RNase H-defective mutants of Escherichia coli: a possible discriminatory role of RNase H in initiation of DNA replication

Summary Mutants of Escherichia coli completely deficient in RNase H activity were isolated by inserting transposon Tn3 into the structural gene for RNase H, rnh, and its promoter. These rnh ^- mutants exhibited the following phenotypes; (1) the mutants grew fairly normally, (2) rnh ^- cells could be transformed with ColE1 derivative plasmids, pBR322 and pML21, though the plasmids were relatively unstable, under non selective conditions, (3) rnh ^- mutations partially suppressed the temperature-sensitive phenotype of plasmid pSC301, a DNA replication initiation mutant derived from pSC101, (4) rnh ^- mutations suppressed the temperature-sensitive growth character of dnaA ^ts mutant, (5) rnh ^- cells showed continued DNA synthesis in the presence of chloramphenicol (stable DNA replication). Based on these findings we propose a model for a role of RNase H in the initiation of chromosomal DNA replication. We suggest that two types of RNA primers for initiation of DNA replication are synthesized in a dnaA/oriC-dependent and-independent manner and that only the dnaA/oriC-dependent primer is involved in the normal DNA replication since the dnaA/oriC independent primer is selectively degraded by RNase H.Abbreviations AP^r ampicillin-resistant - kb kilobase pair(s) - NEM N-ethyl maleimide - Ts temperature-sensitive     

INCORPORATION OF TRITIATED THYMIDINE BY EUCARYOTIC MICROALGAE1

The uptake and incorporation of tritiated thymidine (³H-TdR) by axenic laboratory cultures of marine diatoms and dinoflagellates was measured. ³H-TdR was incorporated into nucleic acids by all four algae examined during a two to six hour period prior to cytokinesis and not during other times of the cell cycle. Between 90-95% of the ³H label incorporated into (cold trichloroacetic acid insoluble) nucleic acids was recovered from DNA. Incorporation of ³H-TdR appears to accurately indicate the timing of DNA synthesis. The incorporation of ³H-TdR by eucaryotic algae during long term (24 h) incubations does not generally preclude using ³H-TdR uptake to estimate bacterial production and growth during short term incubations.     

Selective screening procedure for the isolation of heat- and cold-sensitive, DNA replication-deficient mutants of bacteriophage SPO1 and preliminary characterization of the mutants isolated.

A procedure is described for the selective isolation of temperature-sensitive replication-deficient mutants of Bacillus subtilis phage SPO1. A modification of the procedure permits the isolation of temperature-sensitive mutants in specific cistrons of interest. The applicability of these procedures to other viral systems is discussed. The mutations isolated were assigned to eight replication-deficient cistrons, with the cold-sensitive mutations showing a distribution strikingly different from that of the heat-sensitive mutations. As a preliminary to the identification of initiation-deficient mutants, the mutants were divided into three classes on the basis of their ability to synthesize DNA after a shift to nonpermissive temperature. We also report two incidental results: (i) the SPO1 dUMP hydroxymethylase, like the T4 dCMP hydroxymethylase, may be part of a multifunctional complex; and (ii) mutants were isolated that were replication positive but lysis deficient and failed to complement one of the replication-deficient mutants.     

The effects of incorporated tritium and bromodeoxyuridine on the frequency of sister chromatid exchanges

Cells in third mitosis treated during the first cell cycle with ³H-TdR and during the next two cycles with BrdU (without ³H-TdR) show a typical pattern of chromosome differentiation which allows identification of sister chromatid exchanges occurring during the first (SCE₁, second (SCE₂) and third cycles (SCE₃). Chromosomes labeled only with ³H-TdR had the most SCEs; those labeled only with BrdU, the second highest number; and those labeled with ³H-TdR plus BrdU, the fewest. Since BrdU and ³H-TdR are well known inducers of SCEs, the relatively low frequency of exchanges produced by the combined action of these two compounds is paradoxical. — It is assumed that SCEs are generated by the abnormal recombination of double-strand DNA breaks occurring at the junctions between completely and partially duplicated replicon clusters. Thus, agents that induce absolute blocks to DNA fork displacement will favor the appearance of SCEs because double-strand breaks have more time to occur at junctions. Conversely, agents that inhibit the initiation of replication will decrease the probability of SCEs. Ionizing radiation delays the onset of cluster replication. Therefore, in ³H-TdR plus BrdU-substituted chromosomes the radiation from tritium may inhibit the appearance of BrdU-induced SCEs. Since the inhibition does not exist in chromosomes substituted only with BrdU, the frequency of SCEs in these elements is higher than in double-substituted chromosomes. During the first cell cycle the onset of cluster replication is normal. However, the incorporation of ³H-TdR in the replication fork may enhance the appearance of double-strand breaks, thus inducing a high frequency of SCEs.     

Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis

By examining cytological phenotypes of 587 temperature-sensitive mutants of the fission yeast Schizosaccharomyces pombe, we obtained 18 mutants which cause cell division in the absence of nuclear division. By genetic analyses, these novel nuclear division arrest mutants can be classified into nine complementation groups (designated cut1 – cut9). The cytological phenotype of cut mutants is similar but not identical to that of DNA topoisomerase II mutants (top2). The cut1⁺ gene was cloned by transformation and shown to complement cut2 as well as cut1, indicating a functional relationship between the two genes. The cut genes are required for nuclear division, but their mutant phenotypes differ from most of the previously identified mutants which block nuclear division and also the subsequent cytokinesis. Fluorescence microscopy indicates that the mitotic chromosomes formed in cut mutant cells are abnormal and fail to separate properly. We suggest that cut mutations, like top2, block mitotic chromosome formation and concomitantly nuclear division, but that cytokinesis proceeds independently of the defects in nuclear division, demonstrating uncoordinated mitotic pathways. A novel mutant nuc1 is also described which shows a cytological phenotype similar to the double mutant of DNA topoisomerases I and II but contains normal levels of both DNA topoisomerase activities.     

Isolation and biochemical analysis of a temperature-sensitive scarlet eye color mutant of Drosophila melanogaster

Six new EMS-induced scarlet mutants were selected. Four of these were partially pigmented, with xanthommatin levels ranging from 12% to 45% of normal. In one (st ^754ts), pigment production was temperature sensitive; the level of xanthommatin changed from less than 10% of normal at 29 C to more than 70% at 18 C. In all of the new mutants tested, the level of early pupal 3-hydroxykynurenine was as low as low as that in st ¹. Thus reduced larval accumulation of this metabolite also appears to be a characteristic feature of scarlet mutants. Temperature-pulse and temperature-shift experiments were carried out with st ^754ts to determine the temperature-sensitive period for the scarlet gene during development. The major sensitive period commenced prior to the onset of pigmentation and was over before adult emergence. Thus the initiation of xanthommatin synthesis is not brought about by the activation of the scarlet gene. In similar experiments carried out with a temperature-sensitive white mutant (w ^bl), a similar temperature-sensitive period was obtained.This work was supported by Grant D2 75/15248 from the Australian Research Grants Committee and also by Grant GB 27599 from The National Science Foundation to Professor M. M. Green.     

Mutants of yeast with depressed DNA synthesis

Summary Seven temperature-sensitive mutants have been isolated in Saccharomyces cerevisiae which show a reproducible defect in DNA synthesis at the restrictive temperature. One of these is allelic with rnal1 (Hartwell et al., 1970) but the remaining mutants define six complementation groups and probably represent six different genes. The gene symbol dds (for depressed DNA synthesis) is proposed.At the restrictive temperature, rnal1-2, dds2-1 and dds6-1 show a rapid and almost total cessation of DNA and RNA synthesis, whilst protein synthesis continues for several hours. The remaining dds mutants show a reduced rate of DNA synthesis from the time of temperature shift (dds1, dds3, dds4) or a cessation of DNA synthesis at a later time (dds5). In some cases, RNA synthesis is affected concomitantly with, or soon after, the depression in DNA synthesis. Possible reasons for the phenotypes of these mutants, and for the relative absence of yeast mutants which are unambiguously and specifically affected in DNA synthesis, are discussed.In addition, we report the isolation of seven new alleles of known cdc genes and ten new mutants with a cell cycle phenotype that complement those already known.     

Genetic and enzymatic characterization of conditional lethal mutants of the yeast Schizosaccharomyces pombe with a temperature-sensitive DNA ligase.

