Measurement of Feedback Inhibition In Vivo and Selection of ATCase Feedback Altered Mutants in Salmonella typhimurium

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Aspartate transcarbamoylase (ATCase; encoded by pyrBI genes) is one of the most studied regulatory enzymes in bacteria. It is feedback inhibited by cytidine triphosphate (CTP) and activated by adenosine triphosphate (ATP). Much is known about the catalytic site of the enzyme, not nearly as much about the regulatory site, to which CTP binds. Until now a positive selection for feedback-modified mutants was not available. The selection we have developed involves the use of a pyrA deletion in S. typhimurium. This strain lacks carbamoylphosphate and requires both a pyrimidine and arginine for growth. In this strain citrulline is used to satisfy ... continued below

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v, 109 leaves: ill.

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Bailey, Andrea J., 1952- August 1989.

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  • Bailey, Andrea J., 1952-

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Aspartate transcarbamoylase (ATCase; encoded by pyrBI genes) is one of the most studied regulatory enzymes in bacteria. It is feedback inhibited by cytidine triphosphate (CTP) and activated by adenosine triphosphate (ATP). Much is known about the catalytic site of the enzyme, not nearly as much about the regulatory site, to which CTP binds. Until now a positive selection for feedback-modified mutants was not available. The selection we have developed involves the use of a pyrA deletion in S. typhimurium. This strain lacks carbamoylphosphate and requires both a pyrimidine and arginine for growth. In this strain citrulline is used to satisfy the pyrimidine and arginine requirements. The minimal flow through the pyrimidine pathway from the citrulline-produced carbamoylphosphate is exquisitely sensitive to feedback control of ATCase by CTP. By elevating the CTP pool, via exogenous cytidine, in a strain that also contains a cytidine deaminase mutant (cdd) growth can be stopped completely, indicating 100% inhibition. It was therefore possible to measure in vivo feedback inhibition of ATCase among the citrulline users and to isolate a family of ATCase regulatory mutants with either modified or no response to effectors.

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v, 109 leaves: ill.

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  • August 1989

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  • Aug. 22, 2014, 6 p.m.

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  • Sept. 24, 2015, 2:20 p.m.

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Bailey, Andrea J., 1952-. Measurement of Feedback Inhibition In Vivo and Selection of ATCase Feedback Altered Mutants in Salmonella typhimurium, dissertation, August 1989; Denton, Texas. (digital.library.unt.edu/ark:/67531/metadc330749/: accessed October 23, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; .