Allgemeine Kontrolle der Aminosäurebiosynthese in Mutanten von Candida spec. EH 15/D

The general control of amino acid biosynthesis was investigated in Candida spec. EH 15/D, using single and double mutant auxotrophic strains and prototrophic revertants starved for their required amino acids. These experiments show that starvation for lysine, histidine, arginine, leucine, threonine, proline, serine, methionine, homoserine, asparagine, glutamic acid or aspartic acid can result in derepression of enzymes. A correlation was found between the degree of derepression, growth of strains, and concentration of required amino acids. The amino acids pcol pattern of mutants and revertants is different from that in the wild type strain.     

Regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae

Summary The threonine deaminase gene (ILV1) of Saccharomyces cerevisiae has been designated multifunctional since Bollon (1974) indicated its involvement both in the catalysis of the first step in isoleucine biosynthesis and in the regulation of the isoleucine-valine pathway. Its role in regulation is characterized by a decrease in the activity of the five isoleucine-valine enzymes when cells are grown in the presence of the three branched-chain amino acids, isoleucine, valine and leucine (multivalent repression). We have demonstrated that the regulation of AHA reductoisomerase (encoded by ILV5) and branched-chain amino acid transaminase is unaffected by the deletion of ILV1, subsequently revealing that the two enzymes can be regulated in the absence of threonine deaminase. Both threonine deaminase activity and ILV1 mRNA levels increase in mutants (gcd2 and gcd3) having constitutively derepressed levels of enzymes under the general control of amino acid biosynthesis, as well as in response to starvation for tryptophan and branched-chain amino acid imbalance. Thus, the ILV1 gene is under general amino acid control, as is the case for both the ILV5 and the transaminase gene. Multivalent repression of reductoisomerase and transaminase can be observed in mutants defective in general control (gcn and gcd), whereas this is not the case for threonine deaminase. Our analysis suggests that repression effected by general control is not complete in minimal medium. Amino acid dependent regulation of threonine deaminase is only through general control, while the branched-chain amino acid repression of AHA reducto isomerase and the transaminase is caused both by general control and an amino acid-specific regulation.     

General and specific controls of lysine biosynthesis in Saccharomyces cerevisiae

Summary Six of the eight enzymes of the -aninoadipate pathway for the biosynthesis of lysine in Saccharomyces cerevisiae were examined for repressibility to lysine and for susceptibility to the general control of amino acid biosynthesis. All of the enzymes exhibited a 2 to 4 fold lower level of specific activity in the wildtype strain X2180 when grown in lysine supplemented medium as compared to minimal medium. However, levels of only three of the enzymes, -aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase, were derepressed in the leaky lysine mutant 7305d and leaky arginine mutant 7853-6c when grown in minimal medium. These observations are characteristic of enzymes under general control of amino acid biosynthesis. The remaining three enzymes, homocitrate synthease, homoaconitase and homoisocitrate dehydrogenase were repressed in 7305d cells grown in minimal or lysine supplemented medium.     

Acid phosphatase deficient mutants of Schizosaccharomyces pombe are defective in tyrosine uptake

Summary The uptake of tyrosine and arginine into wild type and acid phosphatase deficient mutants (pho 1) of Schizosaccharomyces pombe was investigated. All 11 pho 1-alleles tested exhibited a reduced tyrosine uptake and impaired uptake cosegregated with the lack of acid phosphatase activity. Kinetic analyses using wild type cells grown in high phosphate medium (acid phosphatase repressed) and low phosphate medium (acid phosphatase derepressed) showed staturation kinetics for tyrosine with a K_M of about 2 × 10^–4 M for both media and a V of about 5 nmol min^–1mg^–1 and 2 nmol min^–1mg^–1 for derepressed and repressed cells respectively. The pho 1–118 strain completely lacked this saturable uptake system for tyrosine. Preliminary evidence suggests that tyrosine uptake may be via a general amino acid permease system and we conclude that mutations in the structural gene of acid phosphatase which abolish enzyme activity lead to a loss of this uptake system. In contrast to tyrosine, arginine uptake seems not to be significantly affected either by different acid phosphatase levels in wild type cells or by the pho 1–118 mutation.     

