Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

August 1991

Abstract

Analysis of E. coli strains SK1047 and SK1048 have shown them to be temperature-sensitive, protein-synthesis deficient. An alteration in ribosomal protein L22 was detected in both strains using two dimensional gel electrophoresis. Protein L22 was purified from both strains by reversed phase high performance liquid chromatography and from two dimensional electrophoretic gels. Purified ribosomal protein L22 was labeled by reductive methylation and used in 23S RNA binding assays with and without ribosomal protein L4. At the permissive temperature, protein L22 from SK1047 bound less efficiently than the control while protein L22 from SK1048 bound as efficiently as the control. At the restrictive temperature, both forms of mutant protein L22 bound less efficiently than the control. In both mutants, temperature sensitivity was mapped to the chromosomal region containing the rplV gene for ribosomal protein L22 using bacteriophage P1 transduction and bacteriophage $\lambda$ complementation. The wild type rplV gene subcloned into plasmid pLF1.0 was also shown to complement temperature sensitivity. The partial diploid nature of strains complemented by $\lambda$fus2 and plasmid pLF1.0 was verified when both wild type and mutant protein L22 were found on two dimensional gels. Reisolation of protein L22 from gels of $\lambda$fus2 complemented cells showed that both forms of protein L22 were in equal proportion irrespective of growth temperature. Reisolation of protein L22 from gels of plasmid pLF1.0 complemented cells showed that incorporation of the mutant protein exceeded the control protein at the permissive temperature; while the reverse was seen at the restrictive temperature. Temperature-shift experiments were conducted on complemented mutant cells to determine the effect of increased gene dosage on the coordinated regulation of ribosomal protein synthesis. Mutants complemented with $\lambda$fus2 exhibited normal cell growth, indicating that regulation was not effected. Cells transformed with plasmid pLF1.0 exhibited a reduction in growth possibly due to the disruption of balanced synthesis. The wild type and both mutant rplV genes were amplified using polymerase chain reaction and the PCR product was sequenced using primer extension. Sequencing of DNA from both mutants revealed the codon CGC for the amino acid arginine at position 8 in the protein chain was mutated to the TGC codon for the amino acid cysteine. The wild type ribosomal protein L22 contains no cysteine residues. The mutation was confirmed by testing control and mutant protein L22 for the presence of sulfhydryls using 4,4$\sp\prime$-dithiodipyridine. Ribosomal protein L22 isolated from both mutant strains was found to contain one cysteine sulfhydryl group.

Document Type

Dissertation - unrestricted

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