Biochemical Mutants Research Articles

Isolation of sugar and antibiotic mutants in Chlorella vulgaris with a new replica plating technique

A new replica plating technique has been described to isolate physiological and biochemical mutants in the unicellular alga, Chlorella. In one operation, 115 different cultures can be inoculated. A pure culture of Chlorella was raised from a single cell which was treated with ethyl methanesulfonate at its LD 50 value to induce mutations. After a prolonged treatment with ethyl methanesulfonate (EMS), 0.87–39.4% mutants were isolated on media with different sugar and antibiotic contents. This technique can be effectively used for isolating physiological and biochemical mutants in other unicellular organisms and also for higher plant cells.

Streptomycin-resistant plants from callus culture of haploid tobacco.

THERE are few reports of the isolation of biochemical mutants from cultured plant cells1–3. We know of none describing plants which have been regenerated from these mutant cell lines and which still carry the mutant trait. We have worked out conditions for the isolation of streptomycin-resistant tobacco cell lines, from which diploid fertile plants carrying the streptomycin-resistant character, were regenerated. Seedlings grown from seeds of self-fertilized resistant Str-r1 plants were also resistant. The progeny obtained from crosses of Str-r1 plants and streptomycin sensitive (Str-s) plants showed non-Mendelian, uniparental transmission of the resistance trait.

Radiation induced biochemical mutation in Colletotrichum gloeosporioides penz. causing anthracnose of guava (Psidium guajava L).

With the aid of radioactive phosphorus and gamma rays from Cobalt60, a number of biochemical mutants were obtained which showed deficiency for amino acids like methionine, leucine and cystine and vitamins like biotin and thiamine. These biochemical mutants could not be distinguished from the wild type when grown on complete or otherwise suitably supplemented media. Pathogenicity tests of some of these biochemical mutants showed that all the five mutants tested are pathogenic on guava variety Allahabad Safeda, though these showed deficiency for different growth factors.

Heterokaryosis in Microsporum gypseum.

The basic properties of heterokaryosis have been studied with the use of morphological and biochemical mutants ofMicrosporum gypseum. A direct proof of heterokaryosis was given with the help of the isolation of hyphal tips. Heterokaryons formed from aconidial components conidiate abundantly. The heterokaryotic constitution is preserved only with the transfer of mycelium; by microconidia the transfer is not effected.

Genetic recombination in Micromonospora.

Biochemical mutants were obtained from Micromonospora chalcea, M. purpurea, and M. echinospora by using ultraviolet radiation or nitrosoguanidine. Crosses carried out between complementary nutritional mutants of the same species showed positive genetic interaction. Data are reported which indicate that the interaction between the crossed strains is due to genetic recombination. No evidence for interspecific genetic recombination was found.

Irradiation induced physiological and morphological variation in Colletotrichum capsici, incitant of anthracnose of chillies

With the aid of radioactive phosphorus and gamma rays, a number of morphological and biochemical mutants ofColletotrichum capsici have been induced and these showed wide range of variability. Biochemical mutants mostly induced by means of p32 irradiation showed deficiency of amino acids or vitamins. Specific deficiency analysis of some of the biochemical mutants revealed requirements for lysine, methionine, glycine or serine and biotin. These biochemical mutants could not be distinguisbed from the wild type in morphological characters when grown on complete or otherwise suitable supplemented media.

THE PATHWAY OF INFECTION OF USTILAGO HORDEI

Biochemical mutants of Ustilago hordei (Pers.) Lagerh were used to infect barley seeds in a study of the pathway of infection and the development of the parasite within the host. Microscopic studies in which the parasite was recovered from pieces of host tissue, identified and grown in vitro confirmed earlier views that the parasite mycelium was present throughout the spike primordia of the barley host.From genetically marked infections, it became evident that diploidization and subsequent recombination and haploidization may follow the dikaryotic stage of the parasite.

Ultraviolet-induced mutation in Streptomyces induces

The wild-type strain Streptomyces indicus Chakrabarty and 4 mutant strains derived from it were irradiated by UV rays of 253.7 nm wavelength at 100 erg/mm 2/sec, for 20, 40, 60 and 80 sec. The survival curve was exponential, and the rate of mutation was parallel with the increasing dosage and was followed later by a decline. The mutants differed from the wild parent in reduced growth rates, changes in the shape and size of the colony, sporulation level, antibiotic activity and variation in the colour of the mycelium. Biochemical mutants were obtained that required histidine, glycine, aspartic acid, lysine, valine, methionine, ornithine, arginine, tyrosine, cysteine, proline, leucine, alanine, threonine, riboflavin, adenine, guanine, inositol, nicotinic acid, hypoxanthine and thymine, either singly, doubly or alternatively. The number of histidine-requiring mutants was maximal. There was marked variation in the antibiotic activity of biochemical mutants: increase, decrease or complete absence of such activity were seen. In some of the auxotrophs, a linking of genetic characters controlling the biosynthesis of amino acids and the production of antibiotic substances was observed.