Genetic basis of destruxin production in the entomopathogen Metarhizium robertsii

Destruxins are among the most exhaustively researched secondary metabolites of entomopathogenic fungi, yet definitive evidence for their roles in pathogenicity and virulence has yet to be shown. To establish the genetic bases for the biosynthesis of this family of depsipeptides, we identified a 23,792-bp gene in Metarhizium robertsii ARSEF 2575 containing six complete nonribosomal peptide synthetase modules, with an N-methyltransferase domain in each of the last two modules. This domain arrangement is consistent with the positioning of the adjacent amino acids N-methyl-l-valine and N-methyl-l-alanine within the depsipeptide structure of destruxin. DXS expression levels in vitro and in vivo exhibited comparable patterns, beginning at low levels during the early growth phases and increasing with time. Targeted gene knockout using Agrobacterium-mediated transformation produced mutants that failed to synthesize destruxins, in comparison with wild type and ectopic control strains, indicating the involvement of this gene in destruxin biosynthesis. The destruxin synthetase (DXS) disruption mutant was as virulent as the control strain when conidial inoculum was topically applied to larvae of Spodoptera exigua, Galleria mellonella, and Tenebrio molitor indicating that destruxins are dispensable for virulence in these insect hosts. The DXS mutants exhibited no other detectable changes in morphology and development.     

Regulation of L-lysine biosynthesis in prototrophic revertants of Corynebacterium glutamicum

The regulation pattern of L-lysine biosynthesis has been investigated in protrophic revertant derived from an initially homoserine-auxotrophic strain of Corynebacterium glutamicum and, additionally, in antimetabolite-resitant mutants. The influence of regulatory important amino acids of the aspartate family on growth and L-lysine biosynthesis and especially on the activity of aspartate kinase was studied. Furthermore the activity of homoserine dehydrogenase in dependence on the growth phase was estimated. From a total number of ninety nine L-lysine forming, homoserine-prototrophic, and S-(2-aminoethyl)-L-cysteine-resistant (AEC) strains the best lysine producers were selected by an emerse cultivation assay as a preselection. Strain No. 132 forming 10 g · 1^?1 lysine was selected for the derivation of α-amino-β-hydroxyvaleric acid (AHV)- and lysine-resistant strains. From 121 double resistant mutants (hse^?rev, AEC^r, AHV^r) sixty two strains (=51.2%) were isolated producing lysine. The best producer, strain 132-IV-37, reached 19.8 g · 1^?1 after 8 optimization steps of the growth medium in comparison to 9.2 g · 1^?1 as the initial value. This higher potential for lysine synthesis in the mutant 132-IV-37 could be attributed to changed regulation of the homoserine dehydrogenase and aspartate kinase. As was shown by the action of threonine or threonine + lysine on the activity of aspartate kinase, a general desensibilization of this key enzyme exists in the mutant strain 132-IV-37, but not in the AEC-resistant parent strain. In addition to isoleucine none of several amino acids tested showed any significant influence on the aspartate kinase. This agrees with the increased lysine formation by this strain in comparison to the parent strain 132.     

Identification of the defects in the hemagglutinin gene of two temperature-sensitive mutants of A/WSN/33 influenza virus

Two temperature-sensitive mutants of WSN influenza virus, ts-61S and ts-134, possess defects in the hemagglutinin (HA) gene. These defects are characterized as a defective intracellular transport of the HA at the nonpermissive temperature and a marked thermolability. The nucleic acid sequences of the HA gene of these two viruses, as well as a series of revertant viruses, were determined. The deduced amino acid sequences demonstrate that the HA of ts-61S varied from the wild type protein by three amino acids while that of ts-134 differed by two residues. For both mutants, analysis of revertant viruses indicated that the phenotype of transport inhibition at the nonpermissive temperature and heat lability were associated with a single amino acid change in the globular portion of the molecule. In the case of ts-61S, the critical change in the HA was the replacement of a serine residue at position 110 with that of a proline. The mutational defect in the HA of ts-134 was due to the substitution of a tyrosine residue at position 159 with that of a histidine residue. Four of five revertants of ts-134 were suppressor revertants, of which some of the compensatory changes did not restore thermostability to the HA.     

