Generation of a human chromosome 18-specific YAC clone collection and mapping of 55 unique YACs by FISH and fingerprinting.
A yeast artificial chromosome (YAC) library was constructed from a somatic cell hybrid line in which the human chromosome content had been reduced by repeated subcloning to one or two copies of chromosome 18. Screening of 4700 primary yeast transformants generated 74 clones containing a YAC with a human DNA insert averaging 190 kb in size. The human YACs were localized to subregions of chromosome 18 by in situ hybridization of biotin-labeled Alu-PCR products obtained using total yeast DNA as a template. Comparisons of interspersed repetitive sequence-PCR and restriction fragment fingerprint patterns identified five sets of identical and three sets of overlapping YACs. Dual-label fluorescence in situ hybridization interphase mapping was used to determine the order of some nonoverlapping YACs. STS (sequence-tagged site) content mapping was carried out with PCR primers for 56 chromosome 18-specific markers. The identification of YACs containing four known genes--encoding the pituitary adenylate cyclase activating polypeptide (PA-CAP), the myelin basic protein (MBP), ferrochelatase (FECH), and SSAV1, an endogenous retroviral element related to the SSAV virus--provides a precise cytological map position for the respective loci. Our final collection of 55 randomly isolated, unique, and regionally localized YACs (D18S107-D18S161) is distributed over the entire chromosome and collectively covers approximately 12 Mb, i.e., 16% of the estimated 77 Mb of DNA in euchromatin of chromosome 18. These YACs provide reagents for the isolation of genes in these regions and represent nucleation points for the generation of STS to increase coverage of the chromosome by YAC contigs.
Chang
E
E
Department of Genetics, Stanford University School of Medicine, California 94305-5428.
Welch
S
S
Luna
J
J
Giacalone
J
J
Francke
U
U
D000262:Adenylyl Cyclases / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D002887:Chromosomes, Human, Pair 18; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis / Q000235:genetics; D005294:Ferrochelatase / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000235:genetics; D009479:Neuropeptides / Q000235:genetics; D009838:Oligodeoxyribonucleotides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D016133:Polymerase Chain Reaction / Q000379:methods; D012190:Retroviridae / Q000235:genetics
C495401:ADCYAP1 protein, human; D004676:Myelin Basic Protein; D009479:Neuropeptides; D009838:Oligodeoxyribonucleotides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D004247:DNA; D000262:Adenylyl Cyclases; D005294:Ferrochelatase