Transformation of Bacillus Subtilis with cloned thymidylate synthetases

Thesis/Dissertation · Tue Jan 01 00:00:00 EST 1980

DOI:https://doi.org/10.2172/5307311· OSTI ID:5307311

Rubin, Edward M. ^[1]

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1. Univ. of Rochester, NY (United States). Dept. of Radiation Biology and Biophysics

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 plasmid 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)

• Research Organization: Univ. of Rochester, NY (United States). Dept. of Radiation Biology and Biophysics
• Sponsoring Organization: USDOE; National Institutes of General Medical Sciences
• DOE Contract Number: AC02-76EV03490
• OSTI ID: 5307311
• Report Number(s): UR-3490-1859
• Country of Publication: United States
• Language: English

Related Subjects

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