Showing papers on "Mutant published in 1982"

Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions.

Abstract: Six Escherichia coli che loci (cheA,-B,-R,-W,-Y, and Z) are located in two adjacent operons that map at minute 42 on the chromosome. Point mutants defective in any of these six functions have aberrant swimming patterns and are generally nonchemotactic. Deletions within the two major che gene operons were isolated in order to examine epistatic interactions among these genes. We first constructed a specialized transducing phage (lambda che22), which carries both of the che operons and their associated promoters. Deleted lambda che22 derivatives were selected by chelating agent inactivation, and these derivatives were characterized by mapping them against a series of host strains with point mutations. Representative nonpolar deletions were then transferred into the E. coli chromosome by homologous recombination. Although the phenotype of cheR mutants (smooth swimming) was expected to be epistatic to that of cheB mutants (tumbly swimming), we found that deletion mutants lacking both of these functions exhibited frequent directional changes or tumbling episodes as they swam. An examination of larger deletions indicated that both the cheA-cheW and cheY-cheZ functions were required for the anomalous tumbling behavior observed in these mutants. Loss of the cheB function was also correlated with an inverted behavioral response to sodium acetate, a strong repellent of wild-type cells. These findings indicate that an important component of the signal transducing machinery may be altered in cheB mutants.

Cold-sensitive cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies

Abstract: We isolated 18 independent recessive cold-sensitive cell-division-cycle (cdc) mutants of Saccharomyces cerevisiae, in nine complementation groups. Terminal phenotypes exhibited include medial nuclear division, cytokinesis, and a previously undescribed terminal phenotype consisting of cells with a single small bud and an undivided nucleus. Four of the cold-sensitive mutants proved to be alleles of CDC11, while the remaining mutants defined at least six new cell-division-cycle genes: CDC44, CDC45, CDC48, CDC49, CDC50 and CDC51.--Spontaneous revertants from cold-sensitivity of four of the medial nuclear division cs cdc mutants were screened for simultaneous acquisition of a temperature-sensitive phenotype. The temperature-sensitive revertants of four different cs cdc mutants carried single new mutations, called Sup/Ts to denote their dual phenotype: suppression of the cold-sensitivity and concomitant conditional lethality at 37 degrees. Many of the Sup/Ts mutations exhibited a cell-division-cycle terminal phenotype at the high temperature, and they defined two new cdc genes (CDC46 and CDC47). Two cold-sensitive medial nuclear division cdc mutants representing two different cdc genes were suppressed by different Sup/Ts alleles of another gene which also bears a medial nuclear division function (CDC46). In addition, the cold-sensitive medial nuclear division cdc mutant csH80 was suppressed by a Sup/Ts mutation yielding an unbudded terminal phenotype with an undivided nucleus at the high temperature. This mutation was an allele of CDC32. These results suggest a pattern of interaction among cdc gene products and indicate that cdc gene proteins might act in the cell cycle as complex specific functional assemblies.

Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Abstract: Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.

Genetic control of heat-shock protein synthesis and its bearing on growth and thermal resistance in Escherichia coli K-12.

Abstract: When Escherichia coli cells grown at 30 degrees C are transferred to 42 degrees C, synthesis of several polypeptides is markedly and transiently induced. A temperature-sensitive nonsense mutant (tsn-K165) of E. coli K-12 is found to be defective in the induction of these proteins. mRNA for one major heat-shock polypeptide (groE protein) tested is induced in the wild type but not in the mutant upon temperature shift. Hence, the mutation defines a (regulatory) gene, designated hin (heat shock induction), whose product is required for active transcription of a set of heat-inducible operons in E. coli. The results reported herein suggest that the heat-shock polypeptides controlled by the hin gene play an important role in cell growth at high temperature. The possible involvement of the hin gene product in protection against thermal killing is also discussed.

Cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis

Abstract: The activity of a cystine transport system in lysosomes prepared from the leukocytes of patients with cystinosis was found to be deficient. In normal subjects, this system was resistant to N-ethylmaleimide and demonstrated saturation kinetics. Lysosomes from individuals heterozygous for cystinosis demonstrated a reduced maximum velocity for cystine egress from lysosomes. The rate of cystine escape from normal lysosomes was enhanced by adenosine triphosphate. The availability of normal and mutant lysosomes provides a means of investigating mechanisms of amino acid transport across lysosomal membranes.

Polycomblike: a gene that appears to be required for the normal expression of the bithorax and antennapedia gene complexes of drosophila melanogaster

Abstract: A newly identified gene is described that is required for the maintenance of normal identities in many of the body segments of the fly. The effects of mutants in this gene, which is called Polycomblike (Pcl), suggest that its wild-type allele functions in the regulation of the bithorax gene complex (BX-C) and the Antennapedia gene complex (ANT-C). Evidence in favor of this idea derives from (1) the close correspondence between segmental transformations caused by Pcl mutants and those caused by dominant gain-of-function mutants in the BX-C and ANT-C, (2) the interactions observed between Pcl mutants and mutants in these complexes, and (3) the dependence upon BX-C and ANT-C dosage of the severity of at least one of the transformations caused by Pcl mutants. Arguments are presented that the control of the BX-C and ANT-C by Pcl+ is negative in nature. The results of clonal analysis experiments indicate that, at least for the BX-C, Pcl+ exerts this control until late in development. Since the wild-type allele of another gene, called Polycomb (Pc), has previously been shown to have many of the same properties as Pcl+, it appears that the BX-C and perhaps also the ANT-C are continuously regulated during development by at least two and probably several other genes.

Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild-type with an antibiotic-supersusceptible mutant.

Abstract: The Pseudomonas aeruginosa mutant Z61 has been shown to be highly supersusceptible to a wide range of antibiotics, including beta-lactams, aminoglycosides, rifampin, tetracycline, and chloramphenicol (W. Zimmerman, Int. J. Clin. Pharmacol. Biopharm. 17:131-134, 1979). Spontaneous revertants were isolated, using gentamicin or carbenicillin as selective agents, and shown to have two patterns of susceptibility to a group of 12 antibiotics. Partial revertants had 2- to 10-fold greater resistance to these antibiotics than mutant Z61, whereas full revertants had antibiotic susceptibilities indistinguishable from those of the wild-type strain K799, from which mutant Z61 had been derived. Uptake of a chromogenic beta-lactam nitrocefin was studied in both uninduced and induced cells of all strains by measuring the steady-state rate of nitrocefin hydrolysis by the inducible, periplasmic beta-lactamase in both whole and broken cells. This demonstrated that outer membrane permeability decreased as antibiotic resistance increased in the series mutant Z61, partial revertants, wild type, and full revertants. The data were consistent with the idea of low outer membrane permeability being caused by a low proportion of open functional porins in the outer membrane as the reason for the high natural antibiotic resistance of wild-type P, aeruginosa strains. In addition, it was observed that levels of benzylpenicillin below the minimal inhibitory concentration for mutant Z61 failed to induce beta-lactamase production. The possibility that this was related to the observed increase in outer membrane permeability is discussed. Preliminary evidence is presented that the pore-forming outer membrane porin protein F is not altered in mutant Z61.

Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Abstract: Saccharomyces cerevisiae MATa cells carrying mutations in either sst1 or sst2 are supersensitive to the G1 arrest induced by alpha factor pheromone. When sst1 mutants were mixed with normal SST+ cells, the entire population recovered together from alpha factor arrest, suggesting that SST+ cells helped sst1 mutants to recover. Complementation tests and linkage analysis showed that sst1 and bar1, a mutation which eliminates the ability of MATa cells to act as a "barrier" to the diffusion of alpha factor, were lesions in the same genes. These findings suggest that sst1 mutants, are defective in recovery from alpha factor arrest because they are unable to degrade the pheromone. In contrast, recovery of sst2 mutants was not potentiated by the presence of SST+ cells in mixing experiments. When either normal MATa cells or mutant cells carrying defects in sst1 or sst2 were exposed to alpha factor for 1 h and then washed free of the pheromone, the sst2 cells subsequently remained arrested in the absence of alpha factor for a much longer time than SST+ or sst1 cells. These observations suggest that the defect in sst2 mutants is intrinsic to the cell and is involved in the mechanism of alpha factor action at some step after the initial interaction of the pheromone with the cell. The presence of an sst2 mutation appears to cause a growth debility, since repeated serial subculture of haploid sst2-1 strains led to the accumulation of faster-growing revertants that were pheromone resistant and were mating defective ("sterile").

Identification of Mycoplasma pneumoniae proteins associated with hemadsorption and virulence.

Abstract: Twenty-two mutants of Mycoplasma pneumoniae spontaneously deficient in hemadsorption were isolated. Examination of mutant protein profiles by one- and two-dimensional polyacrylamide gel electrophoresis permitted the grouping of these mutants into four classes. The largest class of mutants was deficient in four high-molecular-weight proteins (215,000, 210,000, 190,000, and 140,000). A second class of mutants lacked three proteins previously designated A, B, and C (72,000, 85,000, and 37,000, respectively). A single mutant, in addition to lacking proteins A, B, and C, was missing a fourth protein of 165,000 molecular weight. The remaining mutants exhibited protein profiles apparently identical to that of the wild-type strain. All mutant strains attached to the respiratory epithelium of hamster tracheal rings in vitro at reduced levels; however, mutants lacking proteins A, B, and C recognized only neuraminidase-insensitive receptors. None of the mutants tested produced detectable pneumonia in intranasally inoculated hamsters, although one mutant class demonstrated low-level survival in vivo.

DNA sequences necessary for transcription of the rabbit β -globin gene in vivo

Abstract: The DNA sequences required for the expression of the rabbit-beta-globin gene in vivo have been examined. A variety of mutant rabbit beta-globin gene templates were linked to a simian virus 40-plasmid recombinant and introduced into HeLa cells; in these conditions the rabbit beta-globin gene is expressed from its own promoter. Comparison of the level of beta-globin transcripts in a variety of deletion mutants shows that for efficient transcription, both the ATA or Goldberg-Hogness box, and a region between 100 and 58 base pairs in front of the site at which transcription is initiated, are required. Deletion of either of these regions results in a decrease in the level of beta-globin transcripts by an order of magnitude; deletion of the ATA box causes an additional loss in the specificity of the site of initiation of RNA synthesis. The DNA sequences downstream from the ATA box, including the natural beta-globin mRNA cap site, are dispensable for transcription in vivo.

Agrobacterium tumefaciens mutants affected in attachment to plant cells.

Abstract: An analysis of Agrobacterium tumefaciens mutants with Tn5 insertions in chromosomal DNA showed that the chromosome of A. tumefaciens codes for a specific ability of this bacterium to attach to plant cells. This ability is associated with tumorigenesis by A. tumefaciens, the ability of avirulent A. tumefaciens to inhibit tumorigenesis, and the ability to adsorb certain phages. A second class of chromosomal mutations affects tumorigenesis without altering the ability to attach to plant cells. The attachment of A. tumefaciens to plant cells was assayed by mixing radiolabeled bacteria with suspensions of tobacco tissue culture cells or freshly isolated Zinnia leaf mesophyll cells. Under the conditions of this assay, an avirulent Ti plasmid-cured strain attached to the same extent as the same strain containing pTiB6806. Six of eight avirulent mutants with Tn5 insertions in chromosomal DNA showed defective attachment, whereas two retained wild-type attachment ability. In contrast to the strains showing wild-type attachment, the attachment-defective mutants failed to inhibit tumorigenesis when inoculated onto Jerusalem artichoke slices before inoculation of a virulent strain and also showed a loss of sensitivity to two Agrobacterium phages. The loss of phage sensitivity appeared to be due to a loss of ability to adsorb the phages. Staining with Calcofluor indicated that the mutants retained the ability to synthesize cellulose fibrils, which have been implicated in the attachment process. Southern filter hybridizations demonstrated that each mutant contained a single Tn5 insertion, and genetic linkage between the Tn5 insertion in one mutant and the attachment phenotype has also been demonstrated.

