Adenine-requiring Mutants Research Articles

Generation of a Ustilago maydis ade2 Mutant

The need exists to create the auxotrophic mutants for basidiomycete Ustilago maydis to allow selection of gene transformants in minimal growth media. The ADE2 gene was identified by homology with Saccharomyces cerevisiae. Adenine-requiring auxotrophic mutants were effectively created by homologous recombination in protoplasts using a standard plasmid containing the ADE2 locus interrupted by a phleomycin resistance gene, selected on standard complex media. The knockout ade2 mutant produced grows on minimal, defined medium only if supplemented with adenine.

An abnormal cell division cycle in an AIR carboxylase-deficient mutant of the fission yeast Schizosaccharomyces pombe

Adenine-requiring mutant strains of S. pombe enter the stationary phase after depleting a culture medium of adenine or its analogues. Stationary phase cells of six mutants defective at different stages of the purine nucleotide synthetic pathway were examined for cell volume and DNA content, and then compared in these respects with those of a prototrophic wild-type strain. The cell cycle of the wild-type strain was arrested in the G2 phase (2C state) in the nitrogen rich medium, as is evident from DNA content per cell (0.0425 pg) and cell volume (47.7 microns 3). An AIR carboxylase-deficient (ade6) mutant strain was found to have an unusual cell volume (307.4 microns 3) and DNA content (0.1187 pg). By DAPI fluorescence microscopy, each mutant cell was seen to contain only one enlarged nucleus, which indicates the absence of cell populations containing cells in the 4C state of the S phase following nuclear division. It then follows that in ade6 mutant cells, DNA synthesis occurs in the absence of a completed nuclear division. Thus in S. pombe cells, the completion of nuclear division is not necessarily required for the next cycle initiation of DNA synthesis under certain physiological conditions.

Changes in ribosomal properties during adenylate deprivation in the cells of Kluyveromyces lactis.

In an adenine-requiring mutant strain of the yeast, Kluyveromyces lactis, the intracellular content of ATP is one-third to one-fifth that in a prototrophic wild strain under growing conditions. The quantitative differences becomes rather small in resting stationary-phase cells. Temporary changes in the two-dimensional protein patterns of mutant ribosomes occur when the ATP content is lowest during the transition phase of growth. The transfer of exponentially growing cells to a synthetic complete medium void of adenine induces the same changes in mutant ribosomes within several hours. Identification of ribosomal proteins by two-dimensional gel electrophoresis indicated all changeable proteins (at least five proteins) to belong to 40S ribosomal subunits. The mutant ribosomes prepared from the transition-phase cells have much lower activity (below 60%) for poly(U)-directed polyphenylalanine synthesis than those in exponentially growing or resting stationary-phase cells. Thus, changes in ribosomal components associated with the differences in ribosome activity in a cell-free system were noted in the adenylate-deprived cells of K. lactis.

Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene.

Candida albicans is a diploid dimorphic yeast with no known sexual cycle. The development of a DNA transformation system would greatly improve the prospects for genetic analyses of this yeast. Plasmids were isolated from a Candida Sau3A partial library which complements the ade2-1 and ade2-5 mutations in Saccharomyces cerevisiae. These plasmids contain a common region, part of which, when subcloned, produces ade2 complementation. Among the small number of auxotrophs previously isolated in C. albicans, red adenine-requiring mutants had been identified by several groups. In two of these strains, the cloned Candida DNA transformed the mutants to ADE+ at frequencies of 0.5 to 5 transformants per micrograms of DNA. In about 50% of the transformants, plasmid DNA sequences became stably integrated into the host genome and, in the several cases analyzed by Southern hybridization, the DNA was integrated at the site of the ADE2 gene in one of the chromosomal homologs.

Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene.

Candida albicans is a diploid dimorphic yeast with no known sexual cycle. The development of a DNA transformation system would greatly improve the prospects for genetic analyses of this yeast. Plasmids were isolated from a Candida Sau3A partial library which complements the ade2-1 and ade2-5 mutations in Saccharomyces cerevisiae. These plasmids contain a common region, part of which, when subcloned, produces ade2 complementation. Among the small number of auxotrophs previously isolated in C. albicans, red adenine-requiring mutants had been identified by several groups. In two of these strains, the cloned Candida DNA transformed the mutants to ADE+ at frequencies of 0.5 to 5 transformants per micrograms of DNA. In about 50% of the transformants, plasmid DNA sequences became stably integrated into the host genome and, in the several cases analyzed by Southern hybridization, the DNA was integrated at the site of the ADE2 gene in one of the chromosomal homologs.