The DNA ligase activities of wild type and temperature-sensitive lethal cdc 17 mutants of Schizosaccharomyces pombe have been studied by measuring effects on the conversion of relaxed DNA circles containing a single nick to a closed circular form. Such assays have revealed that all cdc 17 mutants have a thermosensitive DNA ligase deficiency, that this deficiency cosegregates 2:2 with their temperature-sensitive cdc-lethality in three tetrads derived from a cross against wild type, and that genetic reversion of the temperature-sensitive cdc^? phenotype is accompanied by a restoration of DNA ligase activity; all of which implies that the temperature-sensitive cdc^? phenotype of cdc 17 mutants is due to a single nuclear mutation causing a DNA ligase deficiency. Both wild type and mutant enzymes have been partially purified by chromatography in heparin/agarose columns. The wild-type enzyme is completely stable in vitro at both permissive (25 °C) and restrictive (35 °C) temperatures, whereas that of two different mutants, though completely stable at 25 °C, is rapidly inactivated at 35 °C, implying that their mutations are located in the structural gene for DNA ligase.     

The inheritance of diastatic power and alpha-amylase contents in spring barley

Summary Nuclei were isolated from various types of donor protoplasts and were transferred into receptor protoplasts in numerous combinations. Five percent uptake was achieved under conditions which did not interfere with viability and subsequent culture of receptor protoplasts. Methodological investigations on nuclei uptake were carried out with cereal and tobacco protoplasts. To look for biological proof of integration and replication of transferred nuclear genes, two complementing, chlorophyll-deficient, light-sensitive mutants of tobacco were used as sources of nuclei and receptor protoplasts. Ca. 5.5 × 10⁷ receptor protoplasts were cultured following transplantation experiments involving these complementing mutants and about 1.8 × 10⁷ of the resulting calli were subjected to selective conditions which discriminate against the parental types. No nuclear hybrids were detected, although in control experiments somatic hybrids were obtained by protoplast fusion. Some explanations for failure of nuclear hybrid formation are discussed together with other possible approaches for selective somatic combination of plant cell genophores.     

Genetic control of DNA initiation in Escherichia coli

We describe the isolation, and properties of a mutant (CT28) of Escherichia coli with a temperature-sensitive defect in DNA initiation that is reversible. The mutation (dna-28) responsible for this defect is shown to be located in the same region of the map as the dnaC group of DNA initiation mutants.A terminalized culture of CT28 initiates DNA synthesis synchronously immediately upon lowering the temperature, and will do so in the presence of chloram-phenicol.During prolonged incubation at the non-permissive temperature, the cells accumulate a capacity to initiate multiple rounds of replication per chromosome.The variation in the susceptibility of the argH^? and thyA^? alleles to reversion by pulse mutagenesis with nitrosoguanidine during a synchronous round of DNA replication, suggests that this round of replication is bidirectional and commences from an origin in the vicinity of 60 to 65 minutes.CT28 contains two temperature-sensitive mutations. These have been mapped and separated into two derivative strains. One of these, CT28-3b, carries the dna-28 mutation of the C locus, and like the parental double mutant is reversibly temperature-sensitive for an initiation function; but it is more temperature-sensitive than either the double mutant or the other single mutant derivative, CT28-1. The other, CT28-1, is not defective in DNA replication or initiation of replication at the non-permissive temperature.     

Temperature-sensitive developmental mutants of Caenorhabditis elegans

About 200 temperature-sensitive mutants of the nematode Caenorhabditis elegans have been isolated. At restrictive temperature, the mutants are blocked in the reproductive life cycle. They have been placed into six broad categories based on their defective phenotypes. The six categories are: (1) mutants blocked in embryogenesis; (2) mutants defective in gonadogenesis; (3) mutants defective in spermatogenesis; (4) mutants that accumulate at an intermediate growth stage; (5) mutants that produce sterile adult progeny; (6) mutants that have a temperature-sensitive morphological defect that interrupts the reproductive life cycle. The critical times of temperature sensitivity have been measured using temperature-shift experiments. Most of the gonadogenesis and spermatogenesis mutants are temperature sensitive during the period of cellular differentiation rather than proliferation. The temperature responses of the gonadogenesis and zygote-defective mutants indicate a common association between functions in gonadogenesis and early embryogenesis. Many of the mutants placed in different categories share other temperature-sensitive phenotypes upon close examination. This implies that many of the functions required for development are general metabolic reactions under increased demand during differentiation and embryogenesis.     

