R. H. Pritchard

Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12

SummaryA strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth was isolated from Escherichia coli K12. Genetic mapping and the molecular weight of the gene product suggest that the mutation is in the cou gene, specifying a sub-unit of DNA gyrase. Nuclear organisation and segregation and placement of septa are grossly abnormal in the mutant at 42°C. RNA synthesis and initiation of DNA replication are also affected at the restrictive temperature but the rate of DNA chain elongation continues almost undisturbed.

The effect of gene concentration and relative gene dosage on gene output inEscherichia coli

SummaryThe differential rate of synthesis of severalEscherichia coli gene products was measured under conditions in which the average number of copies of the corresponding chromosomal gene had been changed by altering the replication velocity of the chromosome. The data show that in steady state exponential cultures the output of genes in a fully repressed, fully derepressed, or non-repressible state is proportional to the average number of copies of the gene per unit mass (gene: mass ratio) and does not depend on the number of copies of the gene relative to all other genes (gene: DNA ratio). In contrast, the output of a gene which was under regulation by endogenously generated effectors was independent of such changes in gene frequency.The relationship found between the number of copies of a gene per unit of cell mass and enzyme output provides a new method for determining the location of the chromosome origin and the direction of replication in bacteria.

A map of four genes specifying enzymes involved in catabolism of nucleosides and deoxynucleosides in Escherichia coli.

SummaryFour genes specifying the enzymes thymidine phosphorylase, purine nucleoside phosphorylase, deoxyribomutase and deoxyriboaldolase were mapped by transduction with phage P1. All pairs show greater than 90 per cent co-transduction. The gene order was found to be dra-tpp-drm-pup, and the gene cluster was shown to lie between the hsp and ser B loci on the chromosome map of Escherichia coli.

A regulatory mutant affecting the synthesis of enzymes involved in the catabolism of nucleosides in Escherichia coli

SummaryA regulatory mutant which leads to constitutive synthesis of enzymes involved in catabolism of nucleosides is described. It is unlinked to the structural genes whose activity is affected. The gene concerned is designated nucR. The amount of thymine required for growth (colony formation) of thy− strains is affected by the nucR mutation. The amount required by a thy−drm−strain is reduced about four fold if it carries the constitutivity mutation. The amount required by a thy−drm+strain is increased at least two fold. These differences in nutritional requirement provide a method for selecting constitutives from non-constitutives and vice versa.

Independence of F replication and chromosome replication inEscherichia coli

SummaryData are presented which show that F replication is not coupled to any stage of the replication cycle of the host chromosome or to cell division, and is probably not related to surface area. It is also shown that the initiation mass of F increases progressively as the growth rate increases, the number of copies of F per unit of mass falling by half between doubling times of 0.8 and 2.7 generations per hour. It is further shown that the presence of an F particle does not influence the initiation mass of the chromosome.

Location and Characterisation of a New Replication Origin in the E. coli K12 Chromosome

SummaryA segment of DNA located in the region of the E. coli K12 chromosome previously identified by the Rac phenotype can function as a self-replicating plasmid. Evidence is presented that this plasmid, the oriJ plasmid, contains the origin of replication of a defective prophage postulated to be located in this chromosomal region by Low (1973). The plasmid can only be maintained in strains in which this postulated prophage has been deleted. In strains which possess the prophage selection for plasmid maintenance permits the isolation of clones containing new deletions which we postulate are the result of prophage excision.

Deoxynucleoside-sensitive mutants ofSalmonella typhimurium

SummaryThymineless mutants ofSalmonella typhimurium which are able to grow with low added concentrations of thymine (20 μM) fall into two classes on the basis of growth on deoxyribose as sole carbon source. Those which can grow are deoxyribomutase negative and those which cannot are deoxyriboaldolase negative. The former class are inhibited by deoxynucleosides and this provides a method for discriminating between different classes oftlr mutants ofEscherichia coli K12, which cannot utilize deoxyribose as a carbon source. It is suggested that the sensitivity of deoxyriboaldolase negative strains is due to the accumulation of deoxyribose-5-phosphate. The data also indicate that deoxyribose-5-phosphate is the inducer of thymidine phosphorylase. It seems that one or both of the deoxyribose phosphates is the toxic compound, and that reversal of inhibition by ribonucleosides is due to inhibition of the enzymes catalysing their formation from deoxynucleosides. We propose that the symbolsdrm anddra be used to denote the structural genes for deoxyribomutase and deoxyriboaldolase respectively.

Fluorouracil and the isolation of mutants lacking uridine phosphorylase in Escherichia coli: location of the gene.

SummaryA selective technique is described for the isolation of mutants of Escherichia coli lacking uridine phosphorylase and the location of the gene specifying this enzyme on the bacterial chromosome is determined. Using strains with appropriate lesions it is shown that there are three routes via which 5-fluorouracil can be converted to compounds which inhibit cell growth.

Chloramphenicol releases a block in initiation of chromosome replication in a dnaA strain of Escherichia coli K12

SummaryDNA-DNA hybridisation experiments show that chloramphenicol induces a burst of initiation from the oriC region of a dnaA46 mutant of Escherichia coli at 36.5° C but not from the isogenic dnaA+ strain. Following this stimulation of initiation, DNA replication proceeds normally towards the terminus. The temporal pattern of the extra initiation is in parallel with the induced stimulation of RNA synthesis caused by chloramphenicol in the same strain. This is consistent with the hypothesis that the stimulation of initiation in the dnaA mutant is the result of the stimulation of the synthesis of an RNA species.

The role of nucleoside phosphorylases in the degradation of deoxyribonucleosides by thymine-requiring mutants of E. coli

SummaryThymine requiring strains of Escherichia coli are known to possess a significant pool of deoxyribose-1-phosphate in contrast to non-mutant strains. In this paper thymine-requiring mutants lacking thymidine phosphorylase, purine nucleoside phosphorylase, and uridine phosphorylase, in various combinations, are used to show that deoxyribose-1-phosphate is a degradation product of pyrimidine deoxynucleosides and that both thymidine phosphorylase and uridine phosphorylase participate in this degradation. Our results confirm an earlier report by Krenitsky, Barclay and Jacquez that uridine phosphorylase has some specificity for deoxyuridine. We also show that this enzyme can degrade bromodeoxyuridine. The data presented here support the hypothesis that breakdown of deoxynucleosides to deoxyribose-1-phosphate is due to an accumulation of the deoxynucleotide precursors of thymidine triphosphate.