Rivka Rudner

Studies on the nucleotide arrangement in deoxyribonucleic acids: X. Frequency and composition of pyrimidine isostichs in microbial deoxyribonucleic acids and in the DNA of E. coli phage T 3

Abstract DNA preparations from Bacillus cereus, Escherichia coli, Salmonella typhimurium and typhosa, Serratia marcescens, and Rhodopseudomonas spheroides and also from bacteriophage T 3 have been studied with respect to the distribution and the composition of the pyrimidine isostichs released by mild acid hydrolysis. Two analytical procedures were employed in these investigations, namely the direct two-dimensional chromatography of the hydrolysate and the fractionation of the fragments on DEAE-cellulose. These DNA specimens showed several common characteristics: (a) The frequencies of the total pyrimidine isostichs of length 1 to 8 are similar in all preparations and correspond to the distribution predicted for a random polynucleotide. (b) The relative frequencies of individual oligonucleotide runs liberated by the various AT- and GC-types of DNA vary greatly from each other and deviate from the calculated random frequencies. (c) There exists a disparity in the relative contribution of cytosine and thymine to the isostichs of length 1 to 5: the shorter sequences are relatively richer in the first pyrimidine, the longer ones in the second. (d) Polycytidylic acid runs are comparatively very rare, whereas the frequency of polythymidylic acid sequences often exceeds random predictions. (e) DNA from genetically related species (E. coli, S. typhimurium and S. typhosa) exhibit very similar patterns of pyrimidine arrangement. The DNA of phage T 3 resembles, also in the frequency of many of its individual pyrimidine sequences, a randomly constructed polynucleotide. Though it has almost the same base composition as the DNA of its host species, E. coli, it differs from the latter in sequence characteristics.

Mutation as an error in base pairing

SummaryReversion patterns of twenty-three mutants at two tryptophan loci (try C andD) inSalmonella typhimurium have been investigated after treatment with three specific mutagens. The compounds used were the base analogues, 5-bromouracil (BU) and 2-aminopurine (AP), and nitrous acid. The mutant alleles showed considerable specificity of response to the mutagens. The increased frequency of mutants after growth in the presence of base analogues was not due to selection of pre-existing mutants as shown by reconstruction experiments. It was determined that BU can be incorporated into the DNA of this organism by replacing thymine. A small amount of unaltered AP was recovered from the DNA of a purine-requiring mutant. These findings suggest that BU and AP induce “basepair transitons” inSalmonella typhimurium as had been previously observed for bacteriophage (Freese 1959).

Studies on the loss and the restoration of the transforming activity of the deoxyribonucleic acid of Bacillus subtilis

Abstract DNA preparations of Bacillus subtilis, were assayed for transforming activity for three unlinked genetic markers (adenine, leucine, methionine) in different stages of de- and renaturation induced by the gradual withdrawal of electrolytes by dialysis and their subsequent restitution. Experiments on denaturation by heat or alkali and thermal renaturation of transforming DNA were also performed. The biological assays were correlated with temperature-absorbance measurements and determinations of the buoyant densities of the preparations. The extent of inactivation by dialysis was related directly to the duration of dialysis and to the DNA concentration undergoing dialysis. The percentages of residual biological activity in denatured DNA were: dialysis, 1–6 %; alkali, 0.6–3 %; heat, 0.7–1.6 %. Irrespective of the method of inactivation, the adenine marker was more resistant than the others, the proportions of residual activities for adenine, leucine, methionine being near 2:1:1. The degree of restoration and the proportions of individual markers recovered by salt addition to transforming DNA that had been inactivated by dialysis depended upon conditions. Dialysed transforming DNA directly supplemented with electrolytes recovered a high percentage of initial activity (75 ± 15%), with marker ratios near those of native DNA. In dialysed DNA, diluted with water before salt addition, restoration was only partial and the marker ratios resembled those in denatured DNA. Subsequent thermal renaturation added only a small constant increment. The average recovery of transforming activity by thermal renaturation amounted to 35 % for DNA inactivated by heat, 48 % for that denatured by alkali. The marker ratios remained as before renaturation. The estimates of recovered secondary structure in renatured DNA based on the hyperchromic rise between 80° and 95° during temperature-absorbance measurements showed a statistically significant agreement with the recoveries of adenine activity. The theoretical implications of these findings are discussed with the help of a scheme illustrating the various structural changes of DNA undergoing dialysis and salt restitution.

