NUTRITIONAL CONTROL OF ASPARAGINE SYNTHETASE

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DESCRIPTION: Nutrient control of gene expression has been analyzed at the molecular level in bacteria and yeast, but less is known in mammalian cells. The applicant has identified several proteins for which synthesis is increased in response the AA deprivation of tissue or cells in culture. One of these is the enzyme AS. The applicant has also shown that the induction of AS is largely or entirely due to enhanced transcription. The overall goals of this proposal are to identify the cis-acting sequences in the AS gene that are responsible for the amino acid-dependent transcription and to test the hypothesis that increased AS activity in asparaginase-resistant leukemia is the result of AA-dependent transcription, possibly involving the AASREs. The search for cis-acting elements in the AS gene will proceed through three specific aims: (1) mapping of DNAse I hypersensitive sites in or near the AS gene in aa fed and starved cells, (2) further in vitro analysis of potential AASREs through the use of deletional and mutational analysis of reporter constructs in transient expression systems, and (3) in vivo footprinting analysis to detect sites (AASREs) that are differentially interacting with trans-acting factors in aa fed versus starved cells. The fourth specific aim will test the hypothesis that asparaginase resistance of leukemia cells results from altered transcriptional control of the AS gene. This will be approached by comparing the DNAse I hypersensitivity and in vivo footprinting of chromatin from asparaginase-sensitive and asparaginase-resistant cells.