Showing papers on "Mutant published in 1983"

Characterization of the herpes simplex virion-associated factor responsible for the induction of alpha genes.

Abstract: Herpes simplex virus (HSV) genes form three groups, alpha, beta, and gamma, whose synthesis is coordinately regulated and sequentially ordered in a cascade fashion. Earlier studies by Post et al. (Cell 24:555-565, 1981) have shown that chimeric genes constructed by fusion of 5' noncoding leader and upstream sequences of alpha genes to the 5' noncoding leader and structural sequences of the viral thymidine kinase (TK), a beta gene, are regulated as alpha genes upon recombination into the viral genome. In cells converted from TK- to TK+ phenotype, these chimeric genes are induced by infection with homologous TK- virus. The induction of the resident chimeric gene does not require viral protein synthesis and is independent of the presence of functional alpha gene 4 product required for the expression of beta genes. In this paper, we report on the properties of the alpha-TK gene chimera resident in converted TK+ murine (L316) and human (I316) cells. Our results were as follows. (i) The pattern of induction of L316 cells exposed to 0.1, 1.0, and 10 PFU per cell suggested that exposure to competent virus is required for induction and that in untreated preparations this virus corresponds to infectious virus. (ii) UV light-irradiated virus was just as effective as untreated virus in inducing alpha-TK chimeras. (iii) HSV-1(HFEM)tsB7 induced the alpha-TK gene chimeras at the nonpermissive (39 degrees C) temperature; at 39 degrees C the parental HSV-1(HFEM)tsB7 capsids accumulate at nuclear pores and do not release viral DNA. (iv) The alpha-TK gene chimeras were not induced by infection with spontaneous TK- mutants of pseudorabies virus and bovine mammillitis virus or with human cytomegalovirus or adenovirus type 2 or by exposure to lysates of HSV-1-infected cells from which the virus was removed by centrifugation. These results indicate that the alpha gene inducer is a virion component located outside the capsid and that its function might be to stimulate the transcription of alpha genes by recognizing regulatory sites on viral DNAs or host cell products or both.

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

Abstract: We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.

Alpha-thalassaemia caused by a polyadenylation signal mutation.

Abstract: Most eukaryotic messenger RNAs have the sequence AAUAAA 11–30 nucleotides from the 3′-terminal poly(A) tract1,2. Since this is the only significant sequence homology in the 3′ non-coding region it has been suggested that it may be a recognition site for enzymes involved in polyadenylation and/or termination of polymerase II transcription2–4. This idea is strengthened by observations on the effect of deletion mutations in or around the AATAAA sequence on polyadenylation of late simian virus 40 (SV40) mRNA; removal of this sequence prevents poly(A) addition3. Naturally occurring variants ofthis hexanucleotide are rare5–11 and hitherto their functional significance has not been assessed. We have now identified a human α2-globin gene which contains a single point mutation in this hexanucleotide (AATAAA → AATAAG). The paired α1 gene on the same chromosome is completely inactivated by a frame-shift mutation. This unique combination has enabled the expression of the mutant α2 gene to be studied in vivo where it has been found that the accumulated level of α2-specific mRNA in erythroid cells is reduced. Furthermore, readthrough transcripts extending beyond the normal poly(A) addition site are detected in mRNA obtained from HeLa cells transfected with cloned DNA from the mutant α2 gene, suggesting that the single nucleotide change in the AATAAA sequence is the cause of its abnormal expression.

X-ray-sensitive mutants of Chinese hamster ovary cell line. Isolation and cross-sensitivity to other DNA-damaging agents.

Abstract: A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D10 values 5–10-fold of wild-type D10 value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D10 values less than 2-fold of wild-type D10 value). The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks.

Herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of alpha (immediate early) viral polypeptides.

