AMPLIFICATION OF AN ADENYLOSUCCINATE SYNTHETASE GENE IN ALANOSINE-RESISTANT MURINE T-LYMPHOMA CELLS - MOLECULAR-CLONING OF A CDNA-ENCODING THE NONMUSCLE ISOZYME

作者：GUICHERIT OM; COOPER BF; RUDOLPH FB; KELLEMS RE

来源：Journal of Biological Chemistry, 1994, 269(6): 4488-4496.

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the initial step in the conversion of IMP to AMP. Two isoforms of this enzyme have been observed in vertebrates. A muscle isozyme is highly abundant in cardiac and skeletal muscle tissue and is thought to play a role in muscle energy metabolism. The non-muscle isozyme, which is present at low levels in most tissues, likely functions in de novo AMP biosynthesis. The analysis of the non-muscle isozyme has been hampered by its low abundance and instability during purification. In this study a genetic selection scheme was used to generate a murine T lymphoma cell line which was at least 100-fold enriched for the non-muscle isozyme, as a result of amplification of the non-muscle synthetase gene. This cell line made possible the purification of the non-muscle isozyme, and the subsequent isolation of isozyme-specific peptides. Based on peptide sequence information a degenerate oligonucleotide probe was designed and used to screen a mouse kidney cDNA library. A 1.5-kilobase cDNA encoding the non-muscle isozyme was cloned and found to contain an open reading frame of 1368 base pairs encoding 456 amino acids. Gene transfer experiments showed that the cDNA encoded a 50-kDa protein, the size expected for mammalian synthetases, that correlated with the presence of high levels of synthetase activity. The deduced amino acid sequence of the mouse non-muscle synthetase is similar to 75% identical to the previously reported mouse muscle synthetase. Southern blot analysis of mouse genomic DNA with the isozyme specific cDNA probes revealed that the synthetase isozymes are encoded by separate genes. The non-muscle gene is expressed in most tissues but is virtually undetectable in striated muscle tissues. Three different transcripts (1.7, 2.8, and 3.4 kilobases) are detected for the nonmuscle isozyme which show a similar tissue distribution. The availability of a cDNA for the non-muscle isozyme of adenylosuccinate synthetase will facilitate further comparative analyses with the previously cloned muscle isozyme.

• 出版日期1994-2-11