title: Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation.
abstract: Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.
authors.affiliation: Laboratory of Gene Transfer, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: J Z
authors.initials: JZ
authors.lastname: Gu
authors.forename: E D
authors.initials: ED
authors.lastname: Carstea
authors.forename: C
authors.initials: C
authors.lastname: Cummings
authors.forename: J A
authors.initials: JA
authors.lastname: Morris
authors.forename: S K
authors.initials: SK
authors.lastname: Loftus
authors.forename: D
authors.initials: D
authors.lastname: Zhang
authors.forename: K G
authors.initials: KG
authors.lastname: Coleman
authors.forename: A M
authors.initials: AM
authors.lastname: Cooney
authors.forename: M E
authors.initials: ME
authors.lastname: Comly
authors.forename: L
authors.initials: L
authors.lastname: Fandino
authors.forename: C
authors.initials: C
authors.lastname: Roff
authors.forename: D A
authors.initials: DA
authors.lastname: Tagle
authors.forename: W J
authors.initials: WJ
authors.lastname: Pavan
authors.forename: P G
authors.initials: PG
authors.lastname: Pentchev
authors.forename: M A
authors.initials: MA
authors.lastname: Rosenfeld
mesh_terms: D000818:Animals; D016466:CHO Cells; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D002887:Chromosomes, Human, Pair 18; D002897:Chromosomes, Human, Pair 7; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D006801:Humans; D051379:Mice; D009542:Niemann-Pick Diseases / Q000235:genetics*
chemical_list: D018076:DNA, Complementary