Abstract: Six mutants isolated from herpes simplex virus type 1 were judged to be defective with respect to the virion-associated function acting to rapidly shut off host polypeptide synthesis in herpes simplex virus-infected cells. The mutants were capable of proper entry into the cells, but, unlike the parent wild-type virus, they failed to shut off host polypeptide syntehsis in the presence of actinomycin D. They were consequently designated as virion-associated host shutoff (vhs) mutants. In the presence of actinomycin D, three of the mutants, vhs1, -2, and -3, failed to shut off the host at both 34 and 39 degrees C, whereas vhs4, -5, and -6 exhibited a temperature-dependent vhs phenotype. Since the mutants were capable of growth at 34 degrees C, it appeared that the vhs function was not essential for virus replication in cultured cells. Temperature-shift experiments performed with the vhs4 mutant showed that an active vhs function was required throughout the shutoff process and that, once established, the translational shutoff could not be reversed. In the absence of actinomycin D, the mutants induced a generalized, secondary shutoff of host translation, which required the synthesis of beta (early) or gamma (late) viral polypeptide(s). The vhs mutants appeared to be defective also with respect to post-transcriptional shutoff of alpha (immediate early) viral gene expression, since (i) cells infected with mutant viruses overproduced alpha viral polypeptides, (ii) there was an increased functional stability of alpha mRNA in the vhs1 mutant virus-infected cells, and (iii) superinfection of vhs1-infected cells with wild-type virus, in the presence of actinomycin D, resulted in a more pronounced shutoff of alpha polypeptide synthesis from preformed alpha mRNA than equivalent superinfection with vhs1 virus. The data suggest that the synthesis of alpha polypeptides in wild-type virus infections is subject to a negative post-transcriptional control involving viral gene product(s) present in infected cell lysates constituting virus stocks. The vhs1 mutant and possibly other vhs mutants contain a mutation in the gene encoding this function.

Cysteine-depleted muteins of biologically active proteins

Abstract: Muteins of biologically active proteins such as IFN-.beta. and IL-2 in which cysteine residues that are not essential to biological activity have been replaced with other amino acids, or in the case of IFN-.beta. deleted or replaced with other amino acids, to eliminate sites for intermolecular crosslinking or incorrect intramolecular disulfide bridge formation. These muteins are made via bacterial expression of mutant genes that encode the muteins that have been synthesized from the genes for the parent proteins by oligonucleotide-directed mutagenesis.

An altered aroA gene product confers resistance to the herbicide glyphosate

Abstract: The hypothesis that the herbicide glyphosate (N-phosphonomethylglycine) acts on plants and microorganisms by inhibiting synthesis of 5-enolpyruvyl-3-phosphoshikimate, a precursor to aromatic amino acids, was tested. Salmonella typhimurium was treated with ethyl methanesulfonate, and mutants mapping at the aroA locus, which encodes 5-enolpyruvyl-3-phosphoshikimate synthetase, were isolated by selection for glyphosate resistance. One of the mutants results in the synthesis of a 5-enolpyruvyl-3-phosphoshikimate synthetase that is resistant to inhibition by glyphosate. The mutant aroA gene and the corresponding wild-type allele were cloned. The mutation confers high resistance to glyphosate when introduced in Escherichia coli in the presence or absence of the wild-type aroA allele.

Identification and characterization of a herpes simplex virus gene product required for encapsidation of virus DNA

Abstract: A mutant of herpes simplex virus type 1, 17tsVP1201, has a temperature-sensitive processing defect in a late virus polypeptide. Immunoprecipitation studies with monoclonal antibodies showed that the aberrant polypeptide in mutant virus-infected cells was the nucleocapsid polypeptide known as p40. Since a revertant, TS(+) for growth, processed the polypeptide normally under conditions restrictive for the mutant, the processing event must be essential for virus replication. Electron microscopic analysis of mutant virus-infected cells grown at the nonpermissive temperature revealed that the nuclei contained large aggregations of empty nucleocapsids possessing some internal structure. Therefore, although the mutant synthesized virus DNA at the nonpermissive temperature, the DNA was not packaged into nucleocapsids. When mutant virus-infected cells were shifted from 39 to 31 degrees C in the presence of cycloheximide, the polypeptide p40 was processed to lower-molecular-weight forms, and full nucleocapsids were detected in the cell nuclei. The aberrant polypeptide of the mutant, however, was not processed in cells mixedly infected with 17tsVP1201 and a revertant at the nonpermissive temperature, suggesting that the defect of the mutant was in the gene encoding p40 rather than in a gene of a processing enzyme.

