RESUMO
Rapid genomic scanning methods are required to identify expressed sequences and we report an efficient, sensitive and specific approach which relies upon hybridization of an amplified, labeled cDNA library to digested cosmid DNA. We identified expressed sequences within a cosmid in the glycerol kinase (GK) "critical region" of Xp21 that had impressive similarity to prokaryotic GKs. We used this genomic sequence information to clone the human hepatic GK cDNA. Independent confirmation of the identity of this gene was obtained by functional complementation of GK deficient E. coli mutants with a construct containing the complete human X-linked GK coding sequence.
Assuntos
Glicerol Quinase/genética , Fígado/enzimologia , Cromossomo X , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Cosmídeos , Escherichia coli/enzimologia , Escherichia coli/genética , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
The adrenal hypoplasia congenita (AHC) and glycerol kinase (GK) loci are telomeric to the Duchenne muscular dystrophy locus in Xp21. We developed a pair of yeast artificial chromosome (YAC) contigs spanning at least 1.2 Mb and encompassing the region from the telomeric end of the Duchenne muscular dystrophy (DMD) locus to beyond YHX39 (DXS727), including the genes for AHC and GK. The centromeric contig consists of 13 YACs reaching more than 600 kb from DMD through GK. The telomeric contig group consists of 8 YACs containing more than 600 kb including the markers YHX39 (DXS727) and QST-59 (DXS319). Patient deletion breakpoints in the region of the two YAC contigs define at least eight intervals, and seven deletion breakpoints are contained within these contigs. In addition to the probes developed from YAC ends, we have mapped eight Alu-PCR probes amplified from a radiation-reduced somatic cell hybrid, two anonymous DNA probes, and one Alu-PCR product amplified from a cosmid end, for a total of 26 new markers within this region of 2 Mb or less. One YAC in the centromeric contig contains an insert encompassing the minimum interval for GK deficiency defined by patient deletion breakpoints, and this clone includes all or part of the GK gene.