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PubMed1998

REMI mutagenesis and identification of pathogenic mutants in blast fungus (Magnaporthe grisea).

Blast fungus' mutant, which is very difficult to obtain, is the key substance in the study of the relation between pathogen and plant. Rice-Aichi-ashahi was used as the host and plasmid pBF101 or pCSN43 as foreign DNA fragment, and REMI mutagenesis was applied to tag the pathogenic sequence in rice blast fungus M.grisea. Three mutants (R2H65, R2H69 and R2B1565) related to pathogenicity were recovered from several hundreds of transformants. R2H69 and R2H65 produced merely strange conidia which could not form appressorium or yield abnormals, and pathogenicity test further confirmed that they were unable to cause disease. R2B1565 possesses the same phenotypes as those of the wild type except that the ability of causing disease was much reduced.

DNA Restriction Enzymes↗
PubMed2001

Identification of a mutant bovine herpesvirus-1 (BHV-1) in post-arrival outbreaks of IBR in feedlot calves and protection with conventional vaccination.

Outbreaks of infectious bovine rhinotracheitis (IBR) have recently been observed in vaccinated feedlot calves in Alberta a few months post-arrival. To investigate the cause of these outbreaks, lung and tracheal tissues were collected from calves that died of IBR during a post-arrival outbreak of disease. Bovine herpesvirus-1 (BHV-1), the causative agent of IBR, was isolated from 6 out of 15 tissues. Of these 6 isolates, 5 failed to react with a monoclonal antibody specific for one of the epitopes on glycoprotein D, one of the most important antigens of BHV-1. The ability of one of these mutant BHV-1 isolates to cause disease in calves vaccinated with a modified-live IBR vaccine was assessed in an experimental challenge study. After one vaccination, the majority of the calves developed humoral and cellular immune responses. Secondary vaccination resulted in a substantially enhanced level of immunity in all animals. Three months after the second vaccination, calves were either challenged with one of the mutant isolates or with a conventional challenge strain of BHV-1. Regardless of the type of virus used for challenge, vaccinated calves experienced significantly (P < 0.05) less weight loss and temperature rises, had lower nasal scores, and shed less virus than non-vaccinated animals. The only statistically significant (P < 0.05) difference between the 2 challenge viruses was the amount of virus shed, which was higher in non-vaccinated calves challenged with the mutant virus than in those challenged with the conventional virus. These data show that calves vaccinated with a modified-live IBR vaccine are protected from challenge with either the mutant or the conventional virus.

Alberta↗
PubMed1981

[Radiosensitive Drosophila lines. II. Identification of the mutant chromosome 2 genes that control mutagen sensitivity].

Genetic control of somatic mutagen sensitivity was studied in a strain of Drosophila melanogaster containing mutations in the second chromosome. The method used allowed to identify the determinants of different kind of sensitivity in the same experiment. It has been shown that the second chromosome of the strain analysed carries two independent mutations, one of them determining supersensitivity of Drosophila larvae to ionizing radiation, the other--to methyl methane-sulfonate. Mutant loci have been located at 59,9 +/- 1,1 and 73,8 +/- 2,5 units of the chromosome genetic map. They have been called rad(2)201G1 and mus(2)201G1, respectively.

Animals↗
PubMed1994

Identification of a mutant human topoisomerase I with intact catalytic activity and resistance to 9-nitro-camptothecin.

Human U-937 myeloid leukemia cells were selected for resistance to increasing concentrations of the camptothecin derivative, 9-nitro-20(S)camptothecin (9-NC). The isolated single cell clone, designated U-937/CR, was approximately 20-fold resistant to 9-NC. Analysis of topoisomerase I (topo I) gene expression in U-937/CR cells demonstrated similar mRNA levels as compared with U-937 cells. Immunoblotting with an anti-topo I serum revealed reactive proteins at 100, 75, and 67 kDa which were expressed at the same level in the parental and 9-NC-resistant clones. These cell lines also demonstrated similar levels of topo I catalytic activity as determined by assaying nuclear extracts for relaxation of supercoiled plasmid DNA. In contrast, catalytic assays performed in the presence of 9-NC demonstrated that topo I activity from U-937/CR cells was approximately 10-fold more resistant than that from U-937 cells. Nucleotide sequencing of topo I cDNAs revealed the substitution of phenylalanine (TTC) at residue 361 in U-937 cells with serine (TCC) in the 9-NC-resistant clone. Expression and partial purification of the mutant topo I protein in Escherichia coli demonstrated resistance of this enzyme to 9-NC in catalytic assays. Taken together, these findings identify a novel mutation in topo I which confers resistance to 9-NC and support the involvement of this region in the interaction between topo I and 9-NC.