Homotypic and heterotypic interference among picornavirus ribonucleic acids.

Simultaneous addition of RNA preparations from two picornaviruses to facilitator compound resulted in greatly reduced numbers of plaques as compared to the number produced by similar concentrations of interferee RNA (that whose plaque formation was not blocked in interference experiments). A similar phenomenon was demonstrated with RNA from biochemical mutants of picornaviruses and their corresponding wild-type viruses. A method of biochemical blocking was shown to be as effective as that using antiserum. Infectivity of RNA preparations was markedly reduced by ultraviolet irradiation, heat, and snake venom phosphodiesterase; however, these agents did not significantly affect interfering capacity of the RNA preparations.

Mutator Factor in Neisseria meningitidis Associated with Increased Sensitivity to Ultraviolet Light and Defective Transformation

A variant of Neisseria meningitidis was found to carry a mutator factor which endowed the bacteria with generalized genetic instability. The reversion frequencies of several biochemical mutants were increased up to 1,000-fold when the factor was introduced. The factor is not unidirectional in preference, since the mutator induced mutants generally reverted with increased frequency in its presence. There could be found no indication of insufficient synthesis of nucleic acid precursors. Attempts to demonstrate an unusual, mutagenic base incorporated in deoxyribonucleic acid (DNA) were negative. Strains carrying the mutator factor had significantly increased sensitivity to ultraviolet light. A mutation to a more ultraviolet-resistant type coincided with a disappearance of the mutator property. The presence of the mutator factor in a competent strain resulted in a reduction of the transformation frequency to between 0.5 and 5% of that in the parental strain. A mutation to the more ultraviolet-resistant type resulted in simultaneous loss of the mutator property and reestablishment of a normal transformation efficiency. It has been suggested that this mutator factor may represent a defect in the DNA repair mechanism, which is also of importance for genetic recombination. The mutator factor showed cotransformation with the locus for streptomycin resistance, but a true linkage could not be proved.

Linked genes arising among multiple mutants in EMS-treated Sacchararomyces.

Previous work has established that EMS treatment of Saccharomyces yields a relatively efficient method for harvesting viable biochemical mutants, and that cells bearing multiple biochemical deficiencies occur with unusually high frequency. In addition a genetic analysis of these multiple mutations has shown a non-random "clustering" of these multiple mutations relative to the total chromosome map.Apparently for reasons which are not immediately clear, EMS-treatment produces an unexpectedly rich abundance of multiple mutations among genetically linked loci. Conceivably, this could occur if only specific chromosomal regions were "open" to the mutagenic action of EMS at any given time.

The Induction of Biochemical and Morphological Mutants in the Moss Physcomitrella patens

The Induction of Biochemical and Morphological Mutants in the Moss Physcomitrella patens

Isolierung von Riboflavin-Mangelmutanten von Saccharomyces cerevisiae

Riboflavin-deficient mutants of S. cerevisiae, not known hitherto, were isolated using a yeast extract-peptone-dextrose medium supplemented with 10 γ riboflavin/ml. After treatment with ethylmethanesulfonate, 0,4% of the surviving cells or 2% of the isolated biochemical mutants were deficient of riboflavin. Seven of 14 riboflavin deficient mutants were tested for temperature dependence but no such dependence was found. Two mutants accumulated a green fluorescing substance. Cross-feeding tests were not successful.

Heterokaryosis in dermatophytes.

Biochemical mutants ofTrichophyton mentagrophytes unable to grow on minimum medium (auxotrophs) have been paired. Growth on minimum medium occurred irregularly for many combinations, but turned out to be caused by syntrophism in most cases. Only for one combination showing the best growth could the formation of a heterokaryon be shown.

Genetic Recombination in Crosses Between Streptomyces aureofaciens and Streptomyces rimosus

Polsinelli, M. (University of Pavia, Pavia, Italy), and Maria Beretta. Genetic recombination in crosses between Streptomyces aureofaciens and Streptomyces rimosus. J. Bacteriol. 91:63-68. 1966.-Biochemical mutants were obtained from Streptomyces rimosus and S. aureofaciens by ultraviolet irradiation. Crosses were performed between auxotrophic strains of S. rimosus and S. aureofaciens with positive results. Data are reported which indicate that the interaction observed in some crosses is due to gene recombination.

RELATION OF BIOCHEMICAL MUTATIONS TO ACTINOMYCIN SYNTHESIS IN STREPTOMYCES ANTIBIOTICUS.