[3H]Amino acid selection of aminoacyl-tRNA synthetase mutants of CHO cells: Evidence of homo- vs. hemizygosity at specific loci

Further application of the tritiated amino acid suicide selection procedure, designed to select conditional protein synthesis mutants, has yielded over 300 aminoacyl-tRNA synthetase mutants of Chinese hamster ovary cells, representing seven complementation groups. A number of these mutants and their revertants were used to examine the extent of X-chromosome linkage or other functional hemizygosity in CHO cells. Our results indicated that none of the seven different aaRSloci tested was X linked, and the the glnRSlocus, unlike the asnRSand leuRSloci, does not appear to be functionally hemizygous in CHO cells.     

Analysis of GPT activity in mammalian cells with a chromosomally integrated shuttle vector containing alteredgpt genes

The molecular mechanisms of reversion in mammalian cells were studied utilizing the pZipGptNeo shuttle vector, with the bacterialgpt gene in the vector integrated into the chromosomal DNA of mouse cells. From mutant cell lines containinggpt genes with single base changes, revertants were selected for the reappearance of GPT activity. The copy number and expression of thegpt genes in such revertants were analyzed, and the GPT activity encoded by revertant genes in both mammalian cells and bacteria characterized. Revertants with wild-type amino acid sequence had, on average, the highest levels of GPT activity. Revertants with amino acid sequences different from the original mutants but not corresponding to wild-type had, on average, approximately half the level of GPT activity as wild-type revertants. Revertants that still contained the original mutation in thegpt gene had even lower levels of activity. These revertants were found to have amplified mutantgpt genes, which, when transferred into bacteria, were seen to encode for GPT polypeptides with partial enzymatic activity. A revertant in which the original mutation that destroyed the AUG translational start codon was retained but in which there was a secondary mutation upstream of the start codon also was characterized. The second mutation generated an in-frame CUG codon that apparently functioned as an alternative, upstream translational start codon.     

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids

The effects of various amino acids on growth and heterocyst differentiation have been studied on wild type and a heterocystous, non-nitrogen-fixing (het⁺ nif^?) mutant of Anabaena doliolum. Glutamine, arginine and asparagine showed maximum stimulation of growth. Serine, proline and alanine elicited slight stimulation of growth of wild type but failed to show any stimulatory effect on mutant strain. Valine, glutamic acid, iso-leucine and leucine at a concentration of as low as 0.1 mM were inhibitory to growth of parent type. Methionine, aspartic acid, threonine, cysteine, and tryptophan did not affect growth at concentrations lower than 0.5 mM. But at 1 mM, these amino acids were inhibitory. In addition to the stimulatory effects of glutamine, arginine and asparagine, the heterocyst frequency was also repressed by these amino acids. Glutamine and arginine at 2 mM completely repressed heterocyst differentiation in the mutant strain; however, other amino acids failed to repress the differentiation of heterocysts. Our results suggest that glutamine and arginine are utilized as nitrogen sources. This is strongly supported from the data of growth and heterocyst differentiation of mutant strain, where at least with glutamine there is good growth without heterocyst formation. Studies with glutamine and arginine on other N₂-fixing blue-green lagae may reveal the regulation of the heterocyst-nitrogenase sub-system.     

