Biochemical Mutants Research Articles

Assay of xylanolytic activity of mutants of Neurospora crassa (Ema 5297) induced with ultra-violet radiation

Mutation was induced in Neurospora crassa (Ema 5297) with UV (having the wave length of 254 nm) irradiation. Three groups of biochemical mutants (leucine, tryptophan and arginine) were obtained. Ema and 7 leucine mutants were used to evaluate whether mutation has any considerable effect for the production of such enzyme. Enzymatic activity was expressed as International Unit (IU). It was found that N. crassa mycelia secrete xylanases to the culture supernatant. Mutant Y10 showed highest xylanase activity (1.663 IU) and wild N. crassa (Ema) showed lowest (0.416 IU) activity.

Recombination in crosses between biochemical mutants of Coprinus lagopus

Recombination in crosses between biochemical mutants of Coprinus lagopus

Repair of CAG/CTG repeat loop in human cells

Trinucleotide repeat (TNR) expansion is the genetic basis for certain neurological and neurodegenerative disorders, including Huntington's disease. Since TNR sequences, e.g., (CAG)n, form a loop or hairpin structure, DNA loop repair is considered a major mechanism for TNR stability. However, the molecular mechanisms of DNA loop repair and protein components involved in this pathway are poorly understood. Using a biochemical approach, where human nuclear extracts were examined for their ability to process a plasmid DNA containing a (CAG)25 or (CTG) 25 loop, we show here that repair of CAG or CTG loop in human cells occurs in a nick‐ or loop‐directed manner. The repair is initiated by an incision at the top of the loop, followed by additional incision and excision, limited repair DNA synthesis, and ligation. Several biochemical mutants derived from fractionations allow us to demonstrate that PCNA, RPA, and FEN1 are essential components involved in TNR loop repair. We also show that MutSβ, a mismatch recognition protein that was previously thought to promote CAG expansion in transgenic mouse model by binding to the CAG loop and inhibiting its repair, has no inhibitory effect on TNR loop repair in human extracts, suggesting that the transgenic animal model may not reflect TNR instability in human cells. Our findings provide molecular insight into the mechanism by which cells maintain TNR stability.

ARE THE STRUCTURAL CHANGES IN ADULT DROSOPHILA MUSHROOM BODIES MEMORY TRACES? STUDIES ON BIOCHEMICAL LEARNING MUTANTS

SUMMARYThe pre-imaginal development of Drosophila mushroom bodies is under the influence of an unknown variable which causes populations of wild-type flies at eclosion to differ in the average number of Kenyon cell fibers. During the first week of adult life the number adjusts to an intermediate level which depends upon the experience of the flies. Under olfactory deprivation or social isolation it reaches a lower level than under favorable rearing conditions (J. Neurogenet., 1 (1984) 113–126). The biochemical learning mutants dunce and rutabaga show no experince-dependent modulation of fiber number (Fig. 2). In both strains the mushroom bodies of young adults seem to develop abnormally; in dunce a loss of aboout 600 fibers is observed, in rutabaga fiber number is low at eclosion and does not increase (Fig. 1a). The following model for long-term memory is proposed: in mushroom bodies outgrowth and decay of Kenyon cell fibers occur simultaneously. The fibers randomly form transient synapses onto extrinsic output neurons of the mushroom bodies and receive synapses from modulating neurons. Experience consolidates certain synapses, thus prolonging survival of the respective Kenyon cell fibers and increasing the steady state level of fiber number (Fig. 3).