Acquisition of Host Cell DNA Sequences by Baculoviruses: Relationship Between Host DNA Insertions and FP Mutants of Autographa californica and Galleria mellonella Nuclear Polyhedrosis Viruses.

Abstract: Mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses, which produce an altered plaque phenotype as a result of reduced numbers of viral occlusions in infected cells, were isolated after passage in Trichoplusia ni (TN-368) cells. These mutants, termed FP (few-polyhedra) mutants, had acquired cell DNA sequences ranging from 0.8 to 2.8 kilobase pairs in size. The insertions of cell DNA occurred in a specific region between 35.0 and 37.7 map units of the A. californica viral genome. A cloned viral fragment containing one of the host DNA inserts was homologous to host DNA inserts in two other mutant viruses and to dispersed, repetitious sequences in T. ni cell DNA. Most of the homology between the cloned insert and cell DNA was contained within a 1,280-base-pair AluI fragment. Marker rescue studies and analysis of infected-cell-specific proteins suggested that the insertion of cell DNA into the viral genomes resulted in the FP plaque phenotype, possibly through the inactivation of a 25,000-molecular-weight protein.

Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli.

Abstract: A phoA-lacZ gene fusion was used to isolate mutants altered in the alkaline phosphatase signal sequence. This was done by selecting Lac+ mutants from a phoA-lacZ fusion strain that produces a membrane-bound hybrid protein and is unable to grow on lactose. Two such mutant derivatives were characterized. The mutations lie within the phoA portion of the fused gene and cause internalization of the hybrid protein. When the mutations were genetically recombined into an otherwise wild-type phoA gene, they interfered with export of alkaline phosphatase to the periplasm. The mutant alkaline phosphatase protein was found instead in the cytoplasm in precursor form. DNA sequence analysis demonstrated that both mutations lead to amino acid alterations in the signal sequence of alkaline phosphatase. Images

Isolation of genomic clones containing the amdS gene of Aspergillus nidulans and their use in the analysis of structural and regulatory mutations.

Abstract: Previous analysis of the amdS gene of Aspergillus nidulans has identified multiple regulatory circuits mediated by trans-acting regulatory genes, cis-acting mutations have been identified and shown to specifically affect individual regulatory circuits. Fine-structure genetic mapping of the amdS regions showed that these cis-acting mutations occur in a complex controlling region adjacent to the amdS structural gene. The amdS gene was cloned by differential hybridization, using cDNA probes derived from a high-level-producing strain and from a strain with a large amdS deletion mutation. RNA blotting experiments showed that a single RNA species of 1,600 to 1,700 base pairs is transcribed from the amdS gene. DNA blotting experiments on a large number of amdS mutant strains, including deletions and translocations, allowed the genetic and physical maps of the gene to be correlated. The controlling region of the gene is situated at the 5' end of the gene and the direction of transcription is toward the centromere of chromosome III. The regulatory mutations in the controlling region were found to be due to small-scale alterations in the DNA rather than to large-scale rearrangements resulting in gene fusions.

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.

Mutations in a new gene, secB, cause defective protein localization in Escherichia coli.

Abstract: We isolated a new class of Escherichia coli mutants with pleiotropic defects in protein secretion. Using a previously described selection procedure (Oliver et al., Ann. Microbiol. [Paris] 133A:105-110, 1982), we obtained a large collection of strains containing mutations that affect protein localization. In many cases, the lesions causing the secretion defects were mapped in or near the previously identified gene, secA (Oliver and Beckwith, Cell 25:765-772, 1981). However, the selection also yielded mutants with mutations in a new locus, which was designated secB. These secB mutants were defective in the localization of maltose-binding protein and, in at least one case, OmpF protein. Double mutants with lesions in both secA and secB had strong defects in the secretion of maltose-binding protein and OmpF protein. The secB locus mapped near cysE at min 80.5 on the E. coli genetic map. The properties of secB mutants suggest that the secB product could be a component of the E. coli secretory apparatus.