Biochemical mutants of Streptomyces antibioticus were isolated and tested for their capacity to produce actinomycin. The yield of actinomycin produced in minimal medium plus the required amino acid, by strains requiring an amino acid not present in the molecule of actinomycin, was not significantly different from that obtained from the wild-type strain. On the other hand, all the strains which required an amino acid, which was also a precursor of the antibiotic, showed on minimal medium a drastic decrease in the production of actinomycin. The results have been interpreted by assuming a different utilization by the cell of exogenous and endogenous amino acid pools for antibiotic and protein synthesis.

THE INFLUENCE OF PRECURSOR POOL SIZE ON MITOCHONDRIAL COMPOSITION IN NEUROSPORA CRASSA.

The chemical composition of mitochondria obtained from exponentially growing Neurospora can be varied by addition of choline or amino acids to the culture medium. The variation affects the phospholipid to protein ratio, and the density of mitochondria as determined by isopycnic centrifugation in sucrose gradients. These variations have been observed in biochemical mutant strains as well as wild type cultures. In a choline-requiring strain, two levels of choline supplementation to the medium have been defined: a low choline concentration just adequate to support maximal logarithmic growth, and a high choline concentration which permits maximal incorporation of radioactive choline into cellular lipids. Mitochondria isolated from cultures growing at the low choline concentration have one-half the phospholipid to protein ratio of those from high choline cultures, and their density is significantly higher. Artificial mixtures of the two types of mitochondria can be resolved into two populations by isopycnic centrifugation. The concentration of cytochromes (measured by mitochondrial difference spectra) and of malate and succinate dehydrogenases (measured by enzyme activity) were the same in both types of mitochondria, on a protein basis. The results suggest that during growth of the mitochondrial mass, the incorporation of phospholipid and protein components can vary independently. Direct kinetic measurements did indeed show that choline, added to a culture growing at low choline concentration, was incorporated into mitochondrial lipids at a rate faster than the incorporation of protein. The mitochondrial phospholipid to protein ratio can also be influenced by the level of leucine supplementation to a leucine-requiring mutant, so that with leucine concentrations above those required for maximal exponential growth, mitochondria of increasing density and decreasing phospholipid to protein ratio are produced. Additions of choline or amino acids to the minimal medium of wild type cultures influence mitochondrial composition in a manner directly comparable to that observed in biochemical mutant strains. The results suggest that mitochondrial composition, in general, is determined by rates of incorporation of the two major components, phospholipid and protein; that these rates can vary independently in response to precursor concentration in the culture medium; and that they normally operate at a precursor (substrate) concentration below saturation level.

VENTURIA INAEQUALIS. XV. HISTOLOGY OF INFECTIONS BY BIOCHEMICAL MUTANTS

Microscopic examination of apple leaves infected by biochemical mutants of Venturia inaequalis showed that all mutants had penetrated the cuticle and had established themselves in the characteristic subcuticular position without an additional source of their required substances from outside the host. Those mutants that were fully as pathogenic as the wild type developed in vivo comparably to it, but those that were nonpathogenic made very limited growth and, with the exception of 1 requiring choline, failed to develop sufficiently to sporulate. However, the nonpathogenic mutants, with the exception of 1 requiring adenine, grew and sporulated in vivo when their required substances were applied to the surface of the inoculated leaves.

CAROTENOID PIGMENTS IN A MUTANT STRAIN OF VERTICILLIUM ALBO-ATRUM REINKE AND BERTHOLD.

THE only pigment known to be produced by wild-type strains of V. albo-atrum is of a dark melanoid type1. In an investigation involving the production of biochemical mutants in this fungus (using a strain originally isolated from wilted lucerne, 1962), an orange pigmented colony, which had a partial requirement for biotin, developed on plates of complete medium spread with conidia irradiated with ultra-violet at a dosage allowing for approximately 5 per cent survival. The irradiation was carried out using a sterile distilled water suspension of conidia in an open Petri dish at a distance of 20 cm from a 2537 A ‘Hanovia’ bactericidal tube model 11 (with ozone reducing filter). The close resemblance of this colony (designated M.5) in morphological details to wild-type led us to conclude that it is a mutant rather than a chance contaminant. The results of a preliminary extraction of M.5 suggested that several different carotenoids were present, and from further work, reported here, we have been able to identify some of these pigments.

THE PROCUREMENT OF BIOCHEMICAL MUTANTS OF SACCHAROMYCES BY THE SYNERGISTIC EFFECT OF ULTRAVIOLET RADIATION AND 2,6-DIAMINO PURINE

Exposure of ultraviolet-irradiated, adenine-dependent haploid yeast to 2,6-diamino purine (an analogue of adenine) consistently gives rise to approximately 0.5% recessive biochemical mutants. This frequency is 22-fold higher than that obtained with either agent alone, and 150-fold higher than the spontaneous frequency. The distribution of mutants into nine discrete nutritional groups attests to the utility of the method for procuring relatively large numbers of different biochemical mutants. Several new gene markers for amino acid requirements are described, including one which controls an apparent single block for both lysine synthesis and respiratory competence.