Amplification of an adenylosuccinate synthetase gene in alanosine-resistant murine T-lymphoma cells. Molecular cloning of a cDNA encoding the "non-muscle" 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 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 approximately 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 non-muscle 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.
Guicherit
O M
OM
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Cooper
B F
BF
Rudolph
F B
FB
Kellems
R E
RE
D000265:Adenylosuccinate Synthase / Q000235:genetics*; D000409:Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D004351:Drug Resistance; D005784:Gene Amplification; D005796:Genes; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D017931:DNA Primers; D007527:Isoenzymes; C000082:alanosine; D000265:Adenylosuccinate Synthase; D000409:Alanine