Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation.
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.
Gu
J Z
JZ
Laboratory of Gene Transfer, National Institutes of Health, Bethesda, MD 20892, USA.
Carstea
E D
ED
Cummings
C
C
Morris
J A
JA
Loftus
S K
SK
Zhang
D
D
Coleman
K G
KG
Cooney
A M
AM
Comly
M E
ME
Fandino
L
L
Roff
C
C
Tagle
D A
DA
Pavan
W J
WJ
Pentchev
P G
PG
Rosenfeld
M A
MA
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*
D018076:DNA, Complementary