Genetic analysis of red, adenine-requiring mutants of Candida albicans.

A number of investigators have described the isolation of red, adenine-requiring mutants of Candida albicans. Other fungi have been shown to give rise to two phenotypically similar, but genetically distinct, types of red, adenine-requiring mutants. This paper is the first indication that the red adenine mutants of C. albicans can similarly be resolved into two distinct classes. It is also believed to be the first report of such a resolution in an imperfect fungus. The resolution of these two classes was achieved by applying three distinct parasexual, analytical methods to this imperfect, naturally diploid yeast. The methods employed were complementation analysis of fused protoplasts and two methods of recombination analysis, induced mitotic crossing over in heterozygous revertants and induced mitotic crossing over in the heterozygous tetraploid products of protoplast fusion. The recombination methods depended on linkage analysis between the ade loci and two loci, met1 (methionine) and arg1 (arginine). The three analytical methods supported the same resolution. The results support the generally accepted view that C. albicans is diploid since they indicate disomic inheritance at the ade1, ade2, and met1 loci.

Growth and Sporulation of Auxotrophs of Bacillus subtilis in a Medium with Limited Nutrients

Growth and sporulation were examined for 30 auxotrophs of Bacillus subtilis in a chemically defined medium with suboptimal amounts of nutrients. All strains except for some adenine-requiring mutants could not overtake sporulation stage II when amino acids, vitamins, or bases were limited, whereas they sporulated fairly well without limitation. Abnormal structures, a cell with thickened cell wall and a cell with several refractile bodies, were found in some strains after the vegetative growth stopped.

Catabolite repression of hydrolases in Aspergillus niger

A comparative study was made on catabolite repression of acid protease and polygalacturonase in an adenine-requiring mutant of Aspergillus niger. Both enzymes are inducible and accumulated extracellularly after cessation of growth caused by adenine starvation. Addition of glucose, fructose, or intermediates of glycolysis, but not tricarboxylic acid cycle intermediates, depressed the formation of both enzymes. Catabolite repression of polygalacturonase by glucose occurred quickly and was almost complete. However, acid protease was only partly repressed even after several hours. Formation of polygalacturonase in the presence of actinomycin S3 or during deinduction was repressed by glucose, whereas that of acid protease was not. In the course of induction of acid protease by peptone, glucose did not affect the length of the induction lag period and the rate of acid protease formation decreased by glucose was not restored by the addition of large amounts of inducer. From these results, we suggest that catabolite repression of polygalacturonase occurs at the level of translation and that of acid protease at transcription.

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism. VI. Enzymatic studies of two mutants unable to convert inosinic acid to adenylic acid.

Ade-H and ade-I are two auxotrophic mutants of Chinese hamster ovary (CHO-K1) cells which specifically require adenine as the purine source to grow. The enzymatic defects of these mutants were examined in cell-free extracts. It was found that ade-H did not have any detectable adenylosuccinate synthetase activity and ade-I was defective in the adenylosuccinate lyase enzyme. The relevance of adenine-requiring mutants to the study of the regulation of purine metabolism in mammalian cells is discussed.

Cell wall analysis of an adenine-requiring mutant of the yeast-like form of Paracoccidioides brasiliensis strain IVIC Pb9.

An adenine-requiring mutant of Paracoccidioides brasiliensis strain IVIC Pb9 was isolated after treatment of the yeast-like (Y) form with nitrosoguanidine. Cell wall analysis of this mutant (strain IVIC Pb141) showed an increase in the amount of alpha-1,3-glucan and a virtual disappearance of the antigenic galactomannan. At the same time, a higher degree of virulence was observed for the mutant. These results agree with the hypothesis presented before (16) about a relationship between cell wall polysaccharides and pathogenicity in P. brasiliensis.

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism. IV. Isolation of a mutant which accumulates adenylosuccinic acid and succinylaminoimidazole carboxamide ribotide

The production, isolation, and characterization of a new complementation group (Ade-I) of adenine-requiring mutant of Chinese hamster cells (CHO-K1) is described. This mutant accumulates two intermediates of purine biosynthesis, both of which contain an aspartate moiety. One of these is shown to be adenylosuccinic acid (AMPS) by chromatographic analysis, while evidence is presented that strongly suggests the other intermediate is succinylaminoimidazole carboxamide ribotide (SAICAR). Thus, Ade-I is most likely lacking the activity of the enzyme adenylosuccinase (EC 4.3.2.2). The use of this and similar mutants for the analysis of regulation of purine biosynthesis in mammalian cells is discussed.

