scholarly journals Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus A20 Gene: Temperature-Sensitive Mutants Have a DNA-Minus Phenotype and Are Defective in the Production of Processive DNA Polymerase Activity

2001 ◽  
Vol 75 (24) ◽  
pp. 12308-12318 ◽  
Author(s):  
Almira Punjabi ◽  
Kathleen Boyle ◽  
Joseph DeMasi ◽  
Olivera Grubisha ◽  
Beth Unger ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Infected Cells ◽  
And Function ◽  
A20 Gene

ABSTRACT Although the vaccinia virus DNA polymerase is inherently distributive, a highly processive form of the enzyme exists within the cytoplasm of infected cells (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168–175, 1997). In the accompanying report we outline the purification of the 49-kDa A20 protein as a stoichiometric component of the processive polymerase complex (N. Klemperer, W. McDonald, K. Boyle, B. Unger, and P. Traktman, J. Virol. 75:12298–12307, 2001). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these, tsA20-6 andtsA20-ER5, have tight temperature-sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with thetsA20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA− phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication.

scholarly journals The A20R Protein Is a Stoichiometric Component of the Processive Form of Vaccinia Virus DNA Polymerase

2001 ◽  
Vol 75 (24) ◽  
pp. 12298-12307 ◽  
Author(s):  
Nancy Klemperer ◽  
William McDonald ◽  
Kathleen Boyle ◽  
Beth Unger ◽  
Paula Traktman
Keyword(s):  
Vaccinia Virus ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Temperature Sensitive ◽  
Physical Interaction ◽  
Native Molecular Mass ◽  
Binding Event ◽  
Vitro Analysis ◽  
A20 Gene

ABSTRACT In vitro analysis of the catalytic DNA polymerase encoded by vaccinia virus has demonstrated that it is innately distributive, catalyzing the addition of <10 nucleotides per primer-template binding event in the presence of 8 mM MgCl2 or 40 mM NaCl (W. F. McDonald and P. Traktman, J. Biol. Chem. 269:31190–31197, 1994). In contrast, cytoplasmic extracts isolated from vaccinia virus-infected cells contain a highly processive form of DNA polymerase, able to catalyze the replication of a 7-kb template per binding event under similar conditions. To study this holoenzyme, we were interested in purifying and characterizing the vaccinia virus processivity factor (VPF). Our previous studies indicated that VPF is expressed early after infection and has a native molecular mass of ∼48 kDa (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168–175, 1997). Using these criteria, we established a six-step chromatographic purification procedure, in which a prominent ∼45-kDa band was found to copurify with processive polymerase activity. This species was identified as the product of the A20 gene. By use of recombinant viruses that direct the overexpression of A20 and/or the DNA polymerase, we verified the physical interaction between the two proteins in coimmunoprecipitation experiments. We also demonstrated that simultaneous overexpression of A20 and the DNA polymerase leads to a specific and robust increase in levels of processive polymerase activity. Taken together, we conclude that the A20 gene encodes a component of the processive DNA polymerase complex. Genetic data that further support this conclusion are presented in the accompanying report, which documents that temperature-sensitive mutants with lesions in the A20 gene have a DNA− phenotype that correlates with a deficit in processive polymerase activity (A. Punjabi et al, J. Virol. 75:12308–12318, 2001).

scholarly journals Chinese hamster ovary cell mutants with temperature-sensitive defects in endocytosis. I. Loss of function on shifting to the nonpermissive temperature.

1986 ◽  
Vol 103 (6) ◽  
pp. 2283-2297 ◽  
Author(s):  
C F Roff ◽  
R Fuchs ◽  
I Mellman ◽  
A R Robbins
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Phenotypic Changes ◽  
Endosomal Acidification