Amino Acid Sequence↗
PubMedDate not supplied

[The induction of mutations in Streptomyces aureofaciens by UV rays and nitrosoguanidine and the identification of the mutants obtained].

Streptomyces aureofaciens 019(8), chlortetracycline producent was subjected to the effect of UV-light and nitrosoguanidine and after that its 121520 colonies have been analyzed by the methods of replicas and 57 mutants were isolated: sensitive to the effect of UV radiation--14; auxotrophs--30; antibiotic-inactive --13. The induction frequency of mutations is within the limits of 0.016 to 0.067%. Auxotrophic mutants manifested genetic instability. Mutants with a block chlortetracyclin biosynthesis are stable.

Chlortetracycline↗
PubMed1985

Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation.

Membrane mutants of animal cells have been isolated by several laboratories, using a variety of selection protocols. The majority are lectin receptor mutants arising from altered glycosylation of membrane molecules. They have been obtained by selection for resistance to cytotoxic plant lectins or by alternative protocols designed, in many cases, to isolate different classes of receptor mutants. The identification of most membrane mutants expressing altered surface carbohydrates is rapidly achieved by determining their resistance to several lectins of different carbohydrate-binding specificities. For Chinese hamster ovary mutants, genetic novelty may subsequently be determined by complementation analysis with selected members of 10 recessive, glycosylation-defective complementation groups defined by this laboratory. In an attempt to identify new complementation groups, 11 Chinese hamster ovary membrane mutants independently isolated in different laboratories have been investigated for their lectin resistance and complementation properties. Only one new complementation group was defined by these studies. The remaining 10 mutants fell into complementation group 1, 2, 3, or 8. Although no evidence for intragenic complementation was observed, indirect evidence for different mutations within some genes was obtained. Seven of the independent isolates fell into complementation group 1, reflecting the high probability of isolating the Lec1 phenotype from Chinese hamster ovary populations. The results emphasize the importance of performing a genetic analysis before biochemical characterization of putative new membrane mutants.

Animals↗
PubMed2006

Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2.

BACKGROUND: Francisella tularensis is a zoonotic intracellular bacterial pathogen that causes tularemia. The subspecies tularensis is highly virulent and is classified as a category A agent of biological warfare because of its low infectious dose by an aerosol route, and its ability to cause severe disease. In macrophages F. tularensis exhibits a rather novel intracellular lifestyle; after invasion it remains in a phagosome for three to six hours before escaping to, and replicating in the cytoplasm. The molecular mechanisms that allow F. tularensis to invade and replicate within a host cell have not been well defined. METHODS: We constructed a stable transposon mutagenesis library of virulent strain Schu S4 using a derivative of the EZ::TN transposon system. Approximately 2000 mutants were screened for the inability to invade, and replicate in the hepatic carcinoma cell line HepG2. These mutants were also tested for replication within the J774.1 macrophage-like cell line. RESULTS: Eighteen mutants defective in intracellular replication in HepG2 cells were identified. Eight of these mutants were auxotrophs; seven had mutations in nucleotide biosynthesis pathways. The remaining mutants had insertions in genes that were predicted to encode putative transporters, enzymes involved in protein modification and turnover, and hypothetical proteins. A time course of the intracellular growth of a pyrB mutant revealed that this mutant was only able to grow at low levels within HepG2 cells but grew like wild-type bacteria in J774.1 cells. This pyrB mutant was also attenuated in mice. CONCLUSION: This is the first reported large-scale mutagenesis of a type A strain of F. tularensis and the first identification of mutants specifically defective in intracellular growth in a hepatic cell line. We have identified several genes and pathways that are key for the survival and growth of F. tularensis in a hepatic cell line, and a number of novel intracellular growth-defective mutants that have not been previously characterized in other pathogens. Further characterization of these mutants will help provide a better understanding of the pathogenicity of F. tularensis, and may have practical applications as targets for drugs or attenuated vaccines.

