Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe.
AUTOR(ES)
Singer-Sam, J
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=393468Documentos Relacionados
- Isolation of a cDNA clone for the human HLA-DR antigen alpha chain by using a synthetic oligonucleotide as a hybridization probe.
- Isolation and DNA sequence of a full-length cDNA clone for human X chromosome-encoded phosphoglycerate kinase.
- MSP RFLP for X-linked proteolipid protein gene (PLP) identified with either rat or human PLP cDNA clone.
- Immunoglobulin variable heavy chain cDNA sequence from a patient with X-linked agammaglobulinemia.
- Detection of a unique human V kappa IV germline gene by a cloned cDNA probe.