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.

scholarly journals The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

2007 ◽  
Vol 189 (24) ◽  
pp. 8793-8800 ◽  
Author(s):  
Christopher J. Rosario ◽  
Mitchell Singer
Keyword(s):  
Dna Replication ◽  
Developmental Process ◽  
Myxococcus Xanthus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Temperature Growth ◽  
Developmental Program

ABSTRACT Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaBA116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaBA116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaBA116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.

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 INVOLVEMENT OF GENE 49 IN RECOMBINATION OF BACTERIOPHAGE T4

Genetics ◽  
1983 ◽  
Vol 104 (1) ◽  
pp. 1-9
Author(s):  
Junichi Miyazaki ◽  
Yeikou Ryo ◽  
Teiichi Minagawa
Keyword(s):  
Dna Replication ◽  
Gene Function ◽  
Bacteriophage T4 ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Recombinant Frequency ◽  
Defective Mutant ◽  
Ts Mutant

ABSTRACT The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants. When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype. However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor. Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvs X, but recombination between rII markers was not.

scholarly journals Retrograde Transport of Golgi-localized Proteins to the ER

1998 ◽  
Vol 140 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Nelson B. Cole ◽  
Jan Ellenberg ◽  
Jia Song ◽  
Diane DiEuliis ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Viral Glycoprotein ◽  
Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

An Alternatively Spliced Survivin Variant Is Positively Regulated by p53 and Sensitizes Leukemia Cells to Chemotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3497-3497
Author(s):  
Ningxi Zhu ◽  
Lubing Gu ◽  
Harry W. Findley ◽  
Fengzhi Li ◽  
Muxiang Zhou
Keyword(s):  
Cell Line ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Doxorubicin Treatment ◽  
Cryptic Exon ◽  
Apoptosis Protein ◽  
Induced Apoptosis

Abstract Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53–143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53–143. When EU-4/p53–143 cell culture was shifted from 37.5°C to the wt-p53-permissive temperature (32.5°C), the expression of survivin and survivin-DEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a pro-apoptotic factor and its expression is upregulated by p53.

scholarly journals Trapping of a Spiral-Like Intermediate of the Bacterial Cytokinetic Protein FtsZ

2006 ◽  
Vol 188 (5) ◽  
pp. 1680-1690 ◽  
Author(s):  
Katherine A. Michie ◽  
Leigh G. Monahan ◽  
Peter L. Beech ◽  
Elizabeth J. Harry
Keyword(s):  
Ring Formation ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Bacterial Cell Division ◽  
Temporal Regulation ◽  
Division Site ◽  
Spiral Form ◽  
Z Ring

ABSTRACT The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.

scholarly journals The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function

2012 ◽  
Vol 199 (1) ◽  
pp. 151-167 ◽  
Author(s):  
Benjamin D. Engel ◽  
Hiroaki Ishikawa ◽  
Kimberly A. Wemmer ◽  
Stefan Geimer ◽  
Ken-ichi Wakabayashi ◽  
...  
Keyword(s):  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Full Length ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Flagellar Beat ◽  
Flagellar Assembly ◽  
Ph Shock ◽  
And Function

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.

scholarly journals The role of ribonuclease II in the maturation of precursor 16S ribosomal ribonucleic acid in Escherichia coli

1974 ◽  
Vol 140 (3) ◽  
pp. 443-450 ◽  
Author(s):  
John R. Dean ◽  
John Sykes
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Maximum Activity ◽  
Exponential Phase ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Experimental Conditions ◽  
Inhibition Of Growth ◽  
Ribosomal Ribonucleic Acid

The suggested involvement of ribonuclease II in the maturation of rRNA has been examined directly by determining the activity of the enzyme and the amount of p16S rRNA in cell-free extracts from Escherichia coli A19 and its temperature-sensitive derivative N464 grown under experimental conditions designed to vary the amounts of enzyme and precursor independently. In strain A19 the enzyme showed maximum activity in circumstances where the amount of p16S rRNA was normal (e.g. exponential-phase cells) or raised eight times (e.g. during inhibition of growth by methionine starvation of the relaxed auxotroph or by chloramphenicol or puromycin treatment). In strain N464 at the non-permissive temperature the ribonuclease II activity may be decreased by 50% without effect upon the amount of p16S rRNA, whereas in methionine starvation of this strain the enzyme activity is at a maximum and the p16S rRNA is eight times that in exponential-phase cells. These observations are discussed in relation to the previously implied role of ribonuclease II in