Showing papers on "Wild type published in 1983"

Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila.

Abstract: The temperature-sensitive Drosophila mutant, shibirets1 (shi), has been shown to exhibit a reversible block in synaptic transmission at 30 degrees C. Various synaptic terminals (neuromuscular, sensory, central) of this mutant were observed by electron microscopy. At 19 degrees C, all terminals of shi showed essentially the same structural features as those of wild-type (Oregon-R) flies, while at 30 degrees C (5 or 10 min of exposure) shi terminals exhibited various structural changes not seen in the wild type. The major structural change observed in all of the various types of terminals was the accumulation of many pitlike structures on the plasma membrane near presynaptic sites. These structures consisted of a spherical head portion, about 50-100 nm in diameter, and a cylindrical neck portion, about 20 nm long and 20-25 nm in diameter. The neck portion was surrounded by a kind of cytoplasmic dense material, about 10 nm thick, reminiscent of a "collar." Thus, these pits are referred to as "collared pits." Similar kinds of pits were observed, although very rarely, in wild-type flies at 19 and 30 degrees C and in shi flies at 19 degrees C. In addition, various degrees of vesicle depletion, and an increase in membranous structures (infoldings and cisternalike or tubulelike structures) often accompanied pit formation. The possibility that these pits are the result of a blocked step in the endocytotic process, which in turn causes vesicle depletion as exocytosis proceeds, is discussed.

Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation.

Abstract: Oligodeoxynucleotide-directed mutagenesis has been used on the gene of tyrosyl-tRNA synthetase from Bacillus stearothermophilus to produce mutant enzymes altered at the adenosine 5'-triphosphate (ATP) binding site. Deliberate attempts were made to alter rather than destroy enzymic activity so that kinetic measurements may be made to identify the subtle roles of the enzyme-substrate interactions in catalysis. Cys-35, the -SH group of which is involved in binding the 3'-OH of the ribose ring of ATP, has been mutated to a serine residue [Winter, G., Fersht, A. R., Wilkinson, A. J., Zoller, M., & Smith, M. (1982) Nature (London) 299, 756-758] or glycine residue. The mutant enzymes are less active than the wild type, and the reduction in activity can be attributed to a decrease in the value of kcat and an increase in KM. Thus, the interaction energy of the side chain of Cys-35 with the substrate is not fully realized in the enzyme-substrate complex but is used preferentially to stabilize the transition state. Relative to its absence in the Gly-35 mutant, the side chain of Cys-35 is calculated to stabilize the transition state for pyrophosphate exchange by 1.2 kcal/mol and the transition state for aminoacylation by 1.0 kcal/mol.

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth.

Abstract: A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO(2) requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO(2) concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O(2). In contrast to a CO(2) compensation concentration of near zero in wild type at all O(2) concentrations examined, the mutant exhibited a high, O(2)-stimulated CO(2) compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of (14)CO(2). At air levels of CO(2) and O(2), the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO(2) concentration. At ambient CO(2), wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO(2) limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO(2) concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO(2), creating a high internal CO(2) concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO(2) concentration.

Generation of adenovirus by transfection of plasmids

Abstract: Biologically active fragments of Adenovirus 5 (Ad5) DNA that span the entire genome have been cloned into plasmids. The covalently attached terminal protein was removed and Eco RI linkers added in a fashion that preserves the Ad5 terminal sequences. When plasmids containing overlapping fragments that represent the entire genome are cotransfected onto 293 cells, infectious virus is obtained. Generation of virus depends upon the release of the 0 or 100 mu Ad5 terminus from pBR322 DNA by Eco RI cleavage. During virus production the modified termini of the transfected fragments are corrected exactly to that of wt viral DNA. The above method for preparing adenovirus recombinants has been used to construct a mutant, Ad5 delta (78.9-84.3), lacking most of the non-essential EIII transcriptional unit. This mutant is phenotypically wild type with respect to burst size and kinetics of growth. Surprisingly, it inhibits wt viral growth upon mixed infections of HeLa or 293 cells, apparently at the level of DNA replication.

The isolation and characterisation of a catalase-deficient mutant of barley (Hordeum vulgare L.).

