Induction of mutation in phage T4 by extracellular treatment with methylene blue and visible light

Summary Photodynamic inactivation with methylene blue and light (MB+Li) of maximally sensitized phages T4 and T4rII nearly followed single-hit kinetics. Not so mutation induction, which in one phage (T4rII N₁₇) tested showed multi-hit kinetics. This difference and the fact that T4rII phages with a GC base pair at the site of the rII-mutation showed no enhanced backmutation when compared to rII phages with an AT base pair or to sign mutants suggests that there is no guanine specificity for MB+Li-induced mutation in phage T4.     

Induction of petite mutation and inhibition of synthesis of respiratory enzymes in various yeasts

A systematic investigation covering a wide diversity of yeast species was made on the appearance of respiratory deficient (petite) mutants after treatment with acriflavine. Petite mutants were obtained from certain species only, but in these species all strains were found to have in common the property of giving rise to petite mutants; such species were designated as petite positive. Species failing to give rise to petite mutants were accordingly called petite negative. The primary action of acriflavine, namely the inhibition of the synthesis of the respiratory system, was shown to occur not only in petite positive yeasts, but also in petite negative ones. Some implications of the results are discussed.     

Replication of bacteriophages in Escherichia coli mutants thermosensitive in DNA synthesis

Summary In E. coli mutants thermosensitive in DNA synthesis the capacity for replication of bacteriophages , P1 and T4 was studied in order to obtain more information about the biochemical lesions in such strains. Two mutant types were used. In one of them DNA synthesis stops immediately at the restrictive temperature (mutant 165/70). In the other type DNA synthesis continues at the elevated temperature for a residual time period before it comes to a halt (mutant 252). The thermolabile synthetic steps involved in both mutant types are presently still unknown.The temperate phages and P1 differ in their ability to replicate in the mutant types at temperatures non-permissive for host cell DNA synthesis. Replication of phage is blocked in 165/70 but can still take place in 252 after host DNA synthesis has come to a halt. Phage P1 shows the opposite behaviour. It grows in the mutant 165/70 but its ability to replicate in 252 at 42° C is restricted to the period of residual host cell DNA synthesis observed in uninfected cells. Replication of phage T4 on the other hand is unimpeded in both mutants at restrictive temperatures.     

Cold-sensitive mutations in the Z gene of prophage P2 that result in increased sensitivity of the lysogens to a low molecular weight product of the host bacteria

Summary Z mutants of bacteriophage P2 form clear plaques and are unable to give rise to stable lysogens in Escherichia coli C. To study the function of the Z gene in lysogenization by P2, temperature-sensitive mutants were isolated. Those that were classified as Z mutants by complementation were all cold-sensitive (cs); they were unable to form lysogens at 30° C, but had wild type phenotype at 42° C. When lysogens carrying such mutants, prepared at 42° C, were shifted to the lower temperature, the bacteria continued to multiply at the normal rate until they reached concentrations of about 5 × 10⁷ per ml, at which point the viable titer began to decrease. Inactivation of the bacteria at even lower concentrations occurred if they were transferred to medium taken from overnight cultures of the same strain, suggesting that they were sensitive to some material that had accumulated in the culture medium.The lethal material was produced not only by csZ lysogens, but by all derivatives of Escherichia coli C tested, including non-lysogens, and at both 30° C and 42° C. Only csZ lysogens were sensitive to it, however, and only at the lower temperature. A preliminary characterization of the material indicates that it is heat-stable, of low molecular weight and does not adsorb to activated charcoal.This work was supported by Research Grant 72 from the Swedish Medical Research Council     

Properties of Bacteriophage T4 Mutants Defective in Gene 30 (Deoxyribonucleic Acid Ligase) and the rII Gene

In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.     

