Regulation Of Nucleic Acid Synthesis Research Articles

ULTRAVIOLET INACTIVATION OF THE MIDI VARIANT OF Qβ RNA: UV‐INDUCED ‘KINETIC BARRIERS’ TO REPLICATION AT TWO TEMPERATURES

Abstract—MDV‐1 RNA is a small variant of bacteriophage Qβ, and is an active template in replication reactions catalyzed by Qβ replicase. The predominant lesion formed when MDV‐1 is irradiated with 254 nm light with a dose of 2500 Jm‐2 is uridine hydrate, which is chemically closely related to the major thymidine products formed in DNA by ionizing radiation. We have found that the replication of irradiated MDV‐1 RNA is inhibited at 37°C as well as 12°C. However, while replication appears to be blocked at the sites of photolesions at 12°C, Qβ replicase ‘pauses’ at the lesions before continuing elongation at 37°C. This effect is not caused by reversion of the photoproducts to parent material at the higher temperature. ‘Pauses’ (caused by ‘kinetic modulators’) during the synthesis of nucleic acids from unirradiated templates have been reported to occur in a number of systems and may play a role in the regulation of transcription and replication. Our results suggest that one consequence of radiation damage to nucleic acid bases may be the introduction of lesions which mimic such modulators, leading to aberrations in the regulation of nucleic acid synthesis.

Regulation of nucleic acid synthesis in Artemia salina nauplii by environmental salinity.

Ribonucleic acid synthesis in Artemia salina nauplii was strongly inhibited by increasing salinity in the surrounding media, as measured by incorporation of tritiated uridine. Although precursor pools varied in both concentration and specific activity with increasing salinity, these changes did not account for the reduction in ribonucleic acid synthesis. Low levels of oxygen at high salt concentrations did not account for this effect. Intracellular ATP concentrations decreased as the salinity of the environment was increased. Levels of ATP were not affected either by increased oxygen or by the presence of ouabain in the medium.

Template-determined, variable rate of RNA chain elongation

Qβ replicase polymerizes MDV-1 RNA at a markedly variable rate. Electrophoretic analyses of partially synthesized strands showed that a few of the elongation intermediates are much more abundant than others, reflecting a variable rate of chain elongation. Our data suggest that at a relatively small number of specific sites in the sequence of this RNA, the progress of the replicase is temporarily interrupted, and then resumes spontaneously, with a finite probability. Since the time spent between these pause sites is negligible compared with the time spent at pause sites, the mean time of chain elongation is well approximated by the sum of the mean times spent at each pause site. Nucleotide sequence analysis of the most prominent elongation intermediates indicated that they all have the potential to form a 3′ terminal hairpin structure. This suggests that the marked variability in the rate of chain elongation is due to the formation of terminal hairpins in the product strand, or the reformation of hairpins in the template strand. A survey of the literature shows that this phenomenon occurs with most, if not all, nucleic acid polymerases. Structure-induced pauses may play a role in the regulation of nucleic acid synthesis.

Nature of the penetration barrier in Escherichia coli K-12: effect of macromolecular inhibition of penetrability in strains containing the envA gene.

The envA mutation in Escherichia coli K-12, which maps at 1.5 min, was previously shown to mediate sensitivity to gentian violet as well as to several antibiotics. Moreover, strains containing the envA gene were recently found to be lysed by lysozyme in the absence of ethylenediaminetetraacetate. It is here reported that the envA mutation mediates an increased uptake of gentian violet. The uptake of the dye was markedly affected by growth with different antibiotics interfering with macromolecular synthesis. Amino acid starvation of a strain containing envA with a stringent control of ribonucleic acid (RNA) synthesis resulted in a decreased uptake of gentian violet. However, no decrease in dye uptake was found during starvation in an envA transductant with a relaxed control of RNA synthesis. Inhibition of deoxyribonucleic acid (DNA) synthesis by nalidixic acid decreased the uptake of gentian violet of envA cells and, in addition, rendered the cells insensitive to the lytic action of lysozyme. Chloramphenicol treatment increased penetrability in wild-type and starved envA cells. In most instances, this effect of chloramphenicol was prevented by selectively interfering with DNA or RNA synthesis. A coordinate regulation of nucleic acid synthesis and penetrability is suggested.

Regulation of nucleic acid synthesis in Lactobacillus acidophillus R-26

Lactobacillus acidophillus R-26 has been used to study the regulation of chromosome replication. RNA synthesis in this organism is stringent in that when amino acids are removed, RNA synthesis is drastically reduced. Synthesis can be restored by the addition of chloramphenicol. Actinomycin D inhibits RNA synthesis completely in concentrations above 2 μg/ml. DNA synthesis proceeds in the absence of all amino acids resulting in an overall increase of 40–50 % in the DNA content per cell. This synthesis can be further increased by the addition of either glutamic or aspartic acids. Thymine or deoxyribose starvation results in a 150 % increase in DNA content upon subsequent restoration of these compounds and incubation in the absence of amino acids. This increase can be inhibited by the presence of actinomycin D or chloramphenicol during the period of deoxyriboside starvation. These results are similar to those observed previously for Escherichia coli and have been explained by assuming that: (a) chromosome replication is completed in the absence of all amino acids; (b) thymine or deoxyriboside starvation induces an extra cycle of chromosome replication. It has been found that actinomycin D will inhibit the extra cycle of DNA replication, if present at concentration of 5 μg/ml. This concentration is much higher than that necessary to inhibit RNA synthesis, but much lower than that necessary to inhibit normal DNA synthesis. It is proposed that actinomycin is, in this case, acting directly upon the initiation of DNA replication.

Artificial heterokaryons of animal cells from different species.

