Dissertations, Theses, and Capstone Projects

Date of Degree

1991

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Rivka Rudner

Committee Members

Marvin S. Friedman

Peter N. Lipke

Sharon D. Cosloy

Charles Tackney

Subject Categories

Biology

Abstract

The nucleotide sequence of the trpE, trpD, and 5’trpC genes of the Bacillus pumilus tryptophan operon was determined for a comparative evaluation. DNA comparisons to the tryptophan operon of a related species, Bacillus subtilis, showed 66% base homology. The overlapping of translational start and stop signals indicate that, similar to Escherichia coli, the trpE, trpD, and trpC genes of the B.pumilus trp operon are translationally coupled. The values for amino acid identity between the two Bacilli showed 68, 62, and 65% for trpE, trpD and 5’trpC genes, respectively. Charge related residues increased the % similarity to 79, 76, and 72%. Amino acid comparisons to the E.coli trpE, trpD, and 5’trpC gene products showed an average of 30 and 50% for % amino acid identity and % amino acid similarity, respectively. Genetic analyzes with a 3.6-kb B.pumilus trp DNA fragment cloned in opposite orientations in plasmids pRR106 and pRR103 revealed the presence of an internal promoter upstream to trpC. A search for consensus-like promoter sequences upstream to trpC did not reveal an obvious promoter sequence. In the trpE gene three potential promoter sequences were identified by homology to the consensus. Two of these tandem promoter-like elements were able to facilitate the expression of β-galactosaidase in E.coli 71-18 when cloned upstream to a plasmid promoterless lacZ gene.

The amidotransferase activity of anthranilate synthetase was assayed from B.subtilis trp mutants transformed with pRR106. The anthranilate synthetase complex showed sensitivity to Trp and to regulation by the mtr locus. Since mtr regulation is believed to act on the trp leader mRNA and the trp leader is not present on pRR106, this result indicates that additional areas or levels of regulation may be involved with mtr.

The 2.733-kb sequenced portion of the trpE, trpD, and 5’trpC genes of B.pumilus was cloned into the integrable plasmid pJH101 as a molecular probe for preliminary experiments into the nature of heterologous integration in Bacillus subtilis.

Comments

Digital reproduction from the UMI microform.

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