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Tolerance of high temperature and antibiotic resistance

Forty one strains ofRhizobium phaseoli were screened for the ability to multiply at high temperatures on yeast extract-mannitol agar. Most strains were tolerant of 30°C, eight strains were tolerant of 45°C and two of 47°C although the rate of multiplication was reduced at 45–47°C. The high temperature-tolerant strains were isolated from Kenyan soils and were fast-growing. Seven of the eight strains tolerant of 45–47°C lost their infectiveness after incubation at high temperature but four strains tolerant of 40°C remained infective after incubation at that temperature.Thirty six strains were resistant to 200 g ml^–1 streptomycin sulphate and 29 strains to 200 g ml^–1 spectinomycin dihydrochloride. Eight strains were resistant to both antibiotics each at 200 g ml^–1. Two of the double-labelled antibiotic-resistant mutants lost their infectiveness onPhaseolus vulgaris. The response to acidity was unaltered and two of the mutants showed a decrease in temperature tolerance. The doublelabelled mutants were recoverable from two Kenyan soils.     

Chromate tolerance in strains of Rhodosporidium toruloides modulated by thiosulfate and sulfur amino acids

Cr(VI) tolerance was studied in four strains of Rhodosporidium toruloides and compared with that of a fifth strain, DBVPG 6662, isolated from metallurgical wastes and known to be Cr(VI) resistant. Tolerance was studied in relation to different species of sulfur (sulfates, thiosulfates, methionine, cysteine) at different concentrations. Djenkolic acid, a poor source of sulfur and an activator of sulfate transport, was also considered. In synthetic medium all strains except the Cr(VI)-resistant one started to be inhibited by 10 g ml^– (0.2 mm) Cr(VI) as K₂Cr₂O₇. DBVPG 6662 was inhibited by 100 g ml^– (2.0 mm) Cr(VI). In Yeast Nitrogen Base without amino acids (minimal medium), supplemented with varying concentrations of chromate, all Cr(VI)-sensitive strains accumulated concentrations of total chromium (from 0.8 to 1.0 g mg^– cell dry wt) after 18 h of incubation at 28 °C. In minimal medium supplemented with 10 g ml^– Cr(VI), the addition of sulfate did not significantly improve the yeast growth. Cysteine at m levels increased tolerance up to 10 g ml^–, whereas methionine only reduced the Cr(VI) toxicity in the strain DBVPG 6739. Additions of djenkolic acid resulted in increased Cr(VI) sensitivity in all strains. The best inorganic sulfur species for conferring high tolerance was thiosulfate at concentrations up to 1 mm. In all cases increased Cr(VI) tolerance was due to a significantly reduced uptake in the oxyanion by the cells and not to the chemical reduction of Cr(VI) to Cr(III) by sulfur compounds.     

Genetic analysis of two bacterial RNA polymerase mutants that inhibit the growth of bacteriophage T7

Summary The Escherichia coli mutants 7009 and BR3 are defective in the growth of bacteriophage T7. We have previously shown that both of these mutant hosts produce an altered RNA polymerase which is resistant to inhibition by the T7 gene 2 protein (De Wyngaert and Hinkle 1979). In both strains, the mutation which prevents T7 growth is closely linked to rifA (rpoB). Both mutants are complemented by transformation with a multicopy plasmid carrying rpoB and rpoC but not by a plasmid carrying only rpoB. This indicates that the mutations reside in rpoC, the structural gene for the subunit of RNA polymerase. When a single copy of the wildtype rpoC allele is introduced into the mutant using the transducing phage drif ^d18, the mutant allele is dominant over wildtype. The drif ^d18 transductant also remains unable to support the growth of T7 in the presence of rifampin. This supports our conclusion that the mutation is in rpoC. We have measured the growth of T7 phage, the kinetics of phage DNA synthesis, and the structure of replicative DNA intermediates in several transductants, and compared these results with those obtained in the original mutant strains.     