DNA-mediated transfer of multiple drug resistance and plasma membrane glycoprotein expression.

Abstract: Colchicine-resistant Chinese hamster ovary (CHO) cell mutants whose resistance results from reduced drug permeability have been isolated previously in our laboratories. This reduced permeability affects a wide range of unrelated drugs, resulting in the mutants displaying a multiple drug resistance phenotype. A 170,000-dalton cell surface glycoprotein (P-glycoprotein) was identified, and its expression appears to correlate with the degree of resistance. In this study we were able to confer the multiple drug resistance phenotype on sensitive mouse L cells by DNA-mediated gene transfer of DNA obtained from the colchicine-resistant mutants. P-glycoprotein was detected in plasma membranes of these DNA transformants by staining with an antiserum raised against membranes of mutant CHO cells. These results are consistent with a causal relationship between P-glycoprotein expression and the multiple drug resistance phenotype.

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function

Abstract: We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.

Mutants blocked in streptomycin production in Streptomyces griseus - the role of A-factor.

Abstract: Ninety-five streptomycin-nonproducing mutants derived from Streptomyces griseus FT-1 by UV-irradiation could be classified into major two classes by cosynthesis tests. Class I mutants (42 strains) were mutants blocked in the pathway of streptomycin biosynthesis while class II mutants (49 strains) required a factor for streptomycin biosynthesis which was excreted by the parental or class I mutant strains. The factor could be replaced by synthetic A-factor (2S-isocapryloyl-3S-hydroxymethyl-γ-butyrolactone) which restored both streptomycin biosynthesis and spore formation in the class II mutants. A-Factor deficient mutants were obtained from several strains of S. griseus and S. bikiniensis at high frequency by treatment with acridine orange or incubation at high temperature. A mutant whose streptomycin biosynthesis was independent of A-factor deficiency was found. The production of A-factor was distributed among various species of actinomycetes.

Mutagenesis in Saccharomyces Cerevisiae

Abstract: Publisher Summary This chapter discusses the mutagenesis in Saccharomyces cerevisiae and compares it with what is known about the same process in E. coli. It also discusses the experimental systems available for mutation research in bakers' yeast, the result of investigations concerning induced and spontaneous mutagenesis in nuclear and mitochondrial genomes, and the conclusions regarding the mechanisms of mutagenesis. Direct screening methods are used for isolating mutants exhibiting either enhanced or diminished levels of spontaneous mutagenesis. A variety of mutations, which reduce spontaneous mutation rates are isolated, one of which is an allele of the REV3 gene. The phenotypes of excision-deficient mutants, that are tabulated, indicate that excision repair in Saccharomyces, as in E. coli, is essentially error-free. The products of at least nine and probably 12 or more genes are concerned with excision repair in yeast. Mutations in nine of these genes prevent the excision of all, or at least a fraction, of pyrimidine dimers and block an early stage in this process, before or at about the incision stage.

Structure and expression of a mouse major histocompatibility antigen gene, H-2Ld

Abstract: A genomic clone encoding H-2Ld, a mouse major transplantation antigen, has been identified and the structure of the H-2Ld gene has been partially determined. We isolated 35 genomic clones from a BALB/c (H-2d) genomic library by hybridization to mouse or human probes. One of these clones encodes H-2Ld as determined by two criteria. First, the gene encodes a protein that is identical at the 76 known amino acid positions for H-2Ld. Second, when introduced into L cells by DNA-mediated gene transfer, a new H-2 antigen is expressed that is recognized by anti-H-2Ld monoclonal antibodies. The sequence of the H-2Ld protein predicted by the DNA sequences shows more than 80% homology to known H-2 antigens. H-2L-like sequences are found in mutant H-2Kb molecules, suggesting that gene conversion or reciprocal recombination may play a role in the development of H-2 polymorphism.