Abstract: A mutant line of barley, R(othamsted)-Pr 79/4, has been isolated which grows poorly in natural air, but normally in air enriched to 0.2% CO2. Analysis of the products of (14)CO2 fixation showed that there was no major block in photosynthetic or photorespiratory carbon metabolism in the mutant and that rates of CO2 fixation were only slightly lower than those measured in the wild type (c.v. Maris Mink). Leaves of the mutant line contained only 10% of the catalase (EC 1.11.1.6) activity found in the wild type; and the two major bands of catalase activity detected after starch-gel electrophoresis of extracts of normal leaves were missing from similar extracts of RPr 79/4. Peroxisomes isolated from mutant leaves contained negligible catalase activity, but normal levels of other enzymes involved in photorespiration. Genetic analysis has shown that the mutation is recessive and that both air-sensitivity and catalase-deficiency segregate together in F2 plants derived from a cross between the mutant and the cultivar Golden Promise. [1-(14)C]Glycollate was not converted to (14)CO2 faster in the mutant leaves than in the normal leaves. Thus there was no evidence that photorespiratory CO2 may be obtained by the chemical action of H2O2 on glyoxylate or hydroxypyruvate.

CAP and RNA polymerase interactions with the lac promoter: binding stoichiometry and long range effects

Abstract: The binding stoichiometries of the complexes formed when the E. coli cyclic AMP receptor protein (CAP) binds to 203 bp lac promoter-operator restriction fragments have been determined. Under quantitative binding conditions, a single dimer of CAP occupies each of two sites in the promoter. Different electrophoretic mobilities are observed for 1:1 complexes formed with L8-UV5 mutant, L305 mutant, and wild type promoter fragments, indicating sequence-specific structural differences between the complexes. The differences in gel mobility between L8-UV5 and wild type complexes disappear when the promoter fragments are cleaved with Hpa II restriction endonuclease. Models in which CAP alters DNA conformation or in which CAP forms a transient intramolecular bridge between two domains of a DNA molecule could account for these observations. The selective binding of RNA polymerase to CAP-promoter complexes is demonstrated: the binding of a single CAP dimer to the promoter is sufficient to stimulate subsequent polymerase binding. Functional CAP molecules are not released from the promoter on polymerase binding.

Reduced Inorganic Carbon Transport in a CO2-Requiring Mutant of Chlamydomonas reinhardii

Abstract: A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO2 concentration for photoautrophic growth Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO2 response of photosynthesis indicated a reduced affinity for CO2 in the mutant At air levels of CO2 (003-004%), O2 inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type Neither strain was significantly affected by O2 at saturating CO2 concentration Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO2 concentration compared to wild type From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO2 concentrating system in C reinhardii photosynthesis

Clonal analysis of heterogeneous crown gall tumor tissues induced by wild-type and shooter mutant strains ofAgrobacterium tumefaciens-expression of T-DNA genes.

Abstract: Tumors were induced by anAgrobacterium tumefaciens strain with a wild-type octopine Ti plasmid and by shooter mutants with a transposon insertion in the auxin-locus of the T-region. Cloning of isolated axenic tumor tissues revealed that in all cases they consisted of tumor cells (10–26%) next to a majority of normal cells. The tumor clones that had been induced by the strain with the wild-type Ti plasmid all grew as amorphous calli. Tumor, clones induced by a shooter mutant were of two different types. One type of clone grew well on phytohormone-free medium. this type invariably regenerated tumorous shoots abundantly on this medium. The other type of clone only grew after the addition of auxin and cytokinin to the culture medium, but slow growth also took place in the presence of only auxin. This type never regenerated shoots spontaneously. After stimulation by a high level of kinetin, however, a few shoots were also obtained from these clones. One of these shoots, like other tumorous shoots, contained the tumor-specific enzyme octopinesynthase (Ocs), but in contrast to other tumorous shoots formed a root-system. The expression of T-DNA genes in shoots proliferating from the cloned tumor tissues induced by a mutant with an insertion in the region for transcript tr. 2 was studied by northern blot hybridization. Except for tr.2 the T-DNA transcripts were detected in the tumorous shoots analysed, including the transcript, tr.1 from the auxin-locus and tr.4 from the cytokinin-locus. This shows that the presence of these transcripts, which are assumed to be responsible for the tumorigenic character of tumor cells, does not interfere with the differentiation of shoot cells. One of the shooty tumor clones (TSO38) showed an unstable character with regard to octopine synthase activity (Ocs±). For, TSO38 and some of its subclones, it was found that only 4% of the regenerated shoots were Ocs+. Northern blot hybridization revealed that the mRNA for octopine synthase was present in extremely low quantity in the population of TSO38 derived shoots. The finding that it was possible to force shoots from clone TSO38 and from subclone TSO38-23− that were Ocs− to become Ocs+, proved that the gene for octopine synthase was present in the Ocs− shoots and that this gene showed unstable expression due to regulation at the level of transcription.

