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

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 o bserved in vertebrates. A muscle isozyme is highly abundant in cardiac and skeletal muscle tissue and is thought to play a role in muscle en ergy metabolism. The non-muscle isozyme, which is present at low level s in most tissues, likely functions in de novo AMP biosynthesis. The a nalysis of the non-muscle isozyme has been hampered by its low abundan ce and instability during purification. In this study a genetic select ion scheme was used to generate a murine T lymphoma cell line which wa s at least 100-fold enriched for the non-muscle isozyme, as a result o f amplification of the non-muscle synthetase gene. This cell line made possible the purification of the non-muscle isozyme, and the subseque nt isolation of isozyme-specific peptides. Based on peptide sequence i nformation 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 fra me of 1368 base pairs encoding 456 amino acids. Gene transfer experime nts showed that the cDNA encoded a 50-kDa protein, the size expected f or mammalian synthetases, that correlated with the presence of high le vels of synthetase activity. The deduced amino acid sequence of the mo use non-muscle synthetase is similar to 75% identical to the previousl y reported mouse muscle synthetase. Southern blot analysis of mouse ge nomic DNA with the isozyme specific cDNA probes revealed that the synt hetase isozymes are encoded by separate genes. The non-muscle gene is expressed in most tissues but is virtually undetectable in striated mu scle tissues. Three different transcripts (1.7, 2.8, and 3.4 kilobases ) are detected for the nonmuscle isozyme which show a similar tissue d istribution. The availability of a cDNA for the non-muscle isozyme of adenylosuccinate synthetase will facilitate further comparative analys es with the previously cloned muscle isozyme.