ABSTRACTA virus, inactivated by ultraviolet light, was used to fuse together cells from different species of vertebrate, and the resulting heterokaryons were examined by autoradiographic and cytological techniques. Heterokaryons could be made with both differentiated and undifferentiated cells: HeLa and Ehrlich ascites cells were studied as examples of undifferentiated cells; rabbit macrophages, rat lymphocytes and hen erythrocytes as examples of differentiated cells. These last three cells were chosen because in them, in varying degrees, the process of differentiation has resulted in suppression of the synthesis of DNA or of both DNA and RNA. This suppression was in all cases found to be reversible: the dormant nuclei could be induced to resume the synthesis of RNA or DNA or both when the differentiated cells were fused with a cell which normally synthesizes RNA and DNA. Observations on heterokaryons in which differentiated cells were fused with HeLa cells and with each other permitted certain general conclusions to be drawn about the regulation of nucleic acid synthesis in the heterokaryon. It was found that if either one of the parent cells normally synthesized RNA, RNA synthesis took place in both types of nuclei in the heterokaryon. If either of the parent cells normally synthesized DNA, DNA synthesis took place in both types of nuclei in the heterokaryon. If neither of the parent cells synthesized DNA, no DNA synthesis took place in the heterokaryon. In all cases where a cell which synthesized a particular nucleic acid was fused with one which did not, the active cell initiated the synthesis of this nucleic acid in the inactive partner. In no case did the inactive cell suppress synthesis in the active partner.The nuclei of heterokaryons in which DNA synthesis took place underwent mitosis, and those nuclei which entered mitosis synchronously usually fused together. This process resulted in the progressive formation of mononucleate hybrid cells, which might thus contain within a single nucleus chromosomal complements derived from different species. These mononucleate hybrid cells were also capable of RNA and DNA synthesis, and many of them in turn underwent mitosis. At metaphase these cells showed, in various combinations, the chromosomal complements of the two parent cells. Mononucleate hybrid cells formed by the fusion of a large number of single cells did not appear to be capable of continued multiplication; but mononucleate cells containing one chromosomal set from each parent cell were still found to be undergoing mitosis many days after cell fusion.

Regulation and Coordination of Purine and Pyrimidine Biosyntheses in Yeast: I. Regulation of Purine Biosynthesis and Its Relation to Transient Changes in Intracellular Nucleotide Levels

The control of purine biosynthesis in a yeast mutant deficient for uracil, adenine, and histidine has been studied in vivo. The adenine mutation causes accumulation of aminoimidazole ribotide in the cells. The control curve relating steady-state purine nucleotide level in the cell to rate of synthesis in the de novo purine synthetic pathway has been determined. Control in the cell depends on a feedback mechanism involving end-product inhibition. The transient responses of the purine nucleotide pool to changes in adenine input have been studied. Under certain conditions the pool overshoots when shifting from one steady-state to another. Transient changes in nucleotide levels are followed by inverse changes in the rate of attempted de novo purine synthesis. A study of the transient responses of specific intracellular nucleotides suggests that inosinic acid controls the rate of attempted purine synthesis. The transient response of nucleic acid synthesis rate to changes in nucleotide levels was studied and the implications for regulation of nucleic acid synthesis discussed.

Possible role of histones in regulation of nucleic acid synthesis

ALTHOUGH this paper will be concerned principally with our studies on the possible role of histones in regulation of nucleic acid synthesis, it seems pertinent to comment first upon the gel electrophoresis of histones. Butler has stated that the most satisfactory way of characterizing histones is the application of starch gel electrophoresis [S, 10, 111. We have found [ 121 that polyacrylamide gels are superior to starch gels in the separation of histones, and Reisfeld et al. [ 131 have reported success with this gel system in the electrophoresis of other cationic proteins. The gels are formed by mixing buffered tetramethy-ethylenediamine catalyst with 60 per cent acrylamide and 0.4 per cent N,N’-methylenebisacrylamide followed by addition of ammonium persulfate. The gels are formed in glass tubes and have a pH of 3 after polymerization. Samples are applied directly upon the gels in a density-stabilized layer, and electrophoresis is conducted in a glycine buffer at pH 4 for 4.5 hr at 15 ma. per tube. The gels are stained in amido black, and excess stain is removed electrically. Patterns obtained by electrophoresis of calf thymus histones are shown diagrammatically in Fig. 1 in comparison with patterns obtained by electrophoresis in starch gels. The bands shown in Fig. 1 C were found consistently by electrophoresis in polyacrylamide gels, and the bands are designated by Roman numerals. Bands II and IV, found in all calf thymus histone preparations examined in this laboratory, stained more heavily than the other bands and provided easily recognized and very reproducible reference points. The inclusion of a sample of unfractionated histone in each electrophoretic run of 6 samples facilitated the assignment of band identities in preparations of histone fractions. Band II was arbitrarily assigned a relative mobility (1~‘)

Regulation of nucleic acid synthesis by androgens.

Castration of adult male mice produced a progressive decrease in the weight of the kidney to a plateau at about 65% of normal within 14 days accompanied by a proportionate decrease in s-RNA. The total DNA also decreased but to a smaller degree while the total RNA decreased to a greater degree than the weight. Similar but greater decreases were observed in the prostate and seminal vesicles. The administration of testosterone propionate, testosterone, 17-methyltestosterone or androstan-17β-ol, 3-one gradually reversed the effects of castration. In addition the total RNA was increased during the period of rapid growth of the tissues to values above normal and then decreased to normal levels as the rate of growth of the tissues decreased. The thymus increased in weight after castration and decreased on androgen administration with concomitant changes in both DNA and RNA. The liver weight decreased slightly with proportionate decreases in s-RNA and RNA but not in DNA. Androgen administration reversed these cha...