HTLV-1 p12I and p30II proteins in viral persistence and pathogenesis

The TP53 gene encodes a nuclear protein implicated in the regulation of the cell cycle, DNA repair, and apoptosis. TP53 mutations and other alterations have been described in numerous types of tumors, and some of these have been associated with poor prognosis. Some reports in the literature have indicated a relationship between TP53 status and prognosis especially in non-small-cell lung cancer, breast cancer and NHL. In order to characterize these molecular abnormalities and their clinical significance in prognosis, we also have analyzed the possible correlation between mutations in TP53 gene, clinical findings, response to chemotherapy and survival in 49 children of our series. The mutations of TP53 gene were analyzed by single-strand conformation polymorphism analysis (SSCP) of exons 5 through 9 and direct sequencing. Mutations of TP53 were detected in 11 of 49 (22.5%) patients and more specifically in 20% of Burkitt’s lymphoma. No significant correlation was found regarding age, gender, clinical stage and LDH level and TP53 gene mutations. The comparison of EFS curves using the Log-Rank test were also not significant. However, the analysis of the effects of mutations on the core p53 structure identified biological and biochemical mutants with phenotypes probably related to different response to chemotherapy. Our data suggest that some types of mutants can alter the protein distinctly and may be associated with a more aggressive phenotype.

Intergeneric somatic hybridization and its application to crop genetic improvement

Related or distant species of cultivated cs are a large pool of many desirable genes. Gene transfer from these species through conventional breeding is difficult owing to post- and pre-zygotic sexual incompatibilities. Somatic hybridization via protoplast fusion is a possible alternative for gene transfer from these species to cultivated crops. Since the early days of somatic hybridization many intergeneric somatic hybrids have been developed through symmetric fusion, asymmetric fusion and microfusion. Somatic hybrids are mainly selected by using markers such as specific media or fusion parents with special features, biochemical mutants, antibiotic resistance and complementation strategy. The hybridity of the regenerants is determined based on morphological, cytological and molecular analysis. The inheritance patterns of nuclear and cytoplasmic genomes in the somatic hybrids are diverse. Nuclear DNA from both fusion parents co-exists congruously in some hybrids with translocation and rearrangement of chromosomes, but spontaneous elimination of chromosomes from either or both fusion parents has been observed very often. In asymmetric fusion, chromosome elimination is an important issue that is a complicated process influenced by many factors, such as irradiation dose, phylogenetic relatedness, ploidy level of fusion parent and regenerants. As for chloroplast genome, uniparental segregation is mainly detected, though co-existence is also reported in some cases. The mitochondrial genome, in contrast to chloroplast, undergoes recombination and very frequent rearrangements. Somatic cell fusion has potential applications for crop genetic improvement by overcoming sexual incompatibility or reproductive barriers, and by realizing novel combinations of nuclear and/or cytoplasmic genomes.

Selection of biochemical mutants of Aspergillus niger with enhanced extracellular ribonuclease production

Combined with u.v. irradiation and the nitrosoguanidine method, selection of biochemical mutants resistant to metabolic inhibitors (2-deoxy-D-glucose, antimycin A, sodium orthovanadate and sodium azide) was a very efficient method for improvement of ribonuclease production by Aspergillus niger. Resistance to sodium azide produced highest RNase production, greatest frequency of positive mutation and shortest sporulation time. The most active strain, Aspergillus niger SA-13-20 resistant to sodium azide, was obtained, which had a 433% increase in RNase production in comparison with the parent strain and had good subculturing stability. Its dynamic characters were similar to those of the parent strain.

Selection of biochemical mutants of Aspergillus niger resistant to some abiotic stresses with increased inulinase production.

The present work aimed at evaluating the usefulness of selecting different kinds of biochemical mutants of Aspergillus niger to increase inulinase production in submerged culture. Conidia of A. niger 13/36, an active producer of inulinase, were subjected to mutagenesis with both u.v. and N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and the products were analysed for inulinase activity with our own diffusion plate method. As a result of mutagenization and selection for obtaining biochemical mutants (e.g. surviving conditions of certain abiotic stresses, good growing on basal medium at 15 and 40 degrees C), A. niger strains resistant to these agents were obtained. Studies of the relationship between a criterion of selection and the frequency of mutation showed that the highest frequency of positive mutations in the second selection (86%) was obtained in mutants characteristic of the best growth at the low temperature (15 degrees C), when compared with the parent culture (28%). The most active mutants grown under stress conditions showed significantly higher inulinase activity (about 1.2-4.5-fold), when compared with the parent strain. The studies presented seem to confirm a high effectiveness of selection in some kinds of biochemical mutants of A. niger with regard to increased inulinase activity. This screening strategy of mutants can be a contribution to modern commercial enzyme production.