Synthesis of ribosomal RNA during speculation in Bacillus subtilis

SPORULATION in Bacillus subtilis is associated with specific changes in RNA metabolism required for the expression of sporulation genes. Although net RNA synthesis stops abruptly at the end of logarithmic growth (T0), active turnover and differential transcription of the genome have been demonstrated1–3. Losick and Sonnenshein4 suggested that modifications of RNA polymerase may play a primary role in the regulation of gene expression during the sporulation process. The expression of ribosomal RNA genes during this period has been subject to conflicting reports1,5–7. The reported turn-off of these genes at the onset of sporulation5,6 has not been observed in similar experiments7. The latter, and the reported changes in template specificity of RNA polymerase4,8 were shown to be strongly dependent on the nature of the sporulation medium and to a lesser extent on the phenotype (sp+ or sp−) of the strain7,8. We have analysed the pattern of synthesis and turnover of stable RNA components in B. subtilis induced to sporulate in the highly defined resuspension medium, SM (ref. 9). We report that rRNA synthesis does continue during sporulation, although at a reduced rate, resulting in the gradual replacement of vegetative rRNA components by similar newly synthesised sporulation species.

Bacterial mutation and the synthesis of macromolecules

SummaryAn analysis was made of the effect of chloramphenicol on mutation in histidine-starvedE. coli from a histidine-dependent to a histidine-independent condition. Although mutations occurred in the absence of chloramphenicol, none could be found in its presence unless it was accompanied by a mixture of amino acids, excluding histidine. The role played by the amino acids was concluded to be twofold: 1.In the absence of chloramphenicol they increased the rate of mutation indirectly by accelerating the rate of gene replication.2.In the presence of chloramphenicol theyenabled gene replication; as a consequence, rare errors or mutations took place at a rate limited by the rate of turnover of the genetic material. The effect of chloramphenicol and amino acids on nucleic acid synthesis is such as to offer an account of this behaviour.

Is DNA replication a necessary condition for mutation

Since mutations occur in non-dividing bacteria (RYA~ 1959), the question arises whether the replication of genetic material is nonetheless required. With bacteriophage T2 it has been shown that mutations can be induced by 5-bromouracil during the DNA synthesis which occurs in the presence or absence of chloramphenicol (LITMA~ and PARDE],~ 1959, BRE~ER and SMIT~ 1959). I t has further been shown in Escherichiacoli that the frequency of mutants induced by caffeine is proportional to the amount of DNA synthesis that occurs when chloramphenicol is present (GLAss and NowcK 1959) and that mutations induced by ultraviolet light arise correlated With subsequent DNA synthesis (LIMB 1959). These findings are consistent with the working hypothesis of FRnnSE (1959) which proposes that some chemical mutagenesis involves the incorporation of abnormal bases followed by pairing errors during subsequent DNA replication. This hypothesis has been tested by the use of populations oi Salmonella typhimurium whose division has been Synchronized by filtration; mutations occur in accordance with DNA replication as the hypothesis predicts (RuD~Etr 1960). This Salmonella system involves mutation f rom tryptophane dependence (try D-I0, here called try-) to t ryptophane independence (try +) induced by 300 #g. 2-aminopurine per ml. I t has the advantage of a low background of spontaneous try + mutants. When trycells are exposed to 20 #g. of chloramphenicol per ml. in the presence or absence of 20 #g. of t ryptophane per ml., within 2 hours there is a net synthesis of 50 per cent more DNA as measured by the Dische reaction, while RNA determined by the orcinol reaction increases almost 3-fold. Thereafter the amounts of these compounds remain constant for as long as 5 hours. If 2-aminopurine is present at the onset of this synthesis, mutations are induced; on the other hand, if the cultures ar9 provided with 2-aminopurine after synthesis has ceased, no mutations aredetectable (Fig. 1). ,Under these conditions, therefore, the synthesis of nucleic acid is required for the induction of mutat ion while the synthesis of protein is not. In the presence of chloramphenicol, although protein synthesis is severely inhibited, a little DNA is replicated and RNA synthesis continues to a somewhat greater extent. When treated cells are plated on minimal medium, this RNA is expected to degrade, as has already been described by NWlD~Am)T and GRos (1957) and HoRowITz, LOMBARD and CHA~GAFF (1958), while the DNA and protein should remain almost constant in amount. In our experiments, when

Phenocopies induced by thymine in Escherichia coli.

SummaryA uracilless strain ofE. coli upon starvation for uracil adapts to synthesize this compound. These adaptations are of two sorts, heritable and non-heritable. The latter are induced by the presence of thymine although little or none of the uracil is synthesized by the demethylation of thymine. The non-heritable adaptations arise in a discontinuous fashion at a rate 10 times as high as the genetic reversions. The non-heritable uracil-independent cells are considered to be phenocopies because they mimic the phenotype of the genetic revertants.