Regulation of chorismate mutase,prephenate dehydrogenase and prephenate dehydratase of Candida maltosa

The enzymes of the phenylalanine-tyrosine pathway were partially purified from Candida maltosa and the regulatory patterns were established. Chorismate mutase (Mr 63,000), prephenate dehydrogenase (M_r 75,000) and prephenate dehydratase (M_r 88,000) were separated from each other. The formation of chorismate mutase was only regulated by a general control of amino acid biosynthesis, whereas the synthesis of prephenate dehydrogenase and prephenate dehydratase was constitutive. Both chorismate mutase and prephenate dehydrogenase were activated by tryptophan and by methylated or fluorinated tryptophan analogues. l-tyrosine as well as α-methyl-dl-tyrosine inhibited the activity of both enzymes. Prephenate dehydratase activity was stimulated by l-tryptophan and by methylated tryptophan derivatives and inhibited by d-tryptophan or 5-fluorotryptophan. In contrast to chorismate mutase, the double-reciprocal curves of substrate saturation of which were hyperbolical (positive cooperativity), prephenate dehydrogenase and prephenate dehydratase showed linear curves of substrate saturation, indicating normal Mi-chaelis kinetics.     

Use of an aberrant polypeptide as a marker in three-factor crosses: Further evidence for independent reassortment as the mechanism of recombination between temperature-sensitive mutants of reovirus type 3

The aberrant μ1 and μ2 viral polypeptide phenotype (designated μ^??) of certain groups of temperature-sensitive mutants of reovirus type 3 has been utilized as a third unselected genetic marker in three-factor crosses. Approximately 50% of the ts⁺ recombinant clones were found to express the wild type μ⁺ phenotype of one of the parents, while the remainder expressed the aberrant μ^?? phenotype of the other. The approximately random distribution of the μ phenotype in two crosses of this type supports the conclusion that recombinants which result from mixed infection with pairs of temperature-sensitive mutants of reovirus type 3 are generated by random assortment of RNA segments. The results of three-factor crosses, furthermore, suggest that the ts mutation of the group A mutant (ts201) is genetically linked to the determinant of the aberrant μ1 and μ2 polypeptide.In addition, ts⁺ revertants of groups B, D, and G retain the μ phenotype of the mutant strain from which they are derived, while the group A (ts201) revertants display a variety of phenotypes.     

Nutzung von Gen5 und Gen6 ts-Mutanten des Phagen T3 für die Anzeige von Hemmstoffen der DNS-Synthese

In an enzyme-specific drug screening system nalidixic acid and 3'FTdR, inhibitors of DNA synthesis, both reduce the growth of wild type and temperature-sensitive point mutants of phage T3 with different efficiencies. The wild type shows the strongest sensitivity against the drugs, while an exonuclease mutant is the most insensitive variant. The DNA polymerase mutants exhibit an intermediate degree of inhibition. The anthracycline antibiotics violamycin BI and adriblastin which preferentially inhibit RNA synthesis show the same degree of inhibition for all mutants. This is true also for the RNA synthesis inhibitor lambdamycin, which is identical with chartreusin. The protein synthesis inhibitors chloramphenicol and o-phenanthroline, a chelating agent, impair all mutants to the same extent. Our data confirm the hypothesis that structural variants of essential viral enzymes, when compared with the wild type should sensitivities against specific inhibitors and show that this T3 system could be used for the indication of specific inhibitors of DNA synthesis.     

Starvation for a specific amino acid induces high frequencies of rho− mutants in Saccharomyces cerevisiae

Auxotrophic yeast cells were starved on solid media for their respective essential amino acid in the course of “adaptive mutation” experiments. Thereby, high proportions of mitochondrially respiratory deficient (rho⁻) mutants accumulated among the cells stressed on selective plates. Using a strain with a plus-four frameshift mutation in a chromosomal gene involved in lysine biosynthesis, we observed that many of the revertant colonies which arose late under the selective pressure were composed of mixtures of rho⁺ and rho⁻ cells, indicating that they originated from founder cells containing intact as well as defective mitochondrial genomes. We show that in spite of the slower growth of rho⁻ cells the late-appearing colonies cannot be interpreted as descending from rho⁻ revertants present before selective plating. Received: 7 November 1996 / 2 February 1997     