Mutagenicity of actinomycin D in Neurospora crassa

Actinomycin D is known to bind to native DNA and is widely used as an antineoplastic agent and inhibitor of DNA-dependent RNA and protein synthesis. We report here the induction by actinomycin D of purple adenine-requiring mutants ( ad-3) in wild-type Neurospora crassa. A significant increase in the frequency of ad-3 mutants was evident when the organism was grown vegetatively in the presence of actinomycin D; the mutation frequency was at least 3.6 per 10 6 survivors. The actinomycin D-induced ad-3 mutants were 29% ad-3A and 71% ad-3B. The ad-3B mutants were classed by complementation pattern as 25% nonpolarized complementing; 14% polarized complementing; and 61% noncomplementing. The spectrum of complementation types of the actinomycin D-induced mutants most closely parallels that of mutants induced by ICR-170, known to induce base-pair insertions or deletions, or that of X ray-induced or spontaneous mutants. It is significantly different from spectra seen following treatment with nitrous acid or N-methyl- N′-nitrosoguanidine, agents known to induce mainly base-pair substitutions.

Relation of growth and protein synthesis to the adenylate energy charge in an adenine-requiring mutant of Escherichia coli.

When Escherichia coli K-12 strain PC 0294, which is unable to synthesize adenine nucleotides, thereonine, proline, and leucine, was starved for adenine, the concentrations of ATP, ADP, and AMP fell rapidly. The adenylate energy charge was not affected until the adenine nucleotide pool fell to about 30% of its normal value. Similarly, cells in an adenine-limited chemostat grew at intracellular adenine nucleotide pool values as low as 30% of normal, but the energy charge in all cases was approximately 0.90. Incorporation of [14C]leucine into protein in adenine-starved cells continued rapidly as long as the energy charge was in or near its normal range, even when the concentration of ATP was 30% to 10% of its normal value. When glucose was added to cells that had been resuspended in medium lacking both glucose and adenine, the energy charge rose rapidly but the concentration of ATP fell, presumably because of nucleic acid synthesis. The rate of [14C]leucine incorporation into protein rose rapidly while adenine nucleotide concentrations fell, and then declined roughly in parallel with the energy charge. At a given value of energy and of total concentration of adenine nucleotides, the rate of protein synthesis may, of course, vary with the concentrations of precursors and modifiers; thus, the rate cannot be predicted from knowledge only of the energy charge. Our results suggest, however, that the rate of protein synthesis and the capacity for growth are much more sensitive to changes in the value of the energy charge than to changes in the concentration of ATP. Growth occurs when the ATP concentration is reduced to one-third of its normal value, but has not been observed when the energy charge has fallen by as much as 10%.

Temperature-sensitive, osmotic-remedial mutants of Neurospora crassa: Osmotic pressure induced alterations of enzyme stability

Three temperature-sensitive, adenine-requiring mutants of Neurospora crassa were found to be osmotic-remedial when non-penetrating solutes were used to increase the osmolarity of the growth medium. The affected enzyme (adenylosuccinase) from one of the mutants (ad-4, 44206t) was found to have higher levels of activity when the organism was grown at non-permissive temperatures under osmotic-remedial conditions than when it was grown with adenine as a nutritional supplement. The enzyme synthesized at 30 degrees C in the presence of adenine exhibited increased sensitivity to inhibition by high salt concentrations and a lowered stability toward heat denaturation, indicating that the remedial effect may be the result of changes in the physical properties of the enzyme molecule. Temperature shift experiments indicate that the enzyme which is synthesized at permissive temperatures or under osmotic-remedial conditions is also stable in vivo under non-permissive conditions. This suggests that the critical period for temperature sensitivity, and conversely osmotic remediability, may be during protein synthesis or during the conformational folding of the protein.

Genetic transformation in yeasts.

SUMMARY: The transformability of 25 strains of yeasts belonging to 23 species in three genera - Saccharomyces, Hansenula and Candida- was investigated with respect to ten characters. Eighteen hour cultures at about the end of the log phase of growth, addition of 10 μg DNA/ml, and an incubation time of 30 min at 30 to 37 °C usually yielded best results. Of the 109 pairs of strains investigated 11 yielded positive results, which included intraspecific, interspecific and intergeneric transformation. Three adenine-requiring mutants of S. cerevisiae were transformed to prototrophy with the DNA of S. cerevisiae var. ellipsoicleus. DNA of the latter strain also transformed three other species of Saccharomyces. In Candida, reciprocal transformation was observed between C. utilis and C. lipolytica. Hansenula petersonii transformed H. jadinii to rhamnose utilization. In intergeneric experiments, H. mrakii was transformed with S. cerevisiae var. ellipsoicleus DNA and C. utilis with the DNA from H. petersonii. Attempts at transformation between Saccharomyces and Candida were unsuccessful. The GC content of DNA from the strains which yielded positive results varied from 36·0 to 37·3 mol %. The implications of these findings for the inter-relationships and taxonomy of yeasts are discussed.