We have isolated three independent Chinese hamster ovary cell mutants (B3853, I223, and M311) with temperature-sensitive, pleiotropic defects in receptor-mediated endocytosis. Activities affected at 41 degrees C include uptake via the D-mannose 6-phosphate receptor, accumulation of Fe from diferric transferrin, uptake of alpha 2-macroglobulin, compartmentalization of newly synthesized acid hydrolases, resistance to ricin, and sensitivity to diphtheria and Pseudomonas toxins and modeccin. The three mutants also displayed decreased sialylation of some secreted glycoproteins at 41 degrees C, reminiscent of the nonconditional mutant DTG1-5-4 that showed both endocytic and Golgi-associated defects (Robbins, A.R., C. Oliver, J.L. Bateman, S.S. Krag, C.J. Galloway, and I. Mellman, 1984, J. Cell Biol., 99:1296-1308). Phenotypic changes were detectable within 30 min after transfer of the mutants to 41 degrees C; maximal alteration of most susceptible functions was obtained 4 h after temperature shift. At 39 degrees C, the mutants exhibited many but not all of the changes manifested at 41 degrees C; resistance to diphtheria and Pseudomonas toxins required the higher temperature. Analysis of cell hybrids showed that B3853 and DTG1-5-4 are in one complementation group ("End1"); M311 and I223 are in another ("End2"). In the End1 mutants, loss of endocytosis correlated with complete loss of ATP-dependent endosomal acidification in vitro; in the End 2 mutants partial loss of acidification was observed. At the nonpermissive temperature, residual levels of endocytic activity in B3853 and M311 were nearly identical; thus, we conclude that the differences measured in endosomal acidification in vitro reflect the different genetic loci affected, rather than the relative severity of the genetic lesions. The mutations in M311 and I223 appear to have different effects on the same protein; in I223 (but not in M311) the full spectrum of phenotypic changes could be produced at the permissive temperature by inhibition of protein synthesis.

scholarly journals Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture.

1983 ◽  
Vol 3 (8) ◽  
pp. 1518-1526 ◽  
Author(s):  
D Boettiger ◽  
R Soltesz ◽  
H Holtzer ◽  
M Pacifici
Keyword(s):  
Rous Sarcoma Virus ◽  
Limb Bud ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Infected Cells

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.

Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture

1983 ◽  
Vol 3 (8) ◽  
pp. 1518-1526
Author(s):  
D Boettiger ◽  
R Soltesz ◽  
H Holtzer ◽  
M Pacifici
Keyword(s):  
Rous Sarcoma Virus ◽  
Limb Bud ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Infected Cells

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.

scholarly journals Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210

1992 ◽  
Vol 117 (5) ◽  
pp. 1041-1053 ◽  
Author(s):  
JR Hamaguchi ◽  
RA Tobey ◽  
J Pines ◽  
HA Crissman ◽  
T Hunter ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Cyclin B1 ◽  
Histone H1 ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.

scholarly journals Selective retention of monoglucosylated high mannose oligosaccharides by a class of mutant vesicular stomatitis virus G proteins.

1989 ◽  
Vol 108 (3) ◽  
pp. 811-819 ◽  
Author(s):  
K Suh ◽  
J E Bergmann ◽  
C A Gabel
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Vesicular Stomatitis Virus ◽  
Plasmid Vector ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
High Mannose

Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H]mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78% of the 3H-mannose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.

scholarly journals New Temperature-Sensitive Alleles of ftsZ in Escherichia coli

2005 ◽  
Vol 187 (1) ◽  
pp. 358-365 ◽  
Author(s):  
Stephen G. Addinall ◽  
Elaine Small ◽  
Duncan Whitaker ◽  
Shane Sturrock ◽  
William D. Donachie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Point Mutations ◽  
Ring Formation ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Z Ring

ABSTRACT We isolated five new temperature-sensitive alleles of the essential cell division gene ftsZ in Escherichia coli, using P1-mediated, localized mutagenesis. The five resulting single amino acid changes (Gly109→Ser109 for ftsZ6460, Ala129→Thr129 for ftsZ972, Val157→Met157 for ftsZ2066, Pro203→Leu203 for ftsZ9124, and Ala239→Val239 for ftsZ2863) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42°C. In each case the division block is associated with loss of Z-ring formation such that fewer than 2% of cells show Z rings at 42°C. The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30°C and therefore cause significant defects in FtsZ polymerization, even at the permissive temperature. Neither purified FtsZ9124 nor purified FtsZ6460 exhibited polymerization when it was assayed by light scattering or electron microscopy, even in the presence of calcium or DEAE-dextran. Hence, both mutations also cause defects in FtsZ polymerization in vitro. Interestingly, FtsZ9124 has detectable GTPase activity, although the activity is significantly reduced compared to that of the wild-type FtsZ protein. We demonstrate here that unlike expression of ftsZ84, multicopy expression of the ftsZ6460, ftsZ972, and ftsZ9124 alleles does not complement the respective lethalities at the nonpermissive temperature. In addition, all five new mutant FtsZ proteins are stable at 42°C. Therefore, the novel isolates carrying single ftsZ(Ts) point mutations, which are the only such strains obtained since isolation of the classical ftsZ84 mutation, offer significant opportunities for further genetic characterization of FtsZ and its role in cell division.

