Akiko Miura

Studies on radiation-sensitive mutants of E. coli

SummaryThe bacterial recA gene participates in the induction by UV irradiation of the clear mutation of phage λ and the Lac- mutation of bacteria. The necessary function is induced by irradiation of Rec+ bacteria and acts upon DNA irradiated with UV light.

A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins

A temperature-sensitive E. coli mutant with a mutation in the spc ribosomal protein operon was found to have a conditional defect in the processing of precursor proteins destined for the periplasmic space or the outer membrane. At high temperatures, significant amounts of precursor proteins having unprocessed signal sequences are detected in the mutant cell by pulse-labeling. The precursors are processed at very slow rates during a subsequent chase. Genetic analysis indicates that the mutation impairs the function of a gene, termed secY, located at the promoter-distal part of the spc operon. The secY gene is distinct from those genes previously known to specify ribosomal proteins, yet it is within the spc operon. It is suggested that the product of the secY gene is a component of the cellular apparatus that is essential for protein secretion across the cytoplasmic membrane. The gene secY is probably identical with prlA, previously identified as a suppressor of signal sequence mutations.

The mutagenic action of sodium bisulfite

Abstract This paper describes the mutagenic activity of sodium bisulfite toward phage λ. The frequency of mutation of the c gene of λ was increased about 10 times as much as that of spontaneous mutation by treatment of the phage with 3 M NaHSO3 solution of pH 5.6 at 37° for 1.5 hrs. The mutagenesis could be related to the cytosine derivative specific reaction of sodium bisulfite.

Structure of nucleohistone. III. Interaction with toluidine blue

Abstract The interaction of toluidine blue with calf-thymus nucleohistone and DNA was investigated spectrophotometrically with special reference to the spectral transition occurring on formation of the complex. The dye was found to bind to nucleohistone and DNA in at least two ways (Process I, II). These binding sites could be estimated separately. It was found by Millipore filtration methods that there were 0.35 and 0.32 sites per unit nucleotide of DNA and nucleohistone respectively for Process I, and 1.00 and 0.48 for Process II. The results indicate that in a nucleohistone molecule about half the phosphate groups are free from basic groups of the histones.

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12.

SummaryTemperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.

Formation of deletion in Escherichia coli between direct repeats located in the long inverted repeats of a cellular slime mold plasmid: Participation of DNA gyrase

SummaryWe constructed a recombinant plasmid containing the 2.1 kb HindIII fragment of plasmid pDG1, isolated from the cellular slime mold (Dictyostelium sp. strain GA11), and using pAG60 as cloning vector. We found that deletions of the recombinant plasmid took place frequently in Escherichia coli wild-type cells. However, the deletion was not observed when the plasmid was introduced into a strain that was an isogenic temperature-sensitive mutant of the gyrA gene. These results suggest that E. coli DNA gyrase is involved in the mechanisms of the deletion formation. It was shown that the 1.0 kb deletant derived from the 2.1 kb HindIII insert was produced by elimination of a 1.1 kb region. Sequence analysis of the deletants showed that cutting and rejoining took place between two out of the six nearly perfect direct repeats [21 bp palindromic sequences; AAAAAA(T/C)GGC(G/C)GCC(A/G)TTTTTT], located near the distal ends of the inverted repeats, preserving one copy of the repeats. These sequences consist of local short inverted repeats, where cutting and rejoining occur at one of the two regions.

Inactivation of bacteriophage λ during the aerobic oxidation of bisulfite

Treatment of bacteriophage lambda with 0.0001 to 0.01 M bisulfite ion in the presence of air and a catalytic amount of Mn(II) ion resulted in a rapid decrease in the plaque-forming activity of the phage. The functions of the coat proteins, i.e., the adsorption to the bacteria and the injection of phage DNA into them, were damaged by the treatment. However, the phage DNA was not affected during this treatment with respect to size and transfection activity. This contrasted to the ease with which naked DNA was damaged by the treatment with bisulfite under comparable conditions. Tryptophan, methionine, and 4-thiouridine, all of which are known to react with the free radicals generated during the aerobic oxidation of bisulfite, protected the phage from the attack of the reagents. The results suggested that proteins may be easily impaired by a combined action of sulfur dioxide and oxygen. Inhalation of sulfur dioxide will offer a situation in which the radicals can be produced near the surface of the respiratory organs. 24 references, tables.

Ultraviolet-induced mutation of bacteriophage λ

SummaryClear mutations of bacteriophage λ are increased within F- recipients in a cros s with ultraviolet (UV) irradiated F′ donors, without a direct UV irradiation of recipients. This mutagenesis occurs when phages are also irradiated with UV. The phenomenon is called an indirect UV induction of the mutation. The indirect UV induction of the mutation is also produced by a cross with UV irradiated Hfr male cells, and does not occur when the recipient is defective in recA gene.