DNA sequence profile of TP53 gene mutations in childhood B-cell non-Hodgkin's lymphomas: prognostic implications.

The TP53 gene encodes a nuclear protein implicated in the regulation of the cell cycle, DNA repair, and apoptosis. TP53 mutations and other alterations have been described in numerous types of tumors, and some of these have been associated with poor prognosis. The aim of this study was to characterize TP53 mutations in childhood B non-Hodgkin's lymphoma, their correlation with clinical prognostic factors and response to therapy. Samples from 49 children with B non-Hodgkin's lymphoma were examined for TP53 alterations by single-strand conformation polymorphism analysis (SSCP) of exons 5-9, direct sequencing and by p53 immunohistochemistry. Mutations of TP53 were detected in 11 of 49 (22.5%) patients and more specifically in 20% of Burkitt's lymphoma. The sequence analysis showed missense mutations in 10 cases and an insertion mutation in one case. Mutations of the transition type occurred more frequently than transversions (seven of 11). Analysis of the spectrum of single-nucleotide substitutions showed a 30% frequency of transition mutations in CpG dinucleotide sequences. The overall frequency of p53 immunostaining positivity was 36% (15 of 41). There was a very good agreement between protein expression and the presence of TP53 mutation (P=0.0005). No significant correlation was found regarding age, gender, clinical stage and LDH level and TP53 mutations. Comparison of EFS curves using the log-rank test were also not significant. However, the analysis of the effects of mutations on the core p53 structure identified biological and biochemical mutants with phenotypes probably related to different response to chemotherapy. Our data suggest that some types of mutants can alter the protein distinctly and may be associated with a more aggressive phenotype. In addition, the impact of TP53 mutations on response to therapy may also be influenced by disruption of other genes.

Manipulação de cereais para acúmulo de lisina em sementes

A lisina é um aminoácido essencial cuja via de biossíntese faz parte da via metabólica do ácido aspártico, pela qual são também sintetizados os aminoácidos treonina, metionina e isoleucina. Além disso, a lisina é o principal aminoácido limitante em todos os cereais e por cerca de 30 anos a via do ácido aspártico tem sido estudada em plantas, com o intuito de desvendar e caracterizar os principais pontos chave na regulação das vias de biossíntese desses aminoácidos. Duas etapas distintas, uma primeira originada a partir do desenvolvimento da cultura de tecidos (anos 70-80) e a segunda a partir do desenvolvimento de técnicas para a transformação de plantas (anos 90), permitiram que mutantes bioquímicos e plantas trangênicas fossem produzidos com alterações específicas em passos metabólicos chave, levando à superprodução e acúmulo de treonina em vários tecidos das plantas. Entretanto, a acumulação de lisina em sementes não foi obtida. Tal fato, associado a estudos bioquímicos da via de degradação da lisina em cereais e em leguminosas, indicou que a manipulação da degradação seria tão ou mais importante que a manipulação da biossíntese de lisina para o acúmulo deste aminoácido em sementes dos cereais . Em milho, o uso e estudo de outros mutantes tais como o opaco-2 e variedades QPM (Quality Protein Maize) contribuíram significativamente para a compreensão dos eventos regulatórios. As estratégias para a obtenção de materiais ricos em lisina e sua relevância à manipulação de outros aminoácidos são revisados.

Direct methods for selecting mutants with increased production of invertase from mutagenized cultures ofAspergillus fumigatus

Aspergillus fumigatus 1, the most effective strain for production of invertase was selected out of moulds and yeast belonging to different species by the method of test-tube microculture. Conidia of the selected strain were further subjected to mutagenesis with both U.V. and N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and the products were analysed for invertase activity with our own diffusion plate method. As a result of mutagenization and selection to obtain biochemical mutants (e.g. resistant to some metabolic inhibitors, surviving conditions of certain stresses, poorly growing on minimal medium containing glycerol or mannose), the Aspergillus fumigatus strains were obtained. Ten of these mutants showed an increased extracellular invertase, the level of which ranged widely for indi-vidual cases from 23.1 to over 96% in comparison with the parental strain. The studies of the relation-ship between a criterion of selection and frequency of mutation showed that the highest frequency of positive mutations was obtained with regard to mutants resistant to phosphomycin (58%) and 2-deoxy-D-glucose (53%). The time course of growth and enzyme production by the most active mutant M-7 showed intra- and extracellular invertase activities to have increased about 2-fold, com-pared with the parental strain.