Animals↗
PubMed1975

Purine mutants of mammalian cell lines. II. Identification of a phosphoribosylpyrophosphate amidotransferase-deficient mutant of Chinese hamster lung cells.

A class of purine auxotrophs blocked early in the purine biosynthetic pathway was examined. The inability of these mutants to accumulate formylglycinamide ribotide (FGAR) in the presence of azaserine suggested that one or more of the first three enzymes of the pathway were either missing or defective. By direct enzyme assay, phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14) was found to be absent in extracts of mutant cells. Thus these cells were unable to convert PRPP to phosphoribosylamine (PRA). By reacting ribose 5-phosphate with ammonium ions, PRA was generated nonenzymatically in the incubation mixture, thus enabling us to test for the presence of the two enzymes required to convert PRA to FGAR. It was demonstrated that sufficient amounts of these enzymes, phosphoribosylglycineamide synthetase (E.C. 6.3.1.3) and phosphoribosylglycineamide formyltransferase (E.C. 2.1.2.2), were present in mutant extracts to allow synthesis of FGAR to occur once PRA was so provided.

Adenine↗
PubMed1994

Identification of biologically active mutants by combinatorial cassette mutagenesis: exclusion of wild-type codon from degenerate codons.

A degenerate codon (N)(N)(G+C) is often used in cassette mutagenesis to encode all 20 natural amino acids at the target mutation site. However, the presence of the wild-type codon in the degenerate codon presents some inconvenience in screening and identification of catalytically active mutants. The wild-type enzyme will always be identified as catalytically active in a screening process and in most cases can only be distinguished from active mutants by DNA sequencing. Sequencing of background wild-type enzyme represents wasted effort in the identification of active mutants. This paper describes a simple approach for exclusion of the wild-type codon in degenerate codons through the synthesis of two or three oligonucleotide mixtures. The minimum number of individual colonies required to achieve a high degree of certainty of including all possible codons for screening of catalytic activity can be estimated using a statistical procedure. The use of degenerate codons that exclude the wild-type amino acid facilitates the screening process and saves time and expense in DNA sequencing.

Amino Acids↗
PubMed1974

Polyene-resistant mutants of Aspergillus fennelliae: identification of sterols.

Four mutants strains of Aspergillus fennelliae resistant to various polyene antibiotics have been analyzed for their sterol content. The mutant strains contained Delta(7,22,24(28))-ergostatrien-3beta-o1 and Delta(7,22)-ergostadien-3beta-o1 as the major sterols, whereas the wild types contained ergosterol, indicating that the mutants contain metabolic blocks for C(5)-C(6) dehydrogenation and C(24)-C(28) reduction in the biosynthesis of ergosterol. Revertant sectors arising from the mutant colonies contained either more Delta(7,22)-ergostadien-3beta-o1 than the parent mutant strain or showed the reappearance of ergosterol. The revertant sectors also showed lower resistance to amphotericin B and a higher rate of sporulation than the parent mutant strain. These results confirm our previous observations that the presence of ergosterol is directly related to the drug susceptibility and the normal rate of sporulation in this species.

Anti-Bacterial Agents↗
PubMed1983

Intrauterine enucleation of normal mice mimics a structural compensatory response in the geniculate of eyeless mutant mice.