Chloroplast gene suppression of defective ribulosebisphosphate carboxylase/oxygenase in Chlamydomonas reinhardii: evidence for stable heteroplasmic genes

Summary The rcl-u-1-18-5B chloroplast mutation results in the absence of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) holoenzyme in the green alga Chlamydomonas reinhardii. The 18-5B mutant strain lacks photosynthesis and displays alight-sensitive, acetate-requiring phenotype. In the present investigations, revertants of 18-5B were recovered that regained photosynthetic competence. These revertants have decreased levels of Rubisco holoenzyme relative to wild type and display heteroplasmicity, segregating wild-type (revertant) and acetate-requiring phenotypes during vegetative growth or through meiosis. One of these revertants, R10-I, was studied further. The heteroplasmicity associated with photoautotrophically-grown R10-I was found to be stable through subcloning and heritable through several crosses. During growth in acetate medium in the dark, where photosynthesis provides no selective advantage, the wild-type phenotype was lost. Acetate-requiring segregants became homoplasmic but wild-type segregants did not. Organellar intergenic-suppression is discussed in light of the observed stable heteroplasmicity.     

Structural characterization of <Emphasis Type="Italic">Tobacco etch virus</Emphasis> coat protein mutants

Summary. The assembly of Tobacco etch potyvirus (TEV) coat protein (CP) and truncated mutants in Escherichia coli was studied. CP from which 28, 63 or 112 amino acids were deleted from the N-terminus polymerized into potyvirus-like particles (PVLPs). These structures were more rigid and progressively smaller in diameter than those produced by full length TEV-CP. CP from which 175 N-terminal amino acids were removed, failed to polymerize. A fragment containing amino acids 131 to 206 of TEV-CP is sufficient for PVLP assembly in E. coli.To determine the function of the highly conserved amino acids Ser152, Arg154, and Asp198 point mutants were generated. The mutant CP63(Asp198Glu) exhibited different spectral properties following circular dichroism analysis showing a lower amount of -helix compared to the wild type molecule. No differences were observed in spectra obtained from fluorescence spectroscopy. The point mutants bind RNA in vitro to the same degree as the wild type protein. However, while the wild type and the Arg154Gln mutant CP were each able to form PVLPs in E. coli, the Asp198Glu and the double mutant Ser152Pro/Arg154Gln mutants did not. These results suggest that the Asp198Glu mutation has an altered secondary structure which affects the capacity of the protein to polymerize but did not affect in vitro protein-RNA interactions.     

Some characteristics of p-fluorophenylalanine-resistant mutants of Penicillium cyclopium

p-Fphe-resistant mutants of Penicillium cyclopium Westling were selected by the gradient method of SZYBALSKI . The strains grow well in the presence of 1 mg p-Fphe/ml¹) nutrient agar. However, sporulation decreases considerably after repeated subcultivation on this medium. Drug resistance in the mutant strains seems to depend on reduced p-Fphe incorporation into proteins. Radioactivity of ³H-p-Fphe added to cultures is incorporated into the proteins of the mutants at a lower rate compared with the wild type strain. Furthermore, in spite of the fact that also in the wild type strain part of the p-Fphe is transformed into phe, the ratio of ³H-p-Fphe to ³H-phe in the proteins after feeding of labelled p-Fphe is lower in the mutant. Addition of 100 μg/ml of p-Fphe to the nutrient solution of the strain res-p-Fphe 11 does not influence hyphal growth but reduces alkaloid production and sporulation. Thus processes of cell specialization in P. cyclopium, as in other microorganisms, are more sensitive to the synthesis of abnormal proteins than processes of basic growth. Simultaneous feeding of p-Fphe and tyr increases the p-Fphe effects due to a stimulated uptake of the drug. In the p-Fphe-resistant mutants the internal level of free phe exceeds that of the wild type strain. Also phe and a small amount of tyr is excreted into the nutrient solution. In spite of the fact that external phe added to the cultures under certain conditions increases alkaloid production in P. cyclopium, mutants and wild type strains produce the same alkaloid amount. This result is discussed with regard to the cellular compartmentation of phe.     