Thymidine metabolism and the monitoring of DNA synthesis in insects

The thymidine degradation pathway established for other organisms is confirmed in insects. When ³H-TdR is used as a marker for DNA synthesis in developing silkmoths, some is incorporated into DNA and some degraded to compounds not incorporated into DNA. After a single injection, ³H-TdR is rapidly cleared from haemolymph and other tissue, resulting in, at most, a 4 hr pulse. In wing tissue, detection of DNA synthesis is possible for a maximum of 4 hr after injection of precursor and for 6 hr in vitro. Continuous monitoring of DNA synthesis can be attained by perfusion, which maintains high levels of circulating ³H-TdR.     

Behavioral mutants of Drosophila melanogaster

Summary Sex-linked behavioral mutants were induced in Drosophila melanogaster with ethyl methanesulfonate (EMS) and isolated by direct visual observation of abnormal phenotypes. The four behavioral phenotypes used were flight-reduction, hyperactivity, hypoactivity and stress-sensitivity, and are easily discernable in either single or small populations of mutant flies. In one screen, forty-two behavioral mutants were recovered from strains derived from 800 mutagen-treated X chromosomes. In a second screen, 139 behavioral mutants were obtained from 2369 X chromosomes. The high rate at which behavioral mutants were recovered in the second screen, when compared to new visibles (28) and new temperature-sensitive lethals (124), suggests that the isolation of behavioral mutations on the autosomes of Drosophila and in the genomes of larger insects should be practical.This research was supported by National Research Council of Canada grant A-1764 to D.T.S.     

Identification and characterization of plasmids in hydrogen uptake positive and hydrogen uptake negative strains ofRhizobium japonicum

Modifications were made of published procedures to allow routine isolation of plasmids fromRhizobium japonicum. The plasmid profiles of a series of H₂ uptake positive and H₂ uptake negative strains were compared. None of the strains ofR. japonicum with high H₂ uptake activities exhibited discernible plasmids, while most of the strains, with little or no H₂ uptake activity, showed plasmids with molecular weights ranging from approximately 49–290 x10⁶. An examination of H₂ uptake negative mutants derived from an H₂ uptake positive parent revealed two discernible plasmid bands in nonrevertible mutants but no detectable plasmids in revertible mutants or in the parent strain from which mutants were derived.     

Sexual differentiation in Mucor: Trisporic acid response mutants and mutants blocked in zygospore development

Spores of a minus strain of Mucor mucedo (Bref.) were treated with 1-methyl-[3-nitro]-1-nitro-soguanidine and mutants were isolated either by testing for zygophore induction with externally supplied trisporic acids (TA) or by mating with wild type plus colonies. Mutants were found defective (Tar^?) or temperature-sensitive (Tar-Ts) in their reaction towards trisporic acids, blocked or temperature-sensitive in their mating with plus strain (Mat^? or Mat-Ts) or temperature-sensitive in zygospore development (Zyg-Ts). The inability to react against externally supplied trisporic acids was not necessarily coupled with an inability to mate with plus strain (phenotype Tar^? Mat⁺). This indicated that the diffusion and uptake of trisporic acids is not a necessary prerequisite to the sexual interaction of Mucor mating types.     

Isolation, propagation and characterization of replication requirements of reiteration mutants of Simian Virus 40.

We have identified five reiteration mutants from serially-propagated, defective stocks of Simian Virus 40 and DAR virus (an SV40³ variant of human origin). The genomes of these mutants contain tandem repeats of specific segments of the SV40 genome. In order to propagate individual reiteration mutants, the monomer DNA segments from the mutant genomes are separated from wild-type SV40 DNA after cleavage by certain bacterial restriction endonucleases which produce short cohesive termini at their cleavage sites. These monomer segments, which are one-third, one-fourth, or one-fifth the size of wild-type SV40 DNA, are then circularized in vitro using bacteriophage T4 polynucleotide ligase and used to infect African green monkey kidney cells in the presence of wild-type or temperature-sensitive mutant DNAs as helpers. While wild-type SV40 and late temperature-sensitive mutants can serve as helpers in the replication and amplification of these minicircular DNAs, early temperature-sensitive mutant genomes are unable to do so at the nonpermissive temperature. The minicircular DNAs are amplified in vivo through an arithmetic series of oligomers. Encapsidation of reiterated molecules between 70 and 100% the size of wild-type SV40 DNA is observed, although reiterated viral DNA molecules much larger than unit size are formed in vivo.     