scholarly journals A New Cistron in the Murine Hepatitis Virus Replicase Gene

2010 ◽  
Vol 84 (19) ◽  
pp. 10148-10158 ◽  
Author(s):  
Helen L. Stokes ◽  
Surendranath Baliji ◽  
Chang Guo Hui ◽  
Stanley G. Sawicki ◽  
Susan C. Baker ◽  
...  
Keyword(s):  
Gene Locus ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Temperature Sensitive ◽  
Replicase Gene ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Murine Hepatitis Virus ◽  
Infected Cells

ABSTRACT We report an RNA-negative, temperature-sensitive (ts) mutant of Murine hepatitis virus, Bristol ts31 (MHV-Brts31), that defines a new complementation group within the MHV replicase gene locus. MHV-Brts31 has near-normal levels of RNA synthesis at the permissive temperature of 33°C but is unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature of 39.5°C. Sequence analysis of MHV-Brts31 RNA indicated that a single G-to-A transition at codon 1307 in open reading frame 1a, which results in a replacement of methionine-475 with isoleucine in nonstructural protein 3 (nsp3), was responsible for the ts phenotype. This conclusion was confirmed using a vaccinia virus-based reverse genetics system to produce a recombinant virus, Bristol tsc31 (MHV-Brtsc31), which has the same RNA-negative ts phenotype and complementation profile as those of MHV-Brts31. The analysis of protein synthesis in virus-infected cells showed that, at the nonpermissive temperature, MHV-Brtsc31 was not able to proteolytically process either p150, the precursor polypeptide of the replicase nonstructural proteins nsp4 to nsp10, or the replicase polyprotein pp1ab to produce nsp12. The processing of replicase polyprotein pp1a in the region of nsp1 to nsp3 was not affected. Transmission electron microscopy showed that, compared to revertant virus, the number of double-membrane vesicles in MHV-Brts31-infected cells is reduced at the nonpermissive temperature. These results identify a new cistron in the MHV replicase gene locus and show that nsp3 has an essential role in the assembly of a functional MHV replication-transcription complex.

scholarly journals Dual Role of the Mitochondrial Chaperone Mdj1p in Inheritance of Mitochondrial DNA in Yeast

1999 ◽  
Vol 19 (12) ◽  
pp. 8201-8210 ◽  
Author(s):  
Marlena Duchniewicz ◽  
Aleksandra Germaniuk ◽  
Benedikt Westermann ◽  
Walter Neupert ◽  
Elisabeth Schwarz ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Optimal Growth ◽  
Polymerase Activity ◽  
Mitochondrial Genomes ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Active Conformation ◽  
Nonpermissive Temperature ◽  
Chaperone Protein

ABSTRACT Mdj1p, a homolog of the bacterial DnaJ chaperone protein, plays an essential role in the biogenesis of functional mitochondria in the yeast Saccharomyces cerevisiae. We analyzed the role of Mdj1p in the inheritance of mitochondrial DNA (mtDNA). Mitochondrial genomes were rapidly lost in a temperature-sensitive mdj1 mutant under nonpermissive conditions. The activity of mtDNA polymerase was severely reduced in the absence of functional Mdj1p at a nonpermissive temperature, demonstrating the dependence of the enzyme on Mdj1p. At a permissive temperature, the activity of mtDNA polymerase was not affected by the absence of Mdj1p. However, under these conditions, intact [rho +] genomes were rapidly converted to nonfunctional [rho −] genomes which were stably propagated in an mdj1 deletion strain. We propose that mtDNA polymerase depends on Mdj1p as a chaperone in order to acquire and/or maintain an active conformation at an elevated temperature. In addition, Mdj1p is required for the inheritance of intact mitochondrial genomes at a temperature supporting optimal growth; this second function appears to be unrelated to the function of Mdj1p in maintaining mtDNA polymerase activity.

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