Error induction and correction by mutant and wild type T4 DNA polymerases. Kinetic error discrimination mechanisms.

The fidelity of DNA synthesis as determined by the misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases. Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased. Thus, the nuclease/polymerase ratio of each of these three DNA polymerases can be controlled. The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration. The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio. The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis with decreasing substrate concentrations is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity. The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs. A value of 1.1 kcal/mol free energy difference, delta G, between adenine: thymine and aminopurine:thymine base pairs is predicted by model analysis of the cocentration dependence of aminopurine misincorporation and removal frequencies. An independent estimate of this free energy difference based on the 6-fold higher apparent Km of T4 DNA polymerase for aminopurine compared to adenine also gives a value of 1.1 kcal/mol. It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase alpha, is 12 to 15%, which is similar to that for the T4 polymerases and consistent with delta G approximately 1.1 kcal/mol.     

Characterization of cyanobacterial glycogen isolated from the wild type and from a mutant lacking of branching enzyme

Cyanobacteria produce glycogen as their primary form of carbohydrate storage. The genomic DNA sequence of Synechocystis sp. PCC6803 indicates that this strain encodes one glycogen-branching enzyme (GBE) and two isoforms of glycogen synthase (GS). To confirm the putative GBE and to demonstrate the presence of only one GBE gene, we generated a mutant lacking the putative GBE gene, sll0158, by replacing it with a kanamycin resistance gene through homologous recombination. GBE in sll0158(-) mutant was eliminated; the mutant strain produced less glucan, equivalent to 48% of that produced by the wild type. In contrast to the wild-type strain that had 74% of the glucan being water-soluble, the mutant had only 14% of the glucan water-soluble. Molecular structures of glucans produced by the mutant and the wild type were characterized by using high-performance size-exclusion and anion-exchange chromatography. The glycogen produced by the wild type displayed a molecular mass of 6.6 x 10(7) daltons (degree of polymerization (DP) 40700) and 10% branch linkages, and the alpha-D-glucan produced by the mutant displayed a molecular mass of 4.7-5.6 x 10(3) daltons (DP 29-35) with slight branch linkages. The results indicated that sll0158 was the major functional GBE gene in Synechocystis sp. PCC6803.     

Differential inhibition of branching enzyme in a morphological mutant and in wild type Neurospora. Influence of carbon source in the growth medium.

1. A morphological mutant of Neurospora crassa, smco 9, (R2508) that exhibits colonial morphology when grown on sucrose or on maltose, showed a partial reversal of this morphology toward that of the wild type when it was grown on potato starch or on isomaltose. 2. A common feature of both potato starch and isomaltose is the presence of alpha-1, 6 glucosidic linkages. This suggested that these morphological effects might be due to differences in alpha-1,4 glucan: alpha-1,4 glucan 6 glycosyltransferase, (EC 2.4.1.18) commonly known as "the branching enzyme". 3. The branching enzyme was purified from wild type, Neurospora crassa, and from the semicolonial mutant, R2508, both grown on sucrose or on potato starch. It has a molecular weight of 140,000 as estimated by gel filtration on a Bio Gel A 1.5 m column. This enzyme plus phosphorylase a in an unprimed reaction catalyzes the synthesis of a branched polysaccharide in vitro. 4. No branching enzyme activity was apparent in extracts of the mutant R2508, grown on potato starch until a thermolabile inhibitor was removed by fractionation on a DEAE column. 5. This inhibitor has a molecular weight greater than 100,000 as estimated on a P-100 polyacrylamide gel column. The specificity of the inhibitor is not absolute in that it inhibits glycogen synthetase in addition to the branching enzyme in Neurospora.     

Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe ‘cytokinin-like’ phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially ‘empty’ leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.     

Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe cytokinin-like phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially empty leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.     

Rootcap structure in wild type and in a starchless mutant of Arabidopsis.

Rootcaps of the wild type (WT) and of a starchless, gravitropic mutant (TC7) of Arabidopsis thaliana L. were examined by electron microscopy to identify cellular polarities with respect to gravity. In columella cells, nuclei are located proximally, and the nuclear envelope is continuous with endoplasmic reticulum (ER) that is in turn connected to nearby plasmodesmata. Impregnation of ER with osmium ferricyanide revealed numerous contacts between columella plastids and ER in both genotypes. ER is present mostly in the outer regions of the columella protoplast except in older columella cells that are developing into peripheral cells. In vertical roots, only columella cells that are intermediate in development (story 2 cells) have a higher surface density (S) of ER in the distal compared to proximal regions of the cell. The distal but not the proximal S of the ER is constant throughout columella development. Plastids are less sedimented in TC7 columella cells compared to those of the WT. It is hypothesized that plastid contact with the ER plays a role in gravity perception in both genotypes.     

