grantor:
University of TorontoAmino acid biosynthetic genes in mammalian cells are responsible for the biosynthesis of non-essential amino acids. Their regulation is poorly understood in spite of their physiological and pathological significance. In bacteria and yeast, amino acid synthesis genes are turned on by amino acid starvation and the molecular mechanisms in these two systems are well understood. In mammalian cells, including Chinese hamster ovary (CHO) cells, the asparagine synthetase (AS) enzymatic activity is up-regulated in response to amino acid starvation. This indicates a conserved adaptation response. The AS gene of CHO cells has been extensively studied at the biochemical level. It provides a model system to study the underlying molecular mechanism of the cellular response to amino acid starvation. The AS gene is a housekeeping gene; its promoter region is devoid of TATA and CAAT consensus sequences, but is GC rich. To investigate AS regulation, we first analyzed a 1.1 kb 5' genomic fragment of the AS gene in wild-type CHO cells under normal growth conditions. The minimal promoter was determined to lie within the -47 to +199 region. Two upstream GC boxes and a GC box in intron 1 were found to correspond to positive cis-elements for AS transcription. Specific DNA-protein interactions were detected within the AS minimal promoter region. Transcriptional regulation of the AS promoter was studied under amino acid starvation conditions. We found that AS promoter constructs were induced by asparagine depletion and that the level of induction was parallel to the level of induced AS enzymatic activity. A novel cis-element 5'-GATGAAACTTCCC-3 ' termed AARECHO was defined by mutational analyses. Chimeric promoter analysis indicated that a 24 bp AARECHO sequence could up-regulate activity of the thymidine kinase (TK) basal promoter upon asparagine depletion. A specific nuclear protein bound to the. AARE CHO containing probe. When AS was up-regulated by amino acid depletion, the uncharged tRNAs appeared to play a role downstream of the amino acid starvation signal and lead to AS up-regulation at the transcriptional level. The data lead us to propose a model for how mammalian cells increase amino acid synthesis upon amino acid starvation.Ph.D