Isolation and properties of Escherichia coli ATPase mutants with altered divalent metal specificity for ATP hydrolysis

A method was devised for isolation of large numbers of energy-transducing ATPase (coupling factor) mutants based on a modification of the procedure of Hong and Ames (Hong, J. and Ames, B. N. (1971) Proc. Natl. Acad. Sci. U.S. 68, 3158–3162) for localized mutagenesis of any small region of the bacterial chromosome using transducing phages. The principle of this procedure is to mutate P1-transducing phage particles carrying the ATPase genes (Unc (uncoupled) DNA) using the strong chemical mutagen hydroxylamine. By transducing ilv^? auxotrophs, a marker closely linked to Unc, to prototrophs, mutated Unc DNA can be introduced into the chromosome. We have used this method in conjunction with suitable selection procedures to isolate about 90 Unc^? strains which have been classified by physiological, genetic, and biochemical criteria into three different phenotypes (Unc A, B, D). Mutants of the Unc D phenotype which were studied in detail were found to have the following properties: (1) aerobic growth yields on glucose are considerably lower than the wild type; growth occurs on glucose under anaerobic conditions; (2) Unc D lesions map near the ilv operon; (3) O₂ uptake is comparable to the rate of wild type; (4) vesicles catalyze respiratory-dependent transhydrogenation, but show very low levels of Ca²⁺ ATP-dependent transhydrogenation; Mg²⁺ is ineffective; (5) oxidative phosphorylation is almost completely blocked irrespective of which metal ion is used; (6) the specific activity of ATPase is only about 20% of the wild type; (7) purified ATPase was found to have a marked specificity for Ca²⁺ as a divalent metal for ATP hydrolysis. A summary of properties of the new Unc mutants is discussed.     

Structural Genes of ATP-dependent Deoxyribonuclease of <Emphasis Type="Italic">Escherichia coli</Emphasis>

RECOMBINATION-deficient (Rec^?) mutants of E. coli express pleiotropic alterations of various phenotypes such as increased ultraviolet light sensitivity, altered patterns of DNA degradation after irradiation, inability to support growth of certain λ phage mutants and many others in addition to reduced recipient ability in mating with Hfr bacteria¹. Yet the primary function of any one of the genes responsible for these alterations has not been elucidated. In this paper, the characteristics of recB and recC mutants having temperature-sensitive functions are described. Particular attention is paid to the properties of the ATP-dependent deoxyribonuclease which is known to be missing in recB and recC mutants^2–5.     

Temperature-Sensitive Modification and Restriction Phenotypes of an Escherichia coli dnaD Mutant

A mutant of Escherichia coli temperature-sensitive for deoxyribonucleic acid synthesis, dnaD, was found to have temperature-sensitive modification and restriction phenotypes. In contrast to the original observation by Carl (1970), the mutant could support the growth of λ phage at 41 C. However, the λ phages thus produced were able to form plaques with normal plating efficiency only on E. coli C, a restriction-less strain, but not on E. coli K. Since the λ phages produced in the mutant at 30 C could form plaques equally well on both E. coli strains, it was concluded that the dnaD mutant has a temperature-sensitive modification phenotype. Furthermore, since the dnaD mutant allowed some growth of unmodified λ·C phages at 41 C but less at 30 C, the mutant is also temperature sensitive in restriction. The relationship, if any, between temperature-sensitive deoxyribonucleic acid synthesis and temperature-sensitive modification-restriction in the dnaD mutant is not known. Similar experiments were done with three dnaC mutants and one dnaA mutant. Two dnaC mutants were found to have altered restriction phenotypes at 41 C, but none of the mutants were defective in modification.     

Failure of tritiated thymidine incorporation into DNA to reflect the autoradiographically demonstrable calcium-induced increase in thymic lymphoblast DNA synthesis

Increasing the extracellular calcium concentration in thymic lymphocyte suspension from 0.6 to 1.8 mM stimulated the proliferation of the lymphoblast subpopulation as measured by increases in the proportion of cells autoradiographically labeled with ³H-TdR and in mitotic activity. However it was not possible to show this increased DNA synthesis by scintillometric measurement of the amount of ³H-TdR incorporated into extracted DNA. On the other hand, calcium did raise the incorporation of ¹⁴C-formate into the thymine residues of DNA, and increased the activity of isolated thymocyte thymidylate synthetase. In contrast to the mitogenic calcium ion, a thymidylate synthetase inhibitor, methotrexate, actually increased the incorporation of ³H-TdR into DNA. It is concluded that calcium increases the endogenous synthesis of thymidylate which in turn prevents the amount of incorporation of exogenous ³H-TdR from accurately reflecting the true level of DNA synthesis.