Characterization of phosphatidylserine synthase from Saccharomyces cerevisiae and a mutant defective in the enzyme

The membrane fraction of exponentially growing cells of Saccharomyces cerevisiae was found to exhibit phosphatidylserine synthase activity. The enzyme was solubilized by Triton X-100 and chromatographed on a Sepharose 6B column. The enzyme had a pH optimum between 8.0 and 8.5. The apparent Km values for CDPdiacylglycerol and L-serine were 0.12 and 13 mM, respectively. Triton X-100 stimulated the enzyme. Mg2+ or Mn2+ was required for the activity. Ca2+ was inhibitory at relatively low concentrations. The enzyme was highly specific to L-serine. Labeling experiments showed that the enzyme synthesized phosphatidylserine by transferring the phosphatidyl moiety to L-serine. A mutant of S. cerevisiae defective in phosphatidylserine synthase was isolated. The strain required ethanolamine for its growth. Ethanolamine could be substituted by choline or high concentrations of L-serine. The mutant showed normal levels of CDPdiacylglycerol-inositol 3-phosphatidyltransferase and phosphatidylethanolamine methyltransferase activities.     

Comparative differential RNA display analysis of arbuscular mycorrhiza in Pisum sativum wild type and a mutant defective in late stage development

In order to analyse gene expression associated with the late stages of arbuscular mycorrhizal development between Pisum sativum and Glomus mosseae, comparative differential RNA display was carried out using wild-type P. sativum and a mutant, RisNod24, where the fungal partner is not able to form functional arbuscules. Comparison of RNA accumulation patterns between controls, G. mosseae-colonized mutant and wild-type roots resulted in the identification of four differentially occurring cDNA fragments. One of the corresponding genes was from the fungus and three of plant origin. One plant gene, Psam4 (P. sativum arbuscular mycorrhiza-regulated), was analysed in more detail. Sequencing of a cDNA clone showed that Psam4 encodes a proline-rich protein. Northern blot analysis and quantitative RT-PCR revealed a higher basal level of Psam4 RNA accumulation in the mutant compared to the wild type. In both pea genotypes, RNA accumulation was reduced after inoculation with mycorrhiza- or nodule-forming symbiotic microorganisms, but enhanced after infection with a root pathogenic fungus.     

Real time enzyme inhibition assays provide insights into differences in binding of neuraminidase inhibitors to wild type and mutant influenza viruses

The influenza neuraminidase (NA) inhibitors zanamivir, oseltamivir and peramivir were all designed based on the knowledge that the transition state analogue of the cleaved sialic acid, 2-deoxy,2,3-dehydro N-acetyl neuraminic acid (DANA) was a weak inhibitor of NA. While DANA bound rapidly to the NA, modifications leading to the improved potency of these new inhibitors also conferred a time dependent or slow binding phenotype. Many mutations in the NA leading to decreased susceptibility result in loss of slow binding, hence this is a phenotypic marker of many but not all resistant NAs. We present here a simplified approach to determine whether an inhibitor is fast or slow binding by extending the endpoint fluorescent enzyme inhibition assay to a real time assay and monitoring the changes in IC(50)s with time. We carried out two reactions, one with a 30 min preincubation with inhibitor and the second without. The enzymatic reaction was started via addition of substrate and IC(50)s were calculated after each 10 min interval up to 60 min. Results showed that without preincubation IC(50)s for the wild type viruses started high and although they decreased continuously over the 60 min reaction time the final IC(50)s remained higher than for pre-incubated samples. These results indicate a slow equilibrium of association and dissociation and are consistent with slow binding of the inhibitors. In contrast, for viruses with decreased susceptibility, preincubation had minimal effect on the IC(50)s, consistent with fast binding. Therefore this modified assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC(50), and critically demonstrates the differential effect of incubation times on the IC(50) and K(i) values of wild type and mutant viruses for each of the inhibitors.     

Cotranslation of activated mutant p53 with wild type drives the wild-type p53 protein into the mutant conformation

Activating mutations of p53 promote tumor progression. The mutant protein adopts a characteristic conformation, which lacks the growth suppressor function of wild-type p53. We show that mutant p53 can drive cotranslated wild-type p53 into the mutant conformation: a similar effect in vivo would block wild-type suppressor function with dominant negative effect. The cotranslational effect of mutant p53 on wild-type conformation depends upon interaction between nascent polypeptides and oligomerization of the full-length proteins. We also show that oligomers of p53 proteins can be induced to change conformation in a cooperative manner. Cell growth stimulation induces a similar conformational change in p53, and our present results indicate that this may involve allosteric regulation.     

Sinapic acid ester metabolism in wild type and a sinapoylglucose-accumulating mutant of arabidopsis.

Sinapoylmalate is one of the major phenylpropanoid metabolites that is accumulated in the vegetative tissue of Arabidopsis thaliana. A thin-layer chromatography-based mutant screen identified two allelic mutant lines that accumulated sinapoylglucose in their leaves in place of sinapoylmalate. Both mutations were found to be recessive and segregated as single Mendelian genes. These mutants define a new locus called SNG1 for sinapoylglucose accumulator. Plants that are homozygous for the sng1 mutation accumulate normal levels of malate in their leaves but lack detectable levels of the final enzyme in sinapate ester biosynthesis, sinapoylglucose:malate sinapoyltransferase. A study of wild-type and sng1 seedlings found that sinapic acid ester biosynthesis in Arabidopsis is developmentally regulated and that the accumulation of sinapate esters is delayed in sng1 mutant seedlings.     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.     