Identification of a compensatory structural pathway for the retinal terminals in the glomerular configuration of the dorsal lateral geniculate nucleus (dLGN) of the adult anophthalmic mutant mouse (ZRDCT-An) led to an experimental analysis of the effect of surgical enucleation on the dLGN of normal embryonic and postnatal C57 mice. Young C57 adults were prenatally enucleated (embryonic days 14-19) or during the early postnatal period (birth to 5 days old). Light and electron microscopic observations on the dLGN of these enucleated mice were compared with the dLGN of two types of mutant mice, the bilaterally eyeless anophthalmic and the unilaterally eyeless naked splotch (NSP). A structural compensatory response, similar to that occurring naturally in these two mutant mice, is activated by prenatal experimental enucleation as shown by the appearance of large replacement terminals (LRTs) for the retinal afferents. The glomeruli in the ipsi- and/or contralateral dLGN in these 3 conditions contain extensive synaptic areas covered by compensatory large axons. This pattern of synaptic thalamic reorganization occurs after experimental prenatal and postnatal enucleation of normal mice. This structural reorganization in experimentally and genetically enucleated mice suggests a common mechanism for replacement of retinal terminals. The precise origin and function of this tract remains to be determined. This evidence demonstrates that the anophthalmic mutant mouse is a valid model system for analysis of thalamic reorganization that occurs in normal mice after perinatal enucleation.

Animals↗
PubMed1975

Nature and biosynthesis of ommochromes in Locusta migratoria cinerascens Fabr. (Orthoptera, Acrididae) and its albino mutant.

The identification and localization of the 4 ommochromes present in locust body were established. 2 new chemical species were isolated: acridiommatins 1 (under crystalline form) and 2 (perhaps a xanthommatin ester). The course of their biosynthesis was studied with 2 different methods: quantitative analysis of the ommochrome content in organs during the 2 last larvel instars and the whole imaginal life and measurement of the tryptophan pyrrolase activities during the same period in the normal strain and in an albino mutant strain. With these 2 methods a cyclic activity related to the moulting cycle was found in the ommochrome pathway without any notable difference beween the 2 strains.

Animals↗
PubMed2003

Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K.

Rheb GTPases represent a unique family of the Ras superfamily of G-proteins. Studies on Rheb in Schizosaccharomyces pombe and Drosophila have shown that this small GTPase is essential and is involved in cell growth and cell cycle progression. The Drosophila studies also raised the possibility that Rheb is involved in the TOR/S6K signaling pathway. In this paper, we first report identification of dominant negative mutants of S. pombe Rheb (SpRheb). Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G1 arrest, and induction of fnx1+, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. We show that transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants blocked nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells.

Amino Acid Sequence↗
PubMed2004

Identification of endogenous glucocorticoid repressed genes differentially regulated by a glucocorticoid receptor mutant able to separate between nuclear factor-kappaB and activator protein-1 repression.

Glucocorticoids are commonly used in the clinic, but long-term treatment is often associated with severe side effects. One way to reduce unwanted effects is to restrict glucocorticoid receptor (GR) signaling through defined pathways. In this study, we examine endogenous target genes regulated by a GR mutant that in contrast to the wild-type GR is unable to repress stimulated nuclear factor-kappaB (NF-kappaB) activity, whereas repression of activator protein-1 (AP-1) activity is maintained. This GR mutant (GRR488Q) harbors a point mutation in the second zinc finger of the DNA binding domain. Its ability to distinguish between NF-kappaB and AP-1 repression is defined using reporter genes regulated by both simple and natural promoters. The inability of GRR488Q to repress NF-kappaB was not related to its inability to activate target genes through a glucocorticoid response element. Furthermore, the discriminating property was observed in three different cell lines, suggesting that this is not a cell-specific effect. These results show that different receptor surfaces or mechanisms are involved in repression of NF-kappaB and AP-1, respectively. It is interesting that the GRR488Q still interacted physically with NF-kappaB. Gene expression profiling of human embryonic kidney 293 cells, which express the wild-type GR and the GRR488Q mutant allowed identification of endogenous genes preferentially repressed by GR interference with NF-kappaB activity. The genes differentially regulated by GRR488Q mutant versus the wild-type GR after 2 h of treatment seem mainly to be involved in control of transcription and cell growth. At 8 h, no such distinction could be seen.

Animals↗