The speB gene of E. coli encodes agmatine ureohydrolase (AUH). AUH catalyses the hydrolysis of agmatine to urea and putrescine in a polyamine biosynthetic pathway. The plasmid pKA5, derived from an E. coli genomic library, was the source of a 2.97 kb restriction fragment containing the speB gene. Sequencing of this fragment revealed three intact open reading frames, ORF1 and ORF2 on one strand and ORF3 on the opposite strand, as well as a truncated open reading frame, ORF4, which terminated 92 kb upstream from ORF3. ORF2 and ORF3 were convergent, and overlapped by 85% of their sequence. ORF1 and ORF3 were separated by a sequence of two imperfect repeats containing four palindromes, three of which were overlapping. ORF3 represented the coding sequence of the speB gene. Two transcripts were detected from the speB gene: a shorter transcript, initiated 101 bp upstream from ORF3, and a polycistronic message, coding for ORF3 and ORF4. The short transcript was abundantly expressed when ORF4 sequences were deleted, but when ORF4 and its upstream sequences were present, the polycistronic message predominated and the amount of the monocistronic message was drastically reduced. The promoter producing the shorter transcript required only a -12 TATACT sequence for activity. Deletion of a 460 bp fragment comprising the 5'-region of ORF1 from a plasmid containing ORF1, ORF2 and speB reduced the activity of AUH by 83%. This fragment contained two divergently oriented promoters. The presence of ORF1 did not stimulate ß-galactosidase encoded by the speB promoter fused to lacΖ. Agmatine induced transcription from speB but not from the ORF4 nor the ORF1 promoters. cAMP caused an 88% reduction in the AUH activity of wild type E. coli K-12 but had no effect on the activity of plasmid encoded AUH. The activity of neither the speB nor the ORF4 promoters fused to lacΖ or phoA were influenced by cAMP; in contrast, the lacZ promoter fused to lacZ or phoA was stimulated by cAMP. Thus, the role of cAMP and CRP on speB expression is indirect and limited to a single copy state. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/54438 |
| Date | January 1989 |
| Creators | Szumanski, Maria B. W. |
| Contributors | Biochemistry and Nutrition, Boyle, S.M., Ebel, Richard E., Potts, Malcolm, Sitz, Thomas O., Storrie, Brian |
| Publisher | Virginia Polytechnic Institute and State University |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | viii, 96 leaves, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 20440540 |
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