Comparative ultrastructure of a filamentous mutant and the wild type ofAgmenellum quadruplicatum

Summary A filamentous variant of the marine coccoid cyanophycean alga,Agmenellum quadruplicatum has been produced after treatment of cells with the chemical mutagen, N-methyl-N-nitro-N-nitrosoguanidine (NTG). The mutation to the filamentous condition is stable and has been compared ultrastructurally with the unicellular wild type. The structural basis for the maintenance of the filamentous state is due to a failure of the homogeneously-structured transverse wall to differentiate into the stratified longitudinal wall. A manifestation of this condition is the continued synthesis of the investment membrane resulting in proliferations at each constriction. The wild type cells are nearly spherical while the filamentous mutant cells are smaller and elongate. An analysis of the structure and morphology of the nucleoplasmic regions is presented for both strains and a correlation between the morphology of these regions and the unicellular and filamentous conditions is made.     

Properties of a baculovirus mutant defective in the protein phosphatase gene.

Autographa california nuclear polyhedrosis virus (AcMNPV) contains a gene, ptp, encoding a protein tyrosine/serine phosphatase, BV-PTP. To investigate the biological function of ptp in the baculoviral replication cycle, we constructed a recombinant baculovirus, vPTPdel, in which the catalytically active site of BV-PTP was deleted. Although the vPTPdel mutant was viable in cell culture, it was partially defective in occluded virus production in SF-21 but not TN-368 cell lines. SF-21 cells infected with vPTPdel were heterogeneous in their ability to support occluded virus production. These results suggest that BV-PTP functions in a cell line-specific and possibly a cell cycle-specific fashion. The yield of occlusion bodies, infectivity (concentration of virus causing 50% mortality) and virulence (the time at which 50% of the cells died) of vPTPdel appeared to be normal in insect larvae. We identified a 35-kDa phosphoprotein as a potential target of the BV-PTP in SF-21 cells.     

Genetic transformation of a Rhizomucor pusillus mutant defective in asparagine-linked glycosylation: production of a milk-clotting enzyme in a less-glycosylated form

Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet (UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5′-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura⁺ transformants with 1 μg pRPPyr4 DNA and 1 × 10⁷ viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4 sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man_0∼1GlcNAc₂ at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains. Received: 1 February 1999 / Received revision: 26 February 1999 / Accepted: 20 March 1999     

Auxotrophic mutant of Staphylococcus aureus interferes with nasal colonization by the wild type

Staphylococcus aureus nasal carriage is a risk factor for infection in humans, particularly in the hospital setting. Bacterial interference was used as an alternative strategy for the prevention of upper respiratory, urogenital and gastrointestinal tract infections. This study was designed to assess if the administration of a live-attenuated aroA mutant of S. aureus is useful as a potential approach to prevent transient staphylococcal nasal carriage by virulent strains. We constructed an aroA mutant of S. aureus Newman strain by homologous recombination. The auxotrophic NK41 mutant was attenuated as determined by the increase of the LD(50) after intraperitoneal challenge. In mice, previous nasal colonization with the NK41 mutant significantly reduced the number of CFU of S. aureus (HU-71 and Hde288) clinical isolates and the parental Newman strain. The NK41 mutant was unable to induce a pro-inflammatory response and to damage the invaded human respiratory epithelial cells. Moreover, the cells previously or simultaneously infected with the NK41 mutant were invaded by virulent strains in a significantly lower degree than those of the control group. In conclusion, the attenuated NK41 mutant interfered with the colonization and establishment of pathogenic strains of S. aureus, which produce severe infections.     

Biosynthesis of secreted beta-hexosaminidase in Tetrahymena thermophila. A comparison of the wild type with a secretory mutant.

The synthesis and secretion of beta-hexosaminidase was studied in wild type and secretion-deficient Tetrahymena thermophila cells by metabolic labelling and immunoprecipitation. beta-Hexosaminidase is synthesized as a Mr 79,000 polypeptide which is within 10 min converted into a Mr 59,000 form. The Mr 59,000 polypeptide is further processed (within 20 min) into at least three major mature forms of Mr 58,000-54,000, which are almost quantitatively secreted into the culture medium within 1-2 h after their synthesis. Both precursor and mature forms contain asparagine-linked oligosaccharide chains which are cleavable by endoglucosaminidase F, but not by endoglucosaminidase H. Neither [32P]orthophosphate nor [35S]sulphate are incorporated into immunoprecipitable precursor and mature beta-hexosaminidases, suggesting the absence of a phosphorylated recognition marker. Biosynthesis and processing of beta-hexosaminidase is apparently unaltered in the secretory mutant MS-1; however the processed polypeptides remain cellular bound in the mutant, indicating that the mutation affects a late event in the secretion pathway of lysosomal enzymes.