The use of ethyl methanesulfonate for the induction of mutants inMycobacterium phlei PA

The mutagenic effect of ethyl methanesulfonate in a concentration of 0.2m on a prototrophic, acid-fast strainMycobacterium phlei PA was studied by following the induction of changes of three genetic markers: prototrophy to auxotrophy and sensitivity to two antituberculosis drugs (INH and STM) to resistence. Ethylmethanesulfonate was found to be a very effective mutagen in all three cases. Thirty auxotrophic strains were obtained, out of which eight exhibited a low frequency of spontaneous reversions and could hence be used for further studies. Of the phenotypes induced the glycine⁻ (serine⁻) type was most frequently isolated and represented more than half of all auxotrophs obtained. Requirements for lysine and purines were also observed. The EMS treatment (1% survival of the basic suspension) resulted in a 74-fold increase of the frequency of INH-resistant mutants and a frequency of STM-resistant mutants about 1.1/2 to almost 2 orders of magnitude higher than the spontaneous values     

Mutagenic effect of ultraviolet radiation on the non-acid-fast strain ofMycobacterium phlei

The mutability of the PN strain ofMycobacterium phlei was examined after induction of auxotrophic mutants and of STM and VM-resistant mutants, by UV irradiation. A total of 30 auxotrophic mutants were isolated, most of them amino acid-dependent five purine-dependent, and one uracil-dependent. To induce the mutants higher UV doses had to be used so that the survival of cells in the original suspension would not exceed a few per cent. For further genetic work use can be made of 8 auxotrophic mutants (PN try^?ura^?, PN arg^?ura^?, PN ileu^?val^?, PN ileu^?, PN leu^?, PN lys^?, PN lys^?-VM^r, PN val^?), these showing a low frequency of spontaneous reversions. No spontaneous auxotrophic mutants have been found. The frequency of STM and VM-resistant mutants is increased upon UV irradiation, a post-irradiation incubation in a liquid medium without the drug being essential for their phenotypic expression. The highest increase of the number of these mutants is attained after 48 h of post-irradiation incubation and it has been found that, within a certain experimental scatter, the same frequency increase is found on using a complete or a minimal liquid medium. The frequency of spontaneous STM-resistant mutants lies within 5.8×10^?6–8.8×10^?6, of those VM-resistant between 3.1×10^?5 and 4.1×10^?5. The highest frequency of induced STM-resistant mutants lies between 3.0×10^?5 and 9.3×10^?5 and of VM-resistant mutants between 1.1×10^?4 and 2.2×10^?4     

The induction of mutants in a non-acid-fast strain ofMycobacterium phlei with nitrous acid

The inactivation and mutagenic effets of nitrous acid on a non-acid-fast strain ofMycobacterium phlei were studied. It was found that 0.017m NaNO₂ at pH 4.4 may be used for the induction of auxotrophic mutants, scotochromogenic and achromogenic mutants and STM-resistant mutants. Three doubly auxotrophic mutants, three mutants requiring amino acids and three mutants depending on vitamins were obtained. One mutant was not classified. Eighteen scotochromogenic mutants were isolated, seventeen of them were orange. Only ten achromogenic mutants were isolated. Twelve scotochromogenic and eight achromogenic mutants could be used in further genetic studies as they did not revert spontaneously to photochromogeny. Six auxotrophic mutants could be used due to their low frequency of spontaneous reversions. The frequency of STM-resistant mutants increased on an average seven-fold after the mutagenic treatment as compared with the spontaneous frequency.     

The mutagenic effect of ethyl methanesulfonate on a non-acid-fast strain ofMycobacterium phlei

Ethyl methanesulfonate was used for the induction of three types of mutants in a non-acidfast strain ofMycobacterium phlei. A total of 20 auxotrophie mutants was isolated. The mutants were isolated mostly when using doses yielding higher survival of the cells of the basic suspension. The auxotrophic mutants isolated required mostly amino acids, two mutants required purines and three mutants required vitamins. By determining the frequency of spontaneous reversions, it was found that 9 auxotrophic mutants could be used for further genetic studies. These included the following phenotypes: isoleucine⁻, leucine⁻, lysine⁻, nicotinic acid⁻, pyridoxine⁻, and xanthine⁻. Seven scotochromogenic mutants were isolated after ethyl methanesulfonate treatment. One was ochre, the remaining six were orange. Six achromogenic mutants were detected. Spontaneous auxotrophic mutants, scotochromogenic and achromogenic mutants were not isolated. The treatment with 0.2m ethyl methanesulfonate resulted in an increase in the frequency of STM-resistant mutants, the maximum phenotypic expression taking place after 72 hours cultivation in a liquid medium without the drug. The frequency of induced STM-resistant mutants varied within the range of 8.6.10⁻⁵–1.0.10⁻⁴ as compared with the frequency of spontaneous mutants 5.8.10⁻⁶–8.8.10⁻⁶.     