Identification of plasmid (pKM101)-coded proteins involved in mutagenesis and UV resistance.

Abstract: Mutagenesis of Escherichia coli by UV and many chemicals is not a passive process and requires the intervention of host functions. The isolation of chromosomal umuC mutants, which are nonmutable by many DNA-damaging agents and are slightly sensitive to them, has led to the model that the umuC gene product(s) functions in a pathway of ‘error-prone repair’1–3. UmuC mutants are suppressed by the introduction of the plasmid pKM1014; pKM101 was derived from the clinically isolated plasmid R46 by in vivo manipulations5. The muc (mutagenesis, UV and chemical) region of pKM101 is required for this suppression and we have suggested that it is an analogue of the chromosomal umuC gene(s)6. We have now subcloned the muc region onto the high-copy number vector pKB354 and identified two protein products by gel electrophoresis of maxicell preparations. Complementation analysis of muc insertion mutations has confirmed the existence of two genes, mucA and mucB, whose protein products are required for pKM101's effects on mutagenesis and resistance to killing. The molecular weights (MWs) of the mucA and mucB proteins are 16,000 and 45,000 respectively.

Fusions of flagellar operons to lactose genes on a mu lac bacteriophage.

Abstract: Previous studies have defined 29 genes necessary for synthesis of the Escherichia coli flagellar apparatus. This study analyzed the transcriptional control of flagellar genes, using Mu d (Apr lac) phage to generate flagellar mutants by insertion. These mutants contained operon fusions of flagellar genes to the lac genes of the Mu d phage and allowed the measurement of flagellar operon expression by detection of beta-galactosidase activity. These fusion mutants expressed the enzyme activity constitutively, and an autogenous regulation mechanism was not revealed. Lambda transducing phages carrying these chromosomal fla-lac fusions were also isolated and used to examine the effect of different fla mutations on expression of each flagellar operon. The results showed that flagellar operons are divided into six classes; (class 1) the flbB operon, which controls all of the other flagellar operons; (class 2) the flaU and flbC operons, which are controlled by the flbB operon gene products and are not required for the expression of other Fla operons; (class 3) the flbA, flaG, flaD, flaN, flaB, and flaA operons, which are under flbB operon control and are required for the expression of other fla operons; (class4) the flaZ operon, which is controlled by the gene products of the group 1 and 3 operons and is required for hag transcription; (class 5) the mocha and flaS operons, which are controlled by the gene products of the group 1 and 3 operons; and (class 6) the hag operon. These results are discussed with respect to the possible assembly sequence of the fla gene products.

A Mutant of Arabidopsis thaliana Which Lacks Activation of RuBP Carboxylase In Vivo.

Abstract: A mutant of Arabidopsis thaliana has been isolated in which ribulose-1,5-bisphosphate carboxylase is present in a nonactivatable form in vivo. The mutation appears to affect carboxylase activation specifically, and not any other enzyme of the photosynthesis or photorespiratory cycles. The effect of the mutation on carboxylase activation is indirect, inasmuch as the properties of ribulose-1,5-bisphosphate carboxylase purified from the mutant are not distinguishable from those of the wild type enzyme. The mutant requires high levels of atmospheric CO2 for growth because photosynthesis is severely impaired in atmospheres containing normal levels of CO2, irrespective of the atmospheric O2 concentration. In this respect, the mutant is distinguished from previously described high-CO2 requiring mutants of Arabidopsis which have defects in photorespiratory carbon or nitrogen metabolism.