S-adenosylmethionine synthetase in methionine regulatory mutants of Salmonella typhimurium

Summary In wild-type bacteria, S-adenosylmethionine (SAM) synthetase activity was repressed by growth in methionine. MetJ regulatory mutants had elevated activities which did not show this repression. Two metK mutants with normal regulation of the methionine biosynthetic enzymes had elevated Km's (methionine) for SAM synthetase while five metK mutants with constitutive methionine enzymes showed no measurable SAM synthetase activity. One mutant, metK ^X 721, similar in phenotype to these five had a wild-type level of SAM synthetase in conditions where SAM decarboxylase activity was blocked. By using an F-factor carrying the metK region of the genome, this mutant was shown to complement six other metK mutants.These results indicate that SAM or a derivative of it, rather than methionine itself, is the co-repressor of the methionine system, regulatory abnormalities resulting from the absence or reduction of the amount of SAM formed by the SAM synthetase reaction. As SAM is essential for bacteria it is likely that there is some alternative biosynthetic route for SAM.     

Plant regeneration in weeping lovegrass, (Eragrostis curvula) through inflorescence culture

Plant regeneration from four genotypes of weeping lovegrass (Eragrostis curvula (Schrad.) Nees), is reported via three developmental pathways: embryogenesis, organogenesis and direct regeneration. Organogenic and embryogenic callus cultures were initiated from young inflorescence segments on Murashige and Skoog's medium supplemented with 2,4-d and BA at different concentrations. The most suitable concentrations of 2,4-d for callus growth and development were 9 and 18 M combined with a BA concentration of 0.044 M. Genotypical differences were observed in the morphogenetic capacity. Direct regeneration was observed under similar culture conditions (culture medium, temperature and photoperiod) but with high light intensity (66 mol m^-2 s^-1). Young plants were successfully transplanted to pots and grown to maturity in the greenhouse.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid     

Caffeine as a comutagen for ethylmethanesulfonate in strains of phage T4

Summary The mutation frequency of DNA polymerase mutants of phage T4 treated with ethyl methanesulfonate (EMS) then incubated in the presence and absence of caffeine was studied using an rII reversion system. The DNA polymerase mutation is shown to be antimutagenic for EMS induction of reversions which occur by a GC to AT transition. Caffeine acts as a comutagen for the induction by EMS of mutant phages and produces a significant increase in the frequency of reversions from rII to r⁺. Caffeine is slightly mutagenic for the phage strain carrying the wild type polymerase and inhibits the action of the 35 exonuclease function of T₄ DNA polymerase as measured in vitro. These findings suggest that caffeine acts by directly influencing nucleotide selection or the editing function of the DNA polymerase.     

Female-specific phages and F-minus strains of Escherichia coli K12

Summary The female-specific phages, I, II, W31 and H (but not T3 and T7) show a low efficiency of plating on all F-minus strains of Escherichia coli K12 except for the thr leu thi mutants descended from strain Y53. The locus responsible is linked to the histidine region and was presumably eliminated from the Y53 line of mutants in the course of mutagenesis.     

Two classes of Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents

Summary Six mutant strains of Bacillus subtilis hypersensitive to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were shown to be deficient in the adaptive response to MNNG and termed ada mutants (Morohoshi and Munakata 1985). All the mutations mapped between the attSPO2 and lin loci on the chromosome. The mutant and wild-type (ada ⁺) cells contained similar constitutive levels of O⁶-methylguanine-DNA methyltransferase activity. Pretreatment with low concentrations of MNNG increased the activity about nine-fold in the ada ⁺ cells, while it uniformly decreased the activity in the ada cells. The pretreatment of three mutants (ada-3, ada-4, and ada-6) as well as ada ⁺, augumented the activity of methylpurine-DNA glycosylase and rendered the cells resistant to the lethal and mutagenic effects of N-propyl- or N-butyl-N-nitro-N-nitrosoguanidine. With the rest of the mutant strains (ada-1, ada-2, and ada-5), neither of such responses was elicited by the pretreatment. Thus, the former ada strains seem to have a defect in the gene specifically involved in the induction of the methyltransferase, while the latter ada strains have a defect in the gene controlling the adaptive response as a whole.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - ENNG N-ethyl-N-nitro-N-nitrosoguanidine - PNNG N-propyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - MMS methyl methanesulphonate     

