalsen_US
dc.subject.meshBlotting, Southern - Methodsen_US
dc.subject.meshChromosomes, Artificial, Yeast - Geneticsen_US
dc.subject.meshCricetinaeen_US
dc.subject.meshDna, Complementary - Analysisen_US
dc.subject.meshGene Expression - Geneticsen_US
dc.subject.meshHumansen_US
dc.subject.meshMolecular Sequence Dataen_US
dc.subject.meshNucleic Acid Hybridization - Methodsen_US
dc.subject.meshRna, Messenger - Isolation & Purificationen_US
dc.subject.meshSequence Analysis, Dnaen_US
dc.subject.meshTranscription, Geneticen_US
dc.titleIsolation of human transcripts expressed in hamster cells from YACs by cDNA representational difference analysisen_US
dc.typeArticleen_US
dc.identifier.emailGuan, XY:xyguan@hkucc.hku.hken_US
dc.identifier.authorityGuan, XY=rp00454en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.pmid10022983-
dc.identifier.scopuseid_2-s2.0-0033046723en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0033046723&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume9en_US
dc.identifier.issue2en_US
dc.identifier.spage182en_US
dc.identifier.epage188en_US
dc.identifier.isiWOS:000078770400010-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridGu, J=7403130063en_US
dc.identifier.scopusauthoridGuan, XY=7201463221en_US
dc.identifier.scopusauthoridAshlock, MA=6603049981en_US

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