Bacteriophage T4 Transfer RNA

Citation

Wilson, John Howard (1972) Bacteriophage T4 Transfer RNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/8JM5-QP49. https://resolver.caltech.edu/CaltechTHESIS:12042017-133901070

Abstract

Two T4-coded nonsense suppressors, psu⁺_a and psu⁺_b, have been isolated and characterized. Both were isolated as pseudo-wild type revertants of phage strains which carry multiple amber mutations. psu⁺_a is an amber suppressor which occurs at a frequency of 10^-11 to 10^-12 and is indistinguishable from wild type phage in its growth on both B and K strains of E. coli bacteria. psu⁺_b may be either an amber or on an ochre suppressor which occurs at a frequency of 10^-7 - 10^-10 and makes small plaques on B strains but grows very poorly or not at all on K strains. Phage with the characteristics of psu⁺_a occure in populations of psu⁺_b phage at a frequency of 10^-4. Both suppressors insert serine in response to the amber codon at an efficiency of about 45%. psu⁺_a and psu⁺_b map less than 0.3 map units apart and are located between genes e and 57 about 8 map units from gene e. On the basis of their initial frequencies of appearance and the frequency of psu⁺_b mutation to psu⁺_a, we speculate that psu⁺_a is derived from the wild type ser-tRNA by two base changes in the anticodon and that psu⁺_b is a one-base-change intermediate.

151 independent suppressor-negative derivatives of psu⁺_b phage have been isolated and characterized. They fall into two complementation groups. One, designated mb (modifier of psu⁺_b phenotype), is unlinked to psu⁺_b and has been located about 10 map units from rII. The second group, designated psu⁺_b, is made up of deletions and single base changes which affect sites within 0.2 map units of the original mutation. Those psu^-_b mutants which still contain the original mutation, psu_b, have been mapped relative to psu_b and each other by a series of two-factor and intragenic three-factor crosses. ³²P-labeles tRNA from mb, psu^-_b and wild type infected cells have been compared by polyacrylamide gel electrophoresis. In mb-infected cells several of the tRNA species are missing, while in psu^-_b-infected cells only the ser-tRNA is clearly absent. These studies suggest there are only two phage genes which are essential for the production of functional ser-tRNA. One is the structural gene for the ser-tRNA and the second plays an undefined role which affects several tRNAs.

E. coli cells infected with phage strains carrying a large deletion of gene e or gene psu⁺_b, are missing most if not all of the phage tRNAs normally present in wild type infected cells. By DNA-RNA hybridization we have demonstrated that the DNA corresponding to the missing tRNAs is absent. Thus the genes for these tRNAs must be clustered in the same region of the genome as the ser-tRNA gene. We have been able to locate and to define a maximum size for the cluster by physically mapping the deletions of genes e and psu⁺_b by examination of heteroduplex DNA in the electron microscope. That such deletions can be isolated indicates that the phage-specific tRNAs from this cluster are dispensable.

Thesis Files