Yeast mutants deficient in protein glycosylation

Abstract: The synthesis of asparagine-linked oligosaccharides involves the formation of a lipid-linked precursor oligosaccharide that has the composition Glc3Man9GlcNAc2. We have used a [3H]mannose suicide selection to obtain mutants in yeast that are blocked in the synthesis of this precursor oligosaccharide. The alg1 mutant accumulated lipid-linked GlcNAc2, alg2 mutants accumulated Man1-2GlcNAc2, alg3 mutants accumulated Man5GlcNAc2, alg4 mutants accumulated Man1-8GlcNAc2, and alg5 and alg6 mutants accumulated Man9GlcNAc2. Some of these mutants appeared to transfer oligosaccharides other than Glc3Man9GlcNAc2 from the lipid carrier to invertase. These aberrant protein-linked oligosaccharides were processed by the addition of outer chain residues in the alg3, alg5, and alg6 mutants. There was virtually no outer chain addition in the alg2 and alg4 mutants. alg4 was the only mutant that failed to secrete invertase.

The structure of a mutant H-2 gene suggests that the generation of polymorphism in H-2 genes may occur by gene conversion-like events.

Abstract: We have cloned a mutant allele of the H–2K b gene known as H–2K bml. DNA sequence analysis of this gene shows that it is the result of an intergenic exchange of DNA (gene conversion) which results in the conversion of a short internal segment of the H–2K b to the corresponding sequence of another H–2 gene. We suggest that this type of gene conversion is widespread in H–2 genes and that it is a major force in the generation of polymorphism in H–2 genes.

Measurement of in vivo mutations in human lymphocytes.

Abstract: A means of measuring in vivo mutations in man would be valuable in assessing the importance of mutation in ageing and human disease and for monitoring individuals exposed to environmental mutagens and carcinogens. The hypoxanthine–guanine phosphoribosyl transferase (HGPRT) locus has been used to study mutagenesis in cultured mammalian (and other) cells1; clones are selected by their ability to grow in the presence of the purine analogue, 6-thioguanine (6-TG). Such clones are almost always HGPRT− and are therefore probably the progeny of a mutant cell. This system has been applied to human lymphocytes by using autoradiography to detect single mutant cells2–6. This approach probably does detect mutant cells, particularly if their number is increased, but the results obtained are variable and rather imprecise4,5. Also, the technique does not allow isolation of clones, thus the mutational origin of thioguanine-resistant (TGr) cells cannot be determined as the gene product, HGPRT, cannot be measured. Previous methods for cloning peripheral blood lymphocytes (PBL) have been so inefficient that detection of TGr lymphocytes has not been practicable. We have cloned PBL by a technique which is highly efficient (20–60% of PBL form a clone) and results in large clones of 103–104 cells that are readily scored using an inverted microscope6,7. We report here that the in vivo mutation frequency is of the order of that observed for human fibroblast lines cultured in vitro1.

Isolation and characterization of Azospirillum mutants excreting high amounts of indoleacetic acid

Abstract: Mutants of Azospirillum brasilense Sp Cd, resistant to 5-fluorotryptophan (FT) excreted 3-indoleacetic acid (IAA), ie, auxin, producing up to 16 μg/mL which was 30 times greater than the wild-typ

Virulence for mice of a proteinase-secreting strain of Candida albicans and a proteinase-deficient mutant.