Comparative studies on soluble protein profiles and isozyme patterns of seven Trichinella isolates

Soluble protein profiles and isozyme patterns of eight enzymes were compared for extracts of muscle stage larvae of the seven Trichinella isolates, using isoelectric focusing in polyacrylamide gel. Soluble protein profiles and isozyme patterns of four enzymes: malic enzyme, glucose-phosphate isomerase, phosphoglucomutase, superoxide dismutase of them were clearly divided into four types. T. pseudospiralis from a racoon and the Polar strain from a polar bear formed type 1 and type 2. The Iwasaki strain from a Japanese black bear and the Yamagata strain from a racoon dog, both from Japan, were type 3. Type 4 consisted of three remaining strains, the Polish strain from a wild pig, the USA strain from a pig and the Thai strain from a human case, which have similar infectivities to pigs. The Thai strain varied a bit electrophoretically from other members of type 4. Zymograms of adenylate kinase and malate dehydrogenase were similar in types 2 and 3. The 6-phosphogluconate dehydrogenase zymogram of type 3, similar to that of type 4, was different from that of type 2. It is assumed from the data that type 3 (Japanese strain) was genetically intermediate to types 2 and 4. T. pseudospiralis and the Polar strain had a common main isozyme of 6-phosphogluconate dehydrogenase. The zymogram of lactate dehydrogenase was common except for T. pseudospiralis.     

RhodosporidiumBANNO: Dosiseffektbeziehungen,Mutageneffektivität und Mutantenspektrum bei der Induktion Auxotrophie verursachender Mutationen durch ultraviolettes Licht und N-Methyl-N′-nitro-N-nitrosoguanidin

The kinetics, efficiency, and specificity of induction of forward mutations to auxotrophy by ultraviolet light (UV) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was examined in stationary phase cells of Rhodosporidium (Rhodotorula) wild strain Rgl. In comparison to the spontaneous level the frequency of auxotrophic mutants was increased more than 1000 times by both mutagens, however, the mutagenic efficiency of MNNG was higher than that of UV. We found that the forward mutation rate is a linear function of the applicated UV and MNNG doses in the range to 600 J m^?2 or 25 mm x min, respectively. The 35 studied biosynthetic pathways to amino acids, purines, pyrimidines, and vitamins are genetically blocked at different frequencies, but there is not any significant difference between UV and MNNG induced frequencies of mutants with a specific requirement. However, in difference to the approximately equal distribution of the MNNG-induced nic mutants among the genetic blocks of the tryptophan-nicotinamide pathway, UV-induced nic mutants occurred with a higher frequency in the genes of the tryptophan pyrrolase and the 3-hydroxykynureninase than in the genes of the other enzymes of the pathway.     

Common virulence factors for Pseudomonas tolaasii pathogenesis in Agaricus and Arabidopsis

Brown blotch of cultivatable mushrooms is a disease caused by the small peptide toxin (tolaasin) secreted by Pseudomonas tolaasii. Here we found that the wild type tolassin-producing P. tolaasii stain 6264 was capable of infection in Arabidopsis thaliana cotyledons, causing chlorotic symptoms and growth arrest as a result of bacterial proliferation. Seven virulence-attenuated mutants of P. tolaasii were isolated from the Agaricus bisporus screen using 2512 mariner-based transposon insertion mutants, and all of them displayed reduced virulence and bacterial proliferation in Arabidopsis infection as well. The transposon was inserted within the genes for tolassin biosynthesis and amino acid biosynthesis, and within an intergenic region between the genes of unknown function. The finding that some virulence factors are commonly required for both Agaricus and Arabidopsis infections suggests that Arabidopsis could be exploited to study the host–pathogen interaction involving P. tolaasii.