Genetic analysis of the proBA genes of Salmonella typhimurium: physical and genetic analyses of the cloned proB+ A+ genes of Escherichia coli and of a mutant allele that confers proline overproduction and enhanced osmotolerance.

Abstract: Because of the fact that proline overproduction relieves the inhibitory effects of high external osmotic strength in a number of procaryotes, we wished to clone a mutant allele, pro-74, that confers proline overproduction and enhanced osmotolerance on Salmonella typhimurium and Escherichia coli. Therefore, the pro-74 allele, originally located on an E. coli episome F'128, was cloned into pBR322. In a parallel experiment, the wild type proB+ A+ genes of E. coli were also cloned from F'128 into pBR322. Both the pro-74 and the proB+ A+ alleles were obtained on a 10.4-kilobase-pair fragment that also contained the unrelated phoE gene. Strains carrying either the wild-type proB+ A+ or the pro-74 alleles on pBR322 grew more slowly, both in minimal medium and media of elevated osmotic strength, than strains carrying the same alleles on the low-copy plasmid, F'128, indicating that some gene in the cloned region is deleterious in high copy. We constructed Tn5 insertion mutations in the proB and the proA genes of E. coli, carried on F'128 in S. typhimurium. Using P22 transduction in S. typhimurium, we transferred these proB and proA::Tn5 insertions from F'128 into the cloned proBA genes on pBR322. From the restriction maps of the plasmids thus generated, we determined the approximate locations of the proB and the proA genes. We also performed complementation tests of S. typhimurium and E. coli proB and proA mutants by using the F'128 proB and proA::Tn5 insertions. These tests revealed that the proBA genes of S. typhimurium form an operon, whose direction of transcription is from proB to proA. They also indicated that in S. typhimurium, as in E. coli, the proB+ gene encodes gamma-glutamyl kinase, and the proA+ gene encodes gamma-glutamyl phosphate reductase. Complementation tests also indicated that the pro-74 mutation is either in the proB structural gene, or its promoter-operator.

DNA sequences of two yeast promoter-up mutants

Abstract: The yeast Saccharomyces cerevisiae has three genetic loci encoding different alcohol dehydrogenase (ADH) isozymes: ADC1, which encodes the classical fermentative isozyme ADHI1; ADR2, which encodes the glucose-repressed isozyme ADHII2; and ADM, which encodes an ADH isozyme found associated with mitochondria. When yeast are grown on glucose, the ADC1 gene is expressed, and the ADR2 gene repressed1,3,4. Conversely, growth on a non-fermentable carbon source such as ethanol or glycerol results in derepression of ADR2, and repression of ADC1. The ADC1 and ADR2 genes have been cloned and sequenced5–8, and a number of cis-acting mutations identified that cause constitutive expression of ADR29,10, and seem to fall into two classes11. The most abundant class consists of mutants that cannot be fully derepressed, and do not revert to wild type at a detectable level: these are caused by the insertion of a transposable element into the 5′-flanking region of the gene6,12. The second class of mutants do revert to a glucose-repressed phenotype at a detectable frequency, and when grown on non-fermentable carbon sources derepress ADR2 to levels up to five times those found in wild-type cells10. We report here the sequencing of the 5′-flanking regions of two such promoter-up, constitutive ADR2 mutants, in both of which the mutant phenotype is associated with an increase in length of a poly(A)·poly(T) tract 222 base pairs (bp) upstream of the gene.

ACTION SPECTRA OF PHOTOGEOTROPIC EQUILIBRIUM IN Phycomyces WILD TYPE AND THREE BEHAVIORAL MUTANTS

Abstract: Photogeotropic equilibrium action spectra in the range from 301 to 740 nm were made for Phycomyces wild type and the three behavioral mutants C47 (madA35), C109 (madBlOl) and LI (madCIIQ), all of which have a raised phototropic threshold. In addition to two broad peaks at 365 and 455 nm, typical for flavins, the wild type action spectrum shows three novel peaks, which have not been observed previously. These peaks are located at 414, 491 and 650 nm. The 650 nm peak has a relative quantum efficiency of 3 × 10−8 compared to the peak at 414 nm. The wavelength dependent shapes of the fluence-response curves of the bending angle and the aiming error angle indicate more than one receptor pigment for phototropism. The shape of the action spectrum of C47 is basically unaltered in comparison to wild type. C109 and LI show substantial differences from the wild type. In the near UV two small peaks at 334 and 365 nm appear; the 414 and 491 nm peaks present in wild type and C47 are missing and two new peaks at 529 nm (not well resolved in C109) and 567 nm are found. None of the three mad mutations affects the 650 nm peak. A model of the sensory transduction chain is presented, which incorporates these and other known features.

