Cloning of rat asparagine synthetase and specificity of the amino acid-dependent control of its mRNA content.
A full-length cDNA clone for rat asparagine synthetase (AS) was isolated from a cDNA library enriched for amino acid-regulated sequences. The AS cDNA was used to investigate the amino acid-dependent repression of AS mRNA content in rat Fao hepatoma cells. In response to complete amino acid starvation, there was an approximately 10-fold increase in the level of AS mRNA. Three species of mRNA, of approx. sizes 2.0, 2.5 and 4.0 kb, were detected and each was simultaneously regulated to the same degree. The expression of AS mRNA increased by 6 h after removal of amino acids, reached a plateau after 9 h, and was blocked by either actinomycin D or cycloheximide. Partial repression of the AS mRNA content was maintained by the presence of a single amino acid in the culture medium, but the degree of effectiveness for each one varied widely. Glutamine showed the greatest ability to repress the AS mRNA content, even at an extracellular concentration 10 times below its plasma level. Other effective repressors included the amino acids asparagine, histidine and leucine, as well as ammonia. Depletion of selected single amino acids from an otherwise complete culture medium also caused up-regulation. In particular, removal of histidine, threonine or tryptophan from the medium, or the addition of histidinol to inhibit histidinyl-tRNA synthetase, resulted in a significant increase in AS mRNA content. The data indicate that nutrient regulation of AS mRNA occurs by a general control mechanism that is responsive to a spectrum of amino acids.
Hutson
R G
RG
Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville 32610-0245.
Kilberg
M S
MS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000172:deficiency / Q000378:metabolism*; D000818:Animals; D001217:Aspartate-Ammonia Ligase / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D006641:Histidinol / Q000494:pharmacology; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000096:biosynthesis; D051381:Rats; D013379:Substrate Specificity; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D000596:Amino Acids; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D006641:Histidinol; D001217:Aspartate-Ammonia Ligase