Mutants with reduced Ca activation inParamecium aurelia

Summary Two heat-sensitive pawn mutants ofParamecium aurelia are capable of avoiding reactions when grown at 23°C but not at 35°C. Electrophysiological analyses show that Ca activation is reduced in the mutants even when they are grown at 23°C. The maximal rate of rise and the peak of the evoked action potential (Ca-spike) in the mutants are smaller than those of wild type in a K-solution. After suppression of K conductance by either TEA⁺ or Ba⁺⁺, the action potentials of the mutants peak at the same level as that of wild type. However, the maximal rate of rise of the mutants remains only about half that of wild type. Thus, the mutations affect Ca activation but not K activation.Incubation at a high temperature (35°C) further reduces Ca activation to almost zero in the mutants but has little or no effect on wild type. This almost complete loss of Ca activation explains the lack of avoiding reactions when the mutants are grown at high temperatures. A double mutant containing two heat-sensitive mutations shows extremely reduced Ca activation even when grown at 23°C.     

Plasmid replication and Hfr formation in strains of Escherichia coli carrying seg mutations.

Summary Several conditional-lethal mutations that do not permit the replication of F-factors ofEscherichia coli K-12 are located at a site calledseg. This gene is located on theE. coli chromosome betweenserB andthr. It is unrelated to other known genes involved in DNA replication. Strains carryingseg mutations were unable to replicate F-lac⁺, several F-gal⁺s, F-his⁺ and bacteriophage at 42°. However, neither phage T4, ColE1, nor any of the R factors tested were prevented from replicating at 42°C.When the kinetics of the loss of F-primes is studied inseg strains, it is found that the rate of curing depends on the size of the plasmid, larger F factors curing faster than smaller ones, and that Hfrs are formed at high frequencies. The Hfrs showed both F-genote enlargement and normal transfer of chromosomal markers. The F-genotes are unstable and segregate chromosomal markers at high frequencies. Some orthodox Hfrs were examined, and two that were known to revert to the F⁺ condition relatively frequently were found to generate enlarged F-genotes on mating, whereas two strains that were very stable with respect to reversion to the F⁺ state did not show F-genote formation.F-genote formation fromseg Hfr strains is dependent of a functionalrecA gene, as F-genote formation was not seen with aseg-2, recA-1 Hfr. This is in contrast to F-genote enlargement shown by both orthodox Hfrs and an Hfr strain constructed by integration of a temperature-sensitive F-gal⁺, whose F-genote enlargement is Rec-independent. Thus there may be more than one mechanism for the formation of enlarged F-genotes.     

Hereditary respiration deficiency in Saccharomycodes ludwigii

Saccharomycodes ludwigii, supposed to be petite-negative, gave rise to respiration-deficient mutants when acriflavine and ultraviolet irradiation, respectively, were applied to this yeast, strain IFO 1194. The frequency of such mutants was very low as compared with that in Saccharomyces cerevisiae and other petite-positive yeasts. Cytochrome composition was characterized by spectrophotometry at the temperature of liquid nitrogen. The respiratory mutants examined contained cytochrome c unaltered in quality and quantity. Cytochrome b was often present only in small amounts though never absent, while cytochrome a+a₃ was either present or absent. The respiratory mutants could form zygotes after conjugation with a wild-type culture of opposite mating type ( vs. a). The hybridization and segregation analysis of spore tetrads showed the inheritance of respiratory mutant character to be either Mendelian or non-Mendelian and similar to that of pet (nuclear) and rho^- (cytoplasmic) mutants, respectively, in Saccharomyces cerevisiae.     