Genetics of somatic mammalian cells: biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism.

Studies are presented on the biochemical genetics of 30 adenine-requiring mutants of the Chinese hamster ovary cell which were induced by mutagenesis and selected by the BrdU-visible light technique. Representative experiments conducted with these mutants include: hybridization with each other; hybridization with normal human cells; nutritional analysis; biochemical analysis with radioactively labeled intermediates; and measurement of reversion frequencies to wild-type phenotype occurring spontaneously and under the influence of selected mutagens. All mutants behave as if having point mutations. These experiments provide information relevant to the determination of dominant-recessive relationships, resolution into different complementation classes, localization of the human chromosomes which carry human genes required by the individual mutants, determination of the point of metabolic block for different mutants, and elucidation of the nature of the underlying DNA changes. These experiments illustrate the range of biochemical-genetic studies now possible with such a family of somatic mammalian cell mutants in vitro. Possible application to problems of human genetic disease are indicated.

Identification of chick chromosomes in cell hybrids formed between chick erythrocytes and adenine-requiring mutants of Chinese hamster cells.

When chick erythrocytes were fused with adenine-requiring mutants of Chinese hamster cells of two different complementation classes and grown in adenine-free medium for 1 week or longer, mitotic cells were observed containing both chick and Chinese hamster metaphase chromosomes within a single cell. Stable hybrid clones were subsequently isolated that possessed, in addition to a complete Chinese hamster genome, a single specific chick chromosome. Hybrids resulting from fusions involving different adenine mutants retained different, identifiable chick chromosomes. Thus, the two chick genes for the endogenous synthesis of adenine were assigned to the respective chick chromosomes.

Conversion of Purine Bases and Their Ribosides to 5'-Inosinic Acid by Bacillus subtilis

Among various nutritional mutants with weak 5'-nucleotidase derived from Bacillus subtilis IAM 1145, the adenine-requiring mutants could convert exogenously added hypoxanthine, guanine, xanthine and their ribosides to 5' inosinic acid (IMP) and accumulate it in the medium. Synthesis of IMP from purine derivatives was observed predominantly in an early stage of the cultivation. The conversion was stimulated by Fe2+ or Mn2+, and markedly depressed by an excess amount of adenine in the production-medium.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes IX. Excess production of photoreactivating enzyme in E. Coli B s-1-160 under different growth conditions, and its suppression by adenine

Summary The extent of photoenzymatic repair resulting from a single light flash is determined by the number of photoreactivating enzyme (PRE) molecules present within cells. This permitted us to study the dependence of PRE production on growth conditions in E. coli B s-1 -160 (an adenine-requiring mutant of strain B s-1 with about a 6-fold increased PRE content), and to compare it with that of the parental strain B s-1 . The excess PRE production was observed only in B s-1 -160 cells entering the stationary phase; their PRE content during exponential growth differed little from that in B s-1 . Supplementation of broth with adenine at concentrations from 40 to 200 μg/ml progressively decreases the amount of PRE present in stationary phase B s-1 -160 cells, but does not affect the PRE content of B s-1 cells. The same effect was observed with several adenine analogues and related substances, some of them unable to support growth of B s-1 -160 in minimal media. The results indicate that excess PRE production occurs only under conditions of severe adenine shortage, i.e. when growth of adenine-requiring cells comes to a halt due to exhaustion of the adenine supply. This suggests that adenine or some related compound is involved in a regulatory mechanism responsible for the low level of PRE production that is characteristic of E. coli .

Conversion of Hypoxanthine Derivatives to Succinyl Adenine Derivatives by Bacillus subtilis

A mutant strain HA-2 was derived from an adenine-requiring mutant of Bacillus subtilis IAM 1145 by transduction with bacteriophage SP-10 and subsequent N-methyl-N′-nitro-N-nitrosoguanidine treatment. The strain could convert exogenously supplemented hypoxanthine or inosine to succinyladenine, succinyladenosine and succinyladenosine 5′-monophosphate (succinyl AMP), and accumulate these compounds in the culture fluid. The strain, however, failed to convert exogenously supplemented inosine 5′-monophosphate to these succinyl compounds. The yields of succinyladenine, succinyladenosine and succinyl-AMP in a production medium supplemented with 17 mm of hypoxanthine were about 4.4 mm, 5.2 mm and 2.2 mm, respectively, after 4 days growth.