Leslie Barnett

More mutant tyrosine transfer ribonucleic acids

Abstract The isolation and properties of several mutants of the su III tyrosine suppressor transfer RNA gene are described. Two mutants with temperature-sensitive suppressor activity specify tRNAs with single base substitutions, giving mispaired bases in the amino acid acceptor arm. In one of these a G · C pair has been replaced by an A · C pair. A second site revertant of this mutant to wild-type suppressor activity specifies a tRNA with an A · U pair at this position. A similar two-step conversion of a G · C pair in the arm of the dihydrouracil loop to an A · U pair is described. Changing either of these base pairs from G · C to A · U does not alter the K m of aminoacylation of the tRNA with tyrosine tRNA synthetase. The tRNA sequence changes associated with three other second-site revertants are described. These have nucleotide substitutions in the dihydrouracil loop and arm. In two instances a C residue is changed to a dihydrouridylic acid residue. Only small amounts of tRNA are synthesised in vivo from the single-step su III mutants, including those specifying mutant tRNAs with A · C pairs in helical regions. In these, restoration of the normal hydrogen bonding by a second mutation results in restoration of the normal levels of tRNA synthesis. A second mutation which increases the synthesis of tRNA in a temperaturesensitive mutant is described. This mutation is linked to the su III − gene but does not lie within the tRNA sequence.

Mutant tyrosine transfer ribonucleic acids.

Three independent mutants of the suIII tyrosine suppressor transfer RNA gene have been isolated. These mutants are shown to produce mutant tRNAs differing from the wild-type suIII+ tRNA molecule in each case by a single base change. A mutant tRNA containing an A residue in place of a G in the “dihydrouracil loop” appears to be defective in a step in protein synthesis occurring after the acylated tRNA is bound to the ribosome. A mutant tRNA having an A residue in place of a G in the “anticodon stem” appears to be defective exclusively in its apparent affinity for the tyrosyl tRNA synthetase. The isolation of mutant tRNAs as well as their biological properties are discussed.

The genes sup-7 X and sup-5 III of C. elegans suppress amber nonsense mutations via altered transfer RNA

The sup-5 III and sup-7 X suppressors in C. elegans have previously been shown to have many genetic properties in common with tRNA nonsense suppressors of microorganisms. We report here the results of two lines of investigation into the molecular basis of these suppressors. In one, which sought to determine the nature of suppressible alleles, we demonstrate through DNA sequencing studies that a suppressible allele, unc-54(e 1300) I, of the myosin heavy chain gene contains a C leads to T substitution, which changes a glutamine codon to amber terminator at residue 1903. In the other approach, which sought to define the nature of the suppressing activity, we show through in vitro translation studies that tRNA fractions from the suppressor strains, but not wild-type, promote the specific readthrough of amber terminators of three different messenger RNAs. We conclude that sup-5 and sup-7 result in readthrough of amber terminators in vivo through an altered tRNA.

Vectors λ200g and λ200c: two useful derivatives of λ2001

Abstract We describe the construction and uses of two new phage λ, replacement vectors. Both are modifications of 2001 which allows selection of cloned inserts by the Spi − phenotype, following the replacement of a gam+ filler fragment. Vector λ 200g has a second gam gene with an amber mutation in its right arm which enables the selection of clones with inserts in the normal way in Su° host bacteria and the maintenance of their genetic stability by subsequent growth in Su+ strains. Vector λ200c is designed for complementation studies. It contains a cat gene and the attP site of λ. Recombinants can be inserted into the bacterial chromosome or into an episome with the aid of appropriate helpers.