The Amino Acid Composition of Human Pituitary Growth Hormone

The amino acid content in human hypophyseal growth hormone has been determined by chromatography on resin columns. On the basis of 29,000 for the molecular weight, the empirical formula of the hormone was obtained: lys₁₃his₅arg₁₄asp₂₇ thr₁₄ser₂₃glu₃₄pro₁₂gly₁₃ala₁₂-(1/2 cys)₆val₁₂met₄-ileu₁₀leu₃₁tyr₁₀phe₁₅try₁(NH₃)₃₂.     

The mutagenic effect of hydroxylamine onMycobacterium phlei strain PA

The mutagenic effect of 0.05m and 1m HA onMycobacterium phlei PA was investigated. To establish the mutagenic effect the inactivating effect was studied under the same experimental conditions. Hydroxylamine at a higher concentration (1m) exhibited relatively high mutagenic effect. This was indicated by about 100-fold and 10-fold higher frequency of INH^r and STM^r mutants, respectively (as compared with spontaneous mutations) and induction of auxotrophic mutants. On the other hand, the mutagenic effect of 0.05m hydroxylamine was low under the same experimental conditions. The inactivating effect of a higher HA concentration (1m under given experimental conditions) was considerably higher when using the given model microorganism than that of the lower one (0.05m under the same experimental conditions). This finding does not agree with literature data obtained in other experimental models.     

Transplantation of isolated nuclei into plant protoplasts

The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca²⁺ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10^-5 to 6·10^-4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration of morphogenetic potential. Welldeveloped shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium.Abbreviations MS Murashige and Skoog (1962) - PEG polyethylene glycol     

The mutagenic effect of nitrosoguanidine onMycobacterium phlei PA

The effect of nitrosoguanidine on the induction of three types of mutagenic changes inMycobacterium phlei PA was studied. The mutagenic changes included: change of prototrophy to auxotrophy, conversion of sensitivity to isoniazide to resistańce and sensitivity to streptomycin to resistance. The induction of auxotrophic mutants was successful especially when using NTG at a concentration of 1000 μg/ml. A total of 100 auxotrophs was obtained out of which only 13 were sufficiently stable to be used in further studies. Amino acid requirements especially the glycine⁻(serine⁻) type characterized more than half of all auxotrophic mutants obtained. A group of mutants requiring purines also included a high number of mutants. A considerable spontaneous reversion frequency was found in both groups of auxotrophs. The kinetics of the induction of INH-resistant mutants by nitrosoguanidine at a concentration of 1000 μg/ml was studied and a high induction of these mutants, particularly at high lethal effect of the mutagen found. The mutability of the STM^r marker was relatively low in the present model microorganism as compared with the two markers mentioned earlier.     

Unstable mutation forms induced by UV irradiation

The growth rate and the stability of prototrophic reversions induced by UV irradiation was investigated in the auxotrophic strainEscherichia coli WP₂ which requires tryptophan for its growth. It was found that in unstable reversions the retarded growth is caused by a limited and variable ability to synthesize the essential nutrient. The irregularity observed in the synthesis of the essential substance and the high frequency of occurrence of auxotrophic forms supports the assumption that these changes are brought about by one of the regions regulating the transmission and expression of genetic information.     

Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5

Drug-resistance element Tn5 coding for kanamycin resistance was used for mutagenesis of Alcaligenes eutrophus strain H16. The vehicle for introducing Tn5 into A. eutrophus was plasmid pJB4JI harboured by Escherichia coli. Kanamycin-resistant transconjugants occurred at a frequency of approximately 5×10^-8. One third of the transconjugants exhibited other plasmid-coded resistances such as gentamycin and spectinomycin. However, the latter markers were not stably maintained in the new host. Among the kanamycin-resistant transconjugants three classes of mutants were found: (i) Auxotrophic mutants occurred at a frequency of 0.8% and showed requirements for histidine, methionine, aspartate orisoleucine. Out of eleven auxotrophic mutants examined eight reverted to prototrophy. However, none of the revertants was kanamycin-sensitive. (ii) Mutants unable to grow with fructose as the carbon source occurred at a frequency of almost 10%. (iii) Mutants which had lost the ability to grow autotrophically with hydrogen and carbon dioxide were found at a frequency of 1%. Further analyses revealed that this class of mutants was either defective in carbon dioxide fixation or impaired in hydrogen metabolism.     