Abstract: SUMMARY: A proteinase-deficient mutant of Candida albicans, M12, was produced by nitrosoguanidine mutagenesis of a proteinase-producing strain, ATCC 28366. The mutant was phenotypically identical to its parent in nearly all biochemical and morphological characteristics except proteinase production. The mutant was considerably less lethal than the parent when inoculated intravenously into mice and lower counts of C. albicans were recovered from the organs of mice infected with the mutant. Both strains were phagocytosed and killed to a similar extent by human and murine polymorphonuclear leukocytes when the yeasts were grown in a medium that did not induce proteinase production. However, in a proteinase-inducing medium, ATCC 28366 was phagocytosed and killed less well than M12. These results indicate that proteinase secretion by C. albicans is one factor determining the virulence of the species, but that other virulence factors are also involved in the pathogenesis of systemic candidosis.

Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene.

Abstract: A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.

Gene in the major cotransduction gap of the Escherichia coli K-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics.

Abstract: In Escherichia coli K-12, amplifiable resistance to tetracycline, chloramphenicol, and other unrelated antibiotics was mediated by at least four spatially separated loci. Tetracycline-sensitive mutants were isolated by Tn5 insertional inactivation of an amplified multiply resistant strain. One of these, studied in detail, showed coordinate loss of expression of all other resistance phenotypes. The Tn5 element in this mutant mapped to 34 min on the E. coli K-12 linkage map. We have designated the locus marA (multiple antibiotic resistance). Tetracycline-sensitive mutants containing marA::Tn5 regained all resistance phenotypes at frequencies of 10(-8) to 10(-7) upon precise excision of Tn5. Moreover, a newly described tetracycline efflux system (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983) was inactivated in tetracycline-sensitive mutants, but recovered in tetracycline-resistant revertants. In merodiploids, F-prime marA+ expressed partial or complete dominance over corresponding mutant chromosomal alleles. Dominance tests also established that a previously amplified host and a mutant marA allele were preconditions for the expression of phenotypic resistances.

Multidrug-resistance phenotype in Chinese hamster ovary cells.

Abstract: Multidrug resistance is a complex pleiotropic phenotype of cross-resistance and collateral sensitivity to unrelated compounds observed in many mammalian cell mutants selected for resistance to single agents. In Chinese hamster ovary cells, colchicine-resistant mutants expressing this phenotype have been characterized extensively. Such mutants arise apparently from a single genetic event, and the basis of this phenotype appears to be localized at the membrane level, resulting in altered drug permeability. Expression of a 170,000-dalton surface glycoprotein (P-glycoprotein) has been identified to correlate with the multidrug-resistance phenotype. Selection of a second mutation in colchicine-resistant mutants, for resistance to phytohemagglutinin, results in an alteration of the carbohydrate moiety in P-glycoprotein and other surface components. This mutation does not noticeably affect the multi-drug-resistance phenotype. The altered permeability of mutant cells to drugs, however, can be modulated by nonionic detergents or metabolic inhibitors. These findings are consistent with a molecular mechanism of multidrug resistance whereby the pleiotropic response of the cell is mediated by an overexpression of a cell-surface protein, the P-glycoprotein.

An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast

Abstract: The peptide mating pheromone alpha-factor and the hydrolytic enzyme invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (Saccharomyces cerevisiae). An in-frame fusion of the structural genes for these two proteins was constructed by connecting the 5'-flanking region and prepro-leader portion of the coding sequence of the alpha-factor gene (MF alpha 1) to a large fragment of the invertase gene (SUC2) lacking its 5'-flanking region and the coding information for the first four amino acids of its signal sequence. Sites that have been implicated in normal proteolytic processing of the alpha-factor precursor have been retained in this construction. The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media. This extracellular invertase appears to contain no prepro-alpha-factor sequences. The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (sec18). Therefore, prior to its efficient proteolytic removal, the alpha-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase. Similar to MF alpha 1, the MF alpha 1-SUC2 fusion is expressed in alpha haploids at levels 65-75 times higher than in a haploids or in a/alpha diploids; also, high-level expression is eliminated in mat alpha 1 mutants but not in mat alpha 2 mutants. Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration. Hence, the 5'-flanking region present in the fusion (about 950 base pairs) is sufficient to confer alpha cell-specific expression to the hybrid gene.