Association of the lethal yellow (Ay) coat color mutation with an ecotropic murine leukemia virus genome.

Abstract: The dilute (d) coat color mutation on chromosome 9 is closely associated with an ecotropic murine leukemia virus (MuLV) genome [Jenkins, N.A., Copeland, N.G., Taylor, B.A. & Lee, B.K. (1981) Nature 293, 370-374]. DBA/2J mice homozygous for the reverse mutation to wild type at the dilute locus (d+2J) lack ecotropic virus-specific sequences, suggesting that the dilute mutation was caused by virus integration. In the experiments described here, we analyzed the ecotropic MuLV DNA content of mice that collectively carry 10 different alleles at the agouti coat color locus (of chromosome 2) to determine whether any of these alleles also are associated with ecotropic virus sequences. Of the 10 alleles analyzed, one allele, lethal yellow (Ay), which is carried congeneically and heterozygously on C57BL/6J, 129/Sv, and LT/Sv mice, was closely associated with an ecotropic MuLV provirus. The close association of this provirus with the Ay allele suggests that this mutation also may be caused by virus integration. Furthermore, this association may be useful for molecular cloning and characterizing the many alleles at this locus.

Deletion and amplification of DNA sequences in melanin-negative variants of Streptomyces reticuli.

Abstract: The sporulating wild type of Streptomyces reticuli produces the pigment melanin. Though the ability to synthesize tyrosinase is frequently lost, it was demonstrated, that the structural gene coding for this enzyme is not located on the extrachromosomal DNA of the wild type strain or melanin-positive variants. Melanin negative variants were found to have lost this gene and to contain amplified nucleotide sequences within their genomes.

Modified plasma-membrane ATPase in mutants of Saccharomyces cerevisiae.

Abstract: Mutations affecting the plasma membrane ATPase of Saccharomyces cerevisiae were obtained by selecting mutants resistant to Dio-9. In a plasma-membrane-enriched fraction of the mutant MG2130, the ATPase activity was resistant to vanadate (50% inhibition by 26 microM in the mutant compared to 1.3 microM in the parental strain). Several catalytic properties of the membrane-bound ATPase were modified by 60-120% in the mutant which had a higher Km for MgATP and was more heatstable, less sensitive to mercurials, and more stimulated by monovalent cations than the parental type. A single mutation is responsible for the phenotypes of four independent allelic mutants. Resistance to Dio-9 in vivo and resistance to vanadate in vitro segregated together in three tetrads issued from a cross between the wild type and mutant. The mutation is semi-dominant as shown by expression of the mutant phenotype in a heterozygous diploid resulting from the cross between the wild type and mutant. It is concluded that the pma locus, affected by these mutations, is the structural gene either for the 100000-Mr subunit of plasma membrane ATPase or for a protein which tightly controls the conformation of the plasma-membrane ATPase within the membrane.

Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen.

Abstract: The synthesis of the H2 uptake system in free-living Rhizobium japonicum SR is repressed both by oxygen and by carbon substrates. Mutants selected for the ability to express hydrogenase in 10.0% partial pressure O2 were also less sensitive than the wild type to repression by carbon substrates such as arabinose, glycerol, gluconate, and succinate. The H2 uptake system in another class of mutants, previously shown to be hypersensitive to repression by O2, is also more sensitive to repression by carbon substrates. The oxygen- and carbon-insensitive mutants express the hydrogen uptake system during heterotrophic growth in the absence of hydrogen and thus can be considered constitutive (Hupc). The amount of cytochromes in the Hupc mutants is similar to that in the wild-type strain; however, the Hupc mutants contain greater methylene blue-dependent and O2-dependent hydrogenase activity, both as free-living cells and as bacteroids. Two-dimensional polyacrylamide gel electrophoresis revealed that during heterotrophic growth the Hupc mutant strain SR470 synthesized at least six peptides not found in the wild-type strain. The concentrations of cyclic AMP and guanosine tetraphosphate were similar in strain SR and the Hupc mutants during heterotrophic growth.