Cloning,expression and directed mutagenesis of the genes for ribulose bisphosphate carboxylase/oxygenase

The dominant natural form of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is composed of large (L) 55-kDa and small (S) 15-kDa subunits. This enzyme (as the L₈S₈ form) is widely distributed among oxygenic photosynthetic species and among chemosynthetic bacteria. Another form lacking small subunits is found as an L₂ dimer in Rhodospirillum rubrum or an L oligomer of uncertain aggregation state from Rhodopseudomonas spharoides. The present article reviews two basically different approaches in cloning the R. rubrum gene for RuBisCO. One results in high level expression of this gene product fused with a limited aminoterminal stretch of -galactosidase and the other results in expression of wild-type enzyme in Escherichia coli. Also reviewed are a number of reports of cloning and assembly of the L₈S₈ enzyme in using E. coli L and S subunit genes from Anacystis nidulans, Anabaena 7120, Chromatium vinosum and Rps. sphaeroides.In vitro oligonucleotide-directed mutagenesis has been applied to the gene for RuBisCO from R. rubrum. In terms of contributing new information to our understanding of the catalytic mechanism for RuBisCO, the most significant replacement has been of lys 166 by a number of neutral amino acids or by arg or his. Results establish that lys 166 is a catalytically essential residue and illustrate the power of directed mutagenesis in understanding structure-function correlates for RuBisCO.Oligonucleotide-directed mutagenesis has also been applied to the first and second conserved regions of the S subunit gene for RuBisCO from A. nidulans. In the latter region, corresponding amino acid changes of trp 55 and trp 58 to phe, singly or together, had little or no effect upon enzyme activity. In contrast, mutagenesis in the first conserved region leading to the following pairs of substitutions: arg10 arg 11 to gly 10 gly11; thr14 phe 15 ser 16 to ala 14 phe 15 ala 16; ser 16 tyr 17 to ala 16 asp 17; or pro 19 pro 20 to ala 19 ala 20, are all deleterious.Advances are anticpated in the introduction and expression of interesting modifications of S (and L) subunit genes in plants. A new method of introducing and expressing foreign genes in isolated etiochloroplasts is identified.Abbreviations RuBisCO ribulose bisphosphate carboxylase/oxygenase - 2-CABP 2-carboxyarabinitol-1,5-bisphosphate - 4-CABP 4-carboxyarabinitol-1,5-bisphosphate     

Antimutagenic effects of caffeine during nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium cells and phages

The effect of caffeine on nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium & nd its P22 and L phages was studied. The detected mutations included phage “clear” mutations, reversions of phage “amber” mutation, and prototrophic reversions of thehis ⁻ auxotroph ofSalmonella typhimurium. Neither therecA mutation of the host nor theerf mutation of the phage genome were found to affect the nitrosoguanidine-induced mutagenesis of the phage during vegetative growth. Beginning with a concentration of 0.2 mg/ml, caffeine decreased the frequency of mutants by 30–60%, attaining a maximum effect at 1.5 mg/ml and retaining this effect even at higher concentrations. A similar antimutagenic effeot was observed with the mutagenesis of the host cells. The nitrosoguanidine-induced mutagenesis does not seem to be related to the function of therecA cell gene or theerf phage gene. The mechanism of mutagenesis by nitrosoguanidine probably has two components, one of them caffeine sensitive, the other caffeine-resistant.     

Genetic analysis of methylotrophic yeastCandida boidinii PLD1

This paper reports the initial experiments for genetic analysis of the haploid methylotrophic yeastCandida boidinii PLD1. The collection of multiply marked auxotrophic mutants was obtained after treatment with UV-light or X-rays. Protoplasts from several mutants were fused by the PEG-CA²⁺ technique and five prototrophic hybrids were isolated. The genetic structure of the hybrids was studied by means of spontaneous and induced mitotic segregation. Our data suggest that hybrids are diploids, heterozygous by parental auxotrophic markers. We obtained genetic linkage between mutationslys2-8-met-3 from one hand andade-17-arg-24 from the other. The genetic maps constructed showed similar characteristics concerning both the order of the markers and their map distances.     

Intraspecific fusion ofAspergillus niger strains producing citric acid using heat-inactivated and living protoplasts

Protoplasts from a prototrophicAspergillus niger strain were first inactivated at 55°C for 12 min with a regeneration frequency of 3.5×10⁻⁶, and then fused with living protoplasts from an auxotrophic strain. The fusion frequency was 1.1×10⁻⁵. Some fusants segregated sectors of prototrophic recombinants Citric acid production in submerged cultivation of these recombinants was examined. More recombinants were obtained by further treating the fusants with (+)-camphor, a diploidization inducer.     