Repressor and rex product of coliphage lambda: lack of collaboration and joint controls

Summary Exposure to 41° C for 10 to 100 minutes rapidly inactivates repressor bearing several ts mutations in the A or B region of gene cI, but does not result in simultaneous rapid loss of the rex function, which restricts phage T4 rII. One may conclude that the rex product does not directly collaborate with the repressor protein. Immediate loss of the rex activity at 47.5° C, observed with most of the cIts mutants and even cI⁺, appears to be unrelated to the repressor inactivation. In tof ⁺ lysogens carrying nonlethal cIts prophage mutants, the prolongation of induction at 41° C ultimately results in irreversible loss of the rex function, but only after about six cell generations. In similar experiments with tof deficient lysogens, loss of the rex activity requires about eleven cell generations and the rex function is regained in less than 30 minutes after return of the lysogen to 30° C. Two methods of rex assay, the more sensitive phage yield method and the infective center method, were employed.     

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Effects of Pb2+, Ni2+, Hg2+ and Se4+ on cultured cells. Analysis of uptake, toxicity and influence on radiosensitivity

Effects of Pb²⁺, Ni²⁺, Hg²⁺ and Se⁴⁺ on cultured human glioma U-343MG cells were investigated considering uptake, toxicity and, in combination with radiation, clonogenic cell survival. The cells were exposed to 0-100 m of the metals for a week before the evaluation. The tests showed a tendency to toxicity with 10 m nickel although not significant (P > 0.05). Selenium, lead and mercury exerted a significant toxicity (P < 0.05) at 2.5 m, 10 m and 1 m, respectively. To challenge the clonogenic cell survival capacity, the cells were irradiated with⁶⁰Co photons after being exposed to the highest nontoxic concentration of the different metals. The clonogenic cell survival tests, after irradiation, showed no significant change if the cells were exposed to 5 m nickel, 0.5 m selenium or 5 m lead compared with those not exposed. Mercury, 0.1 m, gave a relative reduction in survival compared with only irradiated cells of 58 ± 17%. Thus, only mercury affected the radiation-induced damage and/or repair. When exposed to the highest nontoxic concentrations of the different metals, the cultures did not display a significant uptake ratio (metal concentration ratio of exposed cells to control cells) of nickel (3.1 ± 3.3), only a small uptake ratio of selenium (4.0 ± 0.4), while there was a large uptake ratio of both lead (2.6 ± 1.7) x 10² and mercury (1.5 ± 0.2) x 10¹. The results indicated that nickel was neither especially toxic nor influenced the clonogenic cell survival after irradiation. Mercury was more toxic and also influenced the radiation sensitivity. Lead was taken up strongly but did not influence the radiation sensitivity. Selenium accumulated but gave no detectable effect on the radiation sensitivity.     

Genetic study of the role of calcium ions in the cell division cycle of Saccharomyces cerevisiae: a calcium-dependent mutant and its trifluoperazine-dependent pseudorevertants

Summary A cal1-1 mutant of the yeast Saccharomyces cerevisiae showing Ca²⁺-dependent growth was isolated. Its growth continued exponentially in Ca²⁺-rich medium, but stopped in Ca²⁺-poor medium at 37°C. Mg²⁺ ions could not replace Ca²⁺ ions. In Ca²⁺-poor medium, the mutant cells stopped growing homogeneously at the stage of cell division cycle with a tiny bud. The nucleus in these arrested cells was in the G2 stage, judging from observation after nuclear staining and determination of the DNA content. Trifluoperazine-dependent pseudorevertants, which could grow in the presence of 20 M to 80 M trifluoperazine in Ca²⁺-poor medium at 37°C, were obtained from this cal1-1 mutant. The suppressor mutation, tfrl, itself conferred trifluoperazine resistance. Other calmodulin inhibitors structurally unrelated to trifluoperazine had similar effects to trifluoperazine on these pseudorevertants. These results suggest that Ca²⁺ ions and a calmodulin play important roles in the yeast cell division cycle at the stage of bud growth and nuclear division.Abbreviations Tfp trifluoperazine - DAP1 46-diamidino-2-phenylindole - EMS ethyl methanesulfonate - PD parental ditype - NPD nonparental ditype - T tetratype