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Title: Transformation of Bacillus Subtilis with cloned thymidylate synthetases

Bacillus subtilis carries two genes, thyA and thyB, each encoding different protein products, with thymidylate synthetase (TSase) activity. Either of these genes alone is sufficient for thymidine independence in B. subtilis. In addition there exist two B. subtilis temperate bacteriophages which upon infection of thymine requiring auxotrophs results in conversion of the organism to thymine independence. Chimeric plasmids selected for Thy+ transforming activity in E. coli were constructed and then used as a source of defined highly enriched DNA with which to transform competent B. subtilis. These plasmids were studied for their: (1) abiility to transform B. subtilis to thymine independence; (2) site of integration within the B. subtilis chromosome upon transformation; (3) phenotype of Thy+ plasmid generated transformants; and (4) nucleotide sequence homology among the cloned DNA fragments conferring thymine independence. Plasmids containing the two bacteriophage thy genes displayed the phenotype associated with thyA, whereas the plasmids containing the cloned B. subtilis chromosomal genes displayed the phenotype associated with thyB. Utilizing similar technology, the ability of an entirely foreign hybred bacterial plasmiid to transform B. subtilis was examined. In this case the gene from E. coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmidmore » was effective in transforming both E. coli and B. subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine requiring strains of B. subtilis to thymine independence. Although the Thy/sup +/ transformants of E. coli contained plasmid DNA, the Thy+ transformants derived from the transformation of B. subtilis did not contain detectable extrachromosomal DNA. Instead the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis. (ERB)« less
Authors:
 [1]
  1. Univ. of Rochester, NY (United States). Dept. of Radiation Biology and Biophysics
Publication Date:
OSTI Identifier:
5307311
Report Number(s):
UR--3490-1859
DOE Contract Number:
AC02-76EV03490
Resource Type:
Thesis/Dissertation
Research Org:
Univ. of Rochester, NY (United States). Dept. of Radiation Biology and Biophysics
Sponsoring Org:
USDOE; National Institutes of General Medical Sciences
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BACILLUS SUBTILIS; TRANSFORMATIONS; ENZYME ACTIVITY; GENETIC CONTROL; PLASMIDS; HYBRIDIZATION; BACTERIOPHAGES; DNA; EXPERIMENTAL DATA; ISOLATED VALUES; LIGASES; PHENOTYPE; THYMIDYLIC ACID; BACILLUS; BACTERIA; CELL CONSTITUENTS; DATA; DATA FORMS; ENZYMES; INFORMATION; MICROORGANISMS; NUCLEIC ACIDS; NUCLEOTIDES; NUMERICAL DATA; ORGANIC COMPOUNDS; PARASITES; PEST CONTROL; VIRUSES 550400* -- Genetics; 550700 -- Microbiology; 550200 -- Biochemistry