Studies on Bacterial Protease

Some physicochemical properties and amino acid composition of the alkaline protease of B. amylosacchariticus were determined. The molecular weight and sedimentation coefficient were estimated to be 22,700 and 2.89 s, respectively, and the amino terminal amino acid was identified to be alanine. The enzyme contained 15.9% of nitrogen and was composed of 220 residues of amino acid: lys₆, his₅, arg₃, asp₂₀, thr₁₄, ser₃₇, glu₁₂, pro₁₀, gly₂₅ ala₂₇ val₂₀, met₃, isoleu₁₂, leu₁₂, tyr₉, phe₂, try₃ and amide ammonia₁₆ The results indicate that protein nature and chemical properties of the alkaline protease presented here are distinct from those of alkaline proteases obtained from the other strains of B. subtilis, such as subtilopeptidase A, B and BPN’     

Auxotrophic mutants in the selection of the rubomycin producer, Actinomyces coeruleorubidus

A total of 351 auxotrophic mutants with different antibiotic activity, including several mutants with activity higher than that of the parent prototrophic strains were obtained under the effect of gamma-rays from 3 prototrophic strains of Act. coeruleorubidus. It was shown that most of the auxotrophic mutants did not preserve the property of biochemical insufficiency on passages on complete media. A mutant strain 1059-32 with activity 2 times higher than that of the prototrophic strain 2-39 and the parent auxotrophic culture was obtained from the revertants. Requirements in 29 growth factors including 17 amino acids, 4 nitrous bases, 8 vitamins and coenzymes were determined in 46 stable auxotrophic mutants isolated. The effect of the specific and non-specific growth factors on the culture antibiotic production was studied.     

Peptide chain elongation: A possible role of montmorillonite in prebiotic synthesis of protein precursors

Several studies have proven the ability of montmorillonite to catalyse amino acid condensation under assumed prebiotic conditions, simulating wetting-drying cycles. In this work, the oligomerization of short peptides gly₂, gly₃, gly₄ and ala₂ on Ca-and Cu-montmorillonite in drying-wetting cycles at 80 °C was studied. The catalytic effect of montmorillonite was found to be much higher than in the case of glycine oligomerization. From gly₂ after 3 weeks, 10% oligomers (up to gly6, with gly₃ as main products) are formed. Gly₃ and gly₄ give higher oligomers even after 1 cycle. Ala₂ produces both ala₃ and ala₄, whereas ala does not produce any oligomers under these conditions. Heteroologomerization was observed: ala-gly-gly is formed from ala and gly₂. Much higher yields are obtained using Ca-montmorillonite, because copper (II) oxidizes organic molecules. The influence of the reaction mechanism on the preferential oligomerization of oligopeptides is discussed.     

Selection of spontaneous mutants ofYarrowia lipolytica by inositol-less death

Summary A procedure was developed for the selection of spontaneous mutants of the yeastYarrowia lipolytica. An inositol-requiring mutant of a wild-typeY. lipolytica, YB 3-122, was derived by mutagenesis and screening. The mutant had a reversion frequency of less than 6×10^–9. A mutant selection procedure based on inositolless death was then developed using this mutant strain. The selection procedure killed growingY. lipolytica cells and enriched for mutants yielding cultures that consisted of 60–98% spontaneous mutants after two rounds of inositol-less death. The procedure enriched for four classes of mutants, strains that were auxotrophic, metabolite analog sensitive, temperature sensitive, or unable to grow on citric acid as the sole carbon source. Since strain YB 3-122 is now available to yeast researchers, inositol-less death will be useful for the routine isolation of spontaneous mutants ofY. lipolytica.     

Role of Escherichia coli DnaK and DnaJ chaperones in spontaneous and induced mutagenesis and their effect on UmuC stability

The frequency of spontaneous as well as induced reversions of auxotrophic mutations in Escherichia coli AB1157 and its DeltadnaK and DeltadnaKdnaJ derivatives was estimated. The obtained results demonstrate that both mutants tested are characterized by elevated frequency of spontaneous reversions compared to their AB1157 parent. In contrast, the frequency of reversions induced by UV and MMS, i.e. agents inducing the SOS response, is reduced in DeltadnaJ and DeltadnaKdnaJ mutants, pointing to the possible defect of these mutants in error prone repair. Due to the fact that UmuC protein is one of the main players executing the error prone repair, its stability in DeltadnaJ and DeltadnaKdnaJ mutants was also studied. Reduced UmuC stability was demonstrated only in the DeltadnaKdnaJ mutant.