Ts Mutation In Gene Research Articles

Overproduction of the Hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype.

The genes hsdM and hsdS for M. EcoKI modification methyltransferase and the complete set of hsdR, hsdM and hsdS genes coding for R. EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation in hsdS gene, were cloned in pBR322 plasmid and introduced into E. coli C (a strain without a natural restriction-modification (R-M) system). The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained. The hsdSts-1 mutation, mapped previously in the distal variable region of the hsdS gene with C1 245-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42 degrees C. In clones transformed with the whole hsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the mutant hsdS allele was present in the hybrid plasmid. Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of the hsdSts-1 mutation on restriction and modification.

Second-site suppressors of a cold-sensitive prohead accessory protein of bacteriophage phi X174.

This study describes the isolation of second-site suppressors which correct for the defects associated with cold-sensitive (cs) prohead accessory proteins of bacteriophage phi X174. Five phenotypically different suppressors were isolated. Three of these suppressors confer novel temperature-sensitive (ts) phenotypes. They were unable to complement a ts mutation in gene F which encodes the major coat protein of the phage. All five suppressor mutations confer nucleotide changes in the gene F DNA sequence. These changes define four amino acid sites in the gene F protein. Three suppressor mutations placed into an otherwise wild-type background display a cold resistant phenotype in liquid culture infections when compared to a wild-type phi X174 control.

Studies of fowl plague virus temperature-sensitive mutants with defects in synthesis of virion RNA.

Three different types of impairment in the synthesis of virion RNA (vRNA) were detected in three groups of temperature-sensitive (ts) mutants of fowl plague virus (FPV) having ts mutations in genes 1, 3 and 5 respectively. Normal synthesis of poly-(A+) cRNA, poly(A-) cRNA and vRNA was observed under non-permissive conditions early in infection in cells infected with the ts43 mutant having a ts mutation in gene 1 coding for the PB2 protein. However, 4 h after infection synthesis of vRNA ceased, synthesis of poly(A+) cRNA was reduced drastically, but the rate of poly(A-) cRNA synthesis was the same as that in cells infected with wild-type FPV. In cells infected with the ts 166 mutant having a ts mutation in gene 3, coding for the PA polypeptide, a drastic reduction was observed in poly(A+) cRNA synthesis under non-permissive conditions. Synthesis of poly(A-) cRNA was also reduced and synthesis of vRNA was not detected. The ts 60 mutant, having a ts mutation in gene 5 coding for the NP polypeptide, induced synthesis of all types of virus-specific RNA under non-permissive conditions, but the regulation of synthesis of vRNA and poly(A+) cRNA was affected, there being predominant syntheses of RNA segments 5 and 8 late in infection. In cells infected with mutants ts43 and ts 166 synthesis of virus-specific proteins was impaired, which reflected defects in the synthesis of virus-specific RNAs. The data obtained suggest that the PB2 protein may be contained in an enzyme complex responsible for synthesis of vRNA, that different enzyme complexes may be involved in the synthesis of poly(A-) cRNA and vRNA, and that the NP protein plays a significant role in the regulation of vRNA synthesis.

Studies on a temperature-sensitive mutant of fowl plague virus having a mutation in gene 7 coding for the M protein.

A fowl plague virus (FPV) temperature-sensitive mutant, ts 303/1 having a ts mutation in gene 7 coding for the matrix (M) protein has been obtained. The mutant induced synthesis of virus-specific RNA and polypeptides as well as ribonuclear protein (RNP) formation in cells under non-permissive conditions; however, haemagglutinin cleavage was reduced, functionally active haemagglutinin and neuraminidase were absent and virions were not formed. In mutant-infected cells at 36 degrees C haemagglutinin cleavage was also reduced and virions formed had an altered NP:M ratio as well as a decreased haemagglutinin content. A population of virions formed under these conditions was heterogeneous both in morphology and in buoyant density. The data obtained suggest that a mutation in the M proteins of orthomyxoviruses can affect processing of the haemagglutinin and impair final stages of virion morphogenesis.

Extragenic suppression of a ts phenotype during recombination between ts mutants of two fowl plague virus strains with a ts mutation in gene 1.

Fowl plague virus (FPV) ts mutants belonging to six recombination groups and obtained from the Weybridge strain (in the U.S.S.R.) or the Rostock strain (in the U.K.) have been studied in a recombination test. Temperature-sensitive mutants obtained from different FPV strains were revealed which had a ts mutation in gene 1; however, their crossing resulted in ts+ recombinants which appeared with a high frequency. This phenomenon was due not to intragenic complementation but to extragenic suppression, when the expression of a ts phenotype of the Rostock strain mutant gene 1 is suppressed by gene 2 products of the Weybridge strain.

Thermo-inducible expression of cloned early genes of bacteriophage Mu

An EcoRI fragment, containing approx. 5100 base pairs (bp) of the immunity-end of bacteriophage Mu, was inserted into the multicopy plasmid pMB9 by in vitro recombination. The expression of early Mu genes, located on the cloned fragment, is thermo-inducible because of the presence of the ts mutation in gene c. The isolation of a transformant harbouring the recombinant plasmid, pGP1, was possible only when expression of Mu genes was prevented. pGP1 can be maintained at 28°C at high copy number, but at 42°C the pGP1 containing cells are killed due to the expression of the kil gene of Mu. The following Mu genes are present on pGP1: the ner gene, the integration and replication genes A and B, the cim gene, and the kil gene. pGP1 containing cells do not show Gam and Sot activity at 42°C, therefore the leftmost EcoRI site on the Mu DNA is located between genes kil and gam or sot, or within the gam or sot gene.

The UV sensitivity of some early-function temperature-sensitive mutants of phage T4

Temperature-sensitive ( ts) mutants of phage T4 defective in ten early function genes were tested for sensitivity to ultraviolet (UV) light. The mutants were irradiated, plated on Escherichia coli S 6 , and incubated at various temperatures from 18 to 45°. UV sensitivity was shown by ts mutants defective in genes 30 (polynucleotide ligase), 32 (“joint former”), 41 (function unknown), 42 (deoxycytidylate hydroxymethylase), 43 (DNA polymerase), 44 (function unknown), 46 and 47 (deoxyribonucleases). Ts mutations in genes 45 (function unknown) and 56 (dCTP-dUTP nucleotidohydrolase) did not lead to UV sensitivity. The UV sensitivity of mutants defective in genes 30, 43, 46, and 47 is consistent with the model of DNA repair by excision. The observation that eight of ten early function genes contain UV-sensitive mutants is thought to reflect the complexity of the process of UV repair in T4-infected cells.

Repair and recombination in phage T4. I. Genes affecting recombination.

In a previous paper (Bernstein, 1967) temperature-sensitive (ts) mutations in gene 43 (DNA polymerase) and gene 42 (deoxycytidylate hydroxymethylase) of phage T4 were shown to increase recombination severalfold. Evidence is presented here that a ts mutation in gene 30 also increases recombination. Gene 30 has been identified as the structural gene of polynucleotide ligase (Fareed and Richardson, 1967).