Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe.
We have obtained a cDNA clone encoding most of human X-linked 3-phosphoglycerate kinase (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3). Total mRNA was prepared from human adenocarcinoma-derived cell line LS174T and used for cDNA preparation. Double-stranded cDNA was inserted, after tailing with oligo(dC), into the plasmid vector pBR327 and cloned in Escherichia coli K-12. Transformants were screened by colony hybridization with a mixture of 32P-labeled oligodeoxyribonucleotides. A pool of hexadecamers complementary to all 32 possible sequences encoding amino acids 291-296 of X-linked PGK was used for the initial screen. One clone among 2,500 gave a strong positive signal. Plasmid DNA from this clone was purified and characterized by hybridization first to the hexadecamer probe mixture and then to an undecamer probe consisting of a mixture of four sequences. The cloned fragment hybridizes preferentially to DNA from human cells with five X chromosomes. DNA sequence analysis has established that the 1.2-kilobase-pair fragment encodes PGK from amino acid 121 through the COOH terminus.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005260:Female; D006801:Humans; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D012730:Sex Chromosomes; D014960:X Chromosome
D009838:Oligodeoxyribonucleotides; D004247:DNA; D010735:Phosphoglycerate Kinase