Novel use of synthetic oligonucleotide insertion mutants for the study of homologous recombination in mammalian cells

Abstract: Thymidine kinase-deficient mouse L cells have been transformed with plasmid DNAs carrying 8-base-pair Xho I linker insertion mutations in the coding region of the herpes simplex virus type 1 thymidine kinase gene. When the mutant plasmids are introduced individually into LTK- cells, transformation efficiencies are greatly reduced relative to the wild type. However, when two mutant plasmids are cotransferred into the same LTK- recipients, significantly higher frequencies of transformation are observed (30-300 times). Here we demonstrate the usefulness of linker insertions for the study of homologous recombination in detecting the existence of normal thymidine kinase gene sequences (i.e., sequences lacking the insertions after recombination are substantiated by DNA . DNA hybridization). In addition, the frequencies of recombination in the various "crosses" are consistent with the known positions of the mutations.

Isolation of mannan-protein complexes from viable cells of Saccharomyces cerevisiae X2180-1A wild type and Saccharomyces cerevisiae X2180-1 A-5 mutant strains by the action of Zymolyase-60,000.

Abstract: The viable whole cells of Saccharomyces cerevisiae X2180-1A wild type and its mannan mutant strain S. cerevisiae X2180-1A-5, were treated with an Arthrobacter sp. beta-1,3-glucanase in the presence of a serine protease inhibitor, phenyl-methylsulfonyl fluoride. Fractionation of the solubilized materials of each strain with Cetavlon (cetyltrimethylammonium bromide) yielded one mannan-protein complex. Molecular weights of these complexes were almost the same as that of the mannoprotein of the mutant strain prepared by Nakajima and Ballou, which had a molecular weight of 133,000 and were approximately three times larger than those of the mannans isolated from the same cells by hot-water extraction. Each mannan-protein complex contained up to 2% glucose residue, which was not removed by specific precipitation with anti-mannan sera or by affinity chromatography on a column of concanavalin A-Sepharose. Treatment of these complexes with alkaline NaBH4 produced peptide-free mannan containing small amounts of glucose nearly identical to those of the parent complexes. The above findings provide evidence that the glucose residues exist in a covalently linked form to the mannan moiety. Fractionation of the mannan-protein complex of the S. cerevisiae wild-type strain by DEAE-Sephadex chromatography yielded five subfractions of different phosphate content, indicating that these highly intact mannan-protein complexes were of heterogeneous material consisting of many molecular species of different phosphate content.

PHOTOMOVEMENT IN AN “EYELESS” MUTANT OF Chlamydomonas

Abstract: The function of the stigma (“eyespot”) in the green flagellate Chlamydomonas reinhardtii was investigated by comparing the photomovement of the wild-type alga with that of an “eyeless” mutant (ey 627). Movements of individual cells in response to a blue-green stimulus light were recorded using a videomicroscope system and were analyzed using vectorial methods. Cells of the “eyeless” mutant were phototactic; at a high stimulus fluence rate, their swimming paths were directed away from the light source. Although the orientation of the mutant was not as strongly directional as that of the wild type, it was statistically significant. However, the swimming paths of the mutant cells were very erratic in the presence of the stimulus beam, undergoing frequent changes of direction. Despite the differences in their phototactic orientation, cells of mutant and wild type all showed a distinct step-up photophobic response at the onset of stimulation. These results are consistent with the hypothesis that the stigma plays an accessory role in phototaxis, either by shading the photoreceptor or by acting as a quarter-wave reflector.

Isolation and characterization of yeast DNA repair genes : II. Isolation of plasmids that complement the mutations rad50-1, rad51-1, rad54-3, and rad55-3.

Abstract: Plasmids that complement the yeast mutations rad50-1, rad51-1, rad54-3 and rad55-3 were obtained by transforming strains that carried a leu2 marker and the particular rad mutation, with YEp13 plasmids containing near random yeast DNA inserts. Integration of these plasmids or of fragments of these plasmids was accomplished. Genetic studies using the integrants established the presence of the genes RAD51, RAD54 and RAD55 in the respective plasmids. However, a BamHI subclone of the rad50-1 complementing plasmid failed to integrate at the RAD50 locus, indicating that no homology exists between this fragment and the RAD50 gene.A BamHI fragment from the RAD54 plasmid was shown to be internal to the RAD54 gene: its integration within a wild type copy of RAD54 causes the cell to become Rad(-); its excision is X-ray inducible and restores the Rad(+) phenotype. Since cells bearing a disrupted copy of RAD54 are able to survive, we conclude that this gene is not essential.