Recovery and characterization of spontaneous, selenate-resistant mutants of Magnaporthe grisea, the rice blast pathogen

There have been numerous biochemical mutants of Magnaporthe grisea previously described. In this study, selenate-resistant (sel) mutants were recovered and characterized. The mutants were recovered as spontaneous, fast-growing sectors arising from initially restricted colonies grown in the presence of 0.1% sodium selenate. Over 94% of the sectors generated were resistant to selenate, and approximately 70% of the selenate-resistant mutants were unable to utilize sulfate sulfur. The sulfate non-utilizing (sul) mutants could be distinguished from wild type by their thin expansive growth with little or no aerial mycelium on a defined minimal medium with sulfate as the sole sulfur source. However, growth of the sul mutant was comparable to the wild type when given L-methionine as a reduced sulfur source. Sul mutants were recovered from all 36 isolates examined representing four distinct MGR586 DNA fingerprint groups commonly found on rice in Arkansas. The frequency of sul mutants recovered from the 36 isolates varied on different media and among the different isolates examined. Generally, sectoring frequency and the number of sul mutants recovered was much higher on rice bran agar amended with selenate (0.1 to 1.4 sectors per colony) than on a defined minimal medium amended with selenate (0.0 to 0.7 sectors per colony). No sectors were observed on potato dextrose agar amended with 0.1% sodium selenate. The mean sectoring frequency among isolates in MGR586 DNA fingerprint group B was significantly higher than among isolates in three other fingerprint groups examined. Complementation tests with multiple sul mutants from a given isolate and sexual crosses among sul mutants of two fertile laboratory isolates indicated that the sul mutation was at a single locus. The sul mutants tested were pathogenic on rice in greenhouse tests and indistinguishable in their virulence phenotype to the corresponding wild type. The sul mutation was stable and may be useful as a marker in epidemiological studies.

Selection of biochemical mutants of Aspergillus niger with enhanced catalase production.

The production of extracellular catalase in a submerged culture by a number of biochemical mutants has been evaluated. Eight of these mutants showed increased extracellular catalase, the level of which ranged widely in individual cases from 44% to over 94% in comparison with the parental strain. Studies of the relationship between a criterion of selection and the frequency of mutation showed that the highest frequency of positive mutations (15.8% and 24.2%) was obtained with respect to mutants resistant to ethidium bromide (1 mmol/l) and sodium gluconate (45%) respectively. The time course of growth and enzyme production by the most active mutant, AM-20, showed extra- and intracellular catalase activities increasing about 2- and 2.6-fold respectively, compared with the parental strain.

Evaluation of alternative nitrogen and carbon sources for sugarbeet suspension culture platings in development of cell selection schemes

Low molecular weight nitrogenous impurity compounds as well as raffinose are negative quality factors that interfere with efficient processing of sugarbeet (Beta vulgaris L.) for sucrose. In order to identify nutrient media for cell selection of biochemical mutants or transgenics that might have reduced levels of these processing impurities, the ability of 10 endogenous compounds to serve as sole nitrogen or carbon source for suspension plating and subculture callus growth was evaluated. The most productive concentrations of nitrate, ammonium, l-glutamine, l-glutamate, urea, and l-proline as sole nitrogen sources supported plating callus growth at 106, 159, 233, 167, 80, and 52%, respectively, as well as the historical 60 mM mix of nitrate and ammonium in Murashige-Skoog medium. Glycine betaine and choline did not support growth. d(+) Raffinose and d(+) galactose supported plating callus growth only 67 and 25%, respectively, as well as sucrose as sole carbohydrate source. No callus growth occurred on glutamine, glutamate, or glycine betaine as the sole carbon or carbon plus nitrogen source. Platings on either nitrate or ammonium as sole nitrogen source did not differ in sensitivity to the nitrate uptake inhibitor phenylglyoxal, suggesting that phenylglyoxal lacks the specificity for use in selection for mutants of nitrate uptake. The ability of raffinose to be used as the carbon source, and glutamine or glutamate as the nitrogen source, may preclude their use for selection of genetic variants accumulating less of these processing impurities. However, mutants or transgenics able to utilize either glutamine, glutamate, or glycine betaine might be selectable on media containing any one of these as carbon, nitrogen, or carbon plus nitrogen source, respectively, that is incapable of supporting wild-type cell growth.

Genetic analysis of cholinergic nerve terminal function in invertebrates.

Genetic analysis of nerve terminal function is proving fruitful and studies on invertebrates are making a substantial impact. In this survey, particular emphasis has been placed on cholinergic chemical synaptic transmission. The advanced genetics of Drosophila melanogaster and Caenorhabditis elegans with their rich diversity of behavioural and biochemical mutants is providing new insights into the functions of key molecular components of synapses. A 'space-invader' mutant of Periplaneta americana permits investigations of competition between neurons during synaptogenesis and its impact on neurotransmitter release. The growing importance of the C. elegans genome as a major research resource is emphasized in this survey.

Fluorescent actin analogs with a high affinity for profilin in vitro exhibit an enhanced gradient of assembly in living cells.

Constitutive centripetal transport of the actin-based cytoskeleton has been detected in cells spreading on a substrate, locomoting fibroblasts and keratocytes, and non-locomoting serum-deprived fibroblasts. These results suggest a gradient of actin assembly, highest in the cortex at the cytoplasm-membrane interface and lowest in the non-cortical perinuclear cytoplasm. We predicted that such a gradient would be maintained in part by phosphoinositide-regulated actin binding proteins because the intracellular free Ca2+ and pH are low and spatially constant in serum-deprived cells. The cytoplasm-membrane interface presents one surface where the assembly of actin is differentially regulated relative to the non-cortical cytoplasm. Several models, based on in vitro biochemistry, propose that phosphoinositide-regulated actin binding proteins are involved in local actin assembly. To test these models in living cells using imaging techniques, we prepared a new fluorescent analog of actin that bound profilin, a protein that interacts with phosphoinositides and actin-monomers in a mutually exclusive manner, with an order of magnitude greater affinity (Kd = 3.6 microM) than cys-374-labeled actin (Kd > 30 microM), yet retained the ability to inhibit DNase I. Hence, we were able to directly compare the distribution and activity of a biochemical mutant of actin with an analog possessing closer to wild-type activity. Three-dimensional fluorescence microscopy of the fluorescent analog of actin with a high affinity for profilin revealed that it incorporated into cortical cytoplasmic fibers and was also distributed diffusely in the non-cortical cytoplasm consistent with a bias of actin assembly near the surface of the cell. Fluorescence ratio imaging revealed that serum-deprived and migrating fibroblasts concentrated the new actin analog into fibers up to four-fold in the periphery and leading edge of these cells, respectively, relative to a soluble fluorescent dextran volume marker, consistent with the formation of a gradient of actin filament density relative to cell volume. Comparison of these gradients in the same living cell using analogs of actin with high and low affinities for profilin demonstrated that increased profilin binding enhanced the gradient. Profilin and related proteins may therefore function in part to bias the assembly of actin at the membrane-cytoplasm interface.

An overview of the recent advances on the physiology and molecular biology of lignin peroxidases of Phanerochaete chrysosporium

The lignin-degrading white-rot fungus Phanerochaete chrysosporium produces two families of extracellular peroxidases designated lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) which are components of the lignin degradation system of this organism. The number and types of LIP and MNP isozymes produced vary dramatically in response to changes in culture conditions. Protease-mediated degradation of LIPs was shown to be the major cause for the decay of LIP activity in idiophasic cultures of P. chrysosporium. Use of biochemical mutants has not only yielded information on the relative importance of LIPs and MNPs in lignin degradation but has given us insights into the regulation of production of LIPs and MNPs. The genes encoding the major LIPs have been cloned and sequenced and were shown to have a high degree of homology to each other. Karyotyping studies indicated that heterokaryotic strains contain ten chromosomes and that the LIP genes are distributed on at least two chromosomes.

In Vitro Selection of Calli Resistant to a Triazole Cytochrome-P-450-Obtusifoliol-14-Demethylase Inhibitor from Protoplasts of Nicotiana tabacum L. cv Xanthi

The selection of biochemical mutants has been undertaken in order to elucidate regulatory and functional aspects of sterol biosynthesis in plants. 2-(4-Chlorophenyl)-3-phenyl-1-(1H-1,2,4- triazol-1-yl)-2,3-oxidopropane (LAB170250F), an experimental fungicide of the triazole family, was used as a selective agent. Indeed, this compound is a strong inhibitor of the cytochrome-P-450-obtusifoliol-14-demethylase in sterol biosynthesis. The selection strategy consisted of screening large populations of microcalli derived from ultraviolet-mutagenized protoplasts of Nicotiana tabacum L. cv Xanthi for resistance to a lethal concentration of LAB170250F. The best selective conditions were first determined, i.e. strength of the selection pressure as well as the time and duration of its application in the developmental process from protoplast to whole plant. Selection experiments resulted in the recovery of 40 resistant calli. These calli were divided into three classes according to the modification of their sterol content in response to LAB170250F. Some of these calli might be impaired in sterol biosynthesis, but most have a sterol profile identical to that of the control calli. This suggests that the toxic properties of LAB170250F are due to the parallel inhibition of sterol biosynthesis and of at least one additional unidentified target in the plant cell.

Genetic and mutational tools for investigating the genetics and molecular biology of trichothecene production in Gibberella pulicaris (Fusarium sambucinum).

Gibberella pulicaris (Fusarium sambucinum) is a promising organism for studying the genetics and regulation of trichothecene biosynthesis; conditions for obtaining fertile crosses have been defined (Desjardins & Beremand, 1987) and crosses between natural variants have provided some information about the number, location, arrangement, and role of genes which determine trichothecene production (Desjardins & Beremand, 1987; Beremand & Desjardins, 1988). The development of some additional experimental tools and methodologies required for the further genetic analysis of trichothecene production in G. pulicaris are described in the present study. A highly fertile, isogenic line was constructed for G. pulicaris strain R-6380. The ability to readily generate mutants in this strain was also demonstrated. Both biochemical and morphological mutants were obtained following UV-mutagenesis. The inheritance of some of these mutations through meiosis indicated that they will be useful genetic markers for crosses and mapping studies. Since strain R-6380 is also transformable (Salch & Beremand, 1988), it is an excellent choice for transmission and molecular genetic studies involving trichothecene production.

Intraspecific hybridization of Aspergillus awamori var. kawachi by protoplast fusion.

Protoplast fusion was studied to breed a koji-mold, Aspergillus awamori var. kawachi, used for making a Japanese spirit, shochu. Protoplasts were obtained from mycelia of two biochemical mutants using an enzyme system from Trichoderma viride consisting of mainly β-1, 3-glueanase and chitinase. Approximately 10-20% of these protoplasts regenerated on a solid culture medium with a soft agar overlay. When the two types of protoplasts were treated with a fusogen including 35% polyethylene glycol, they fused effectively and formed a heterokaryon. A strain obtained from the hetrokaryon using d-camphor showed high stability (98.8%) and numerical sporulation, and produced the same levels of amylase and citric acid as those by the original strain. From these results and the formation of recombinants, the new strain was assumed to be a heterozygous diploid. The appearance frequency of the diploid was 2.9 × 10-2. The existence of a parasexual cycle in this fungus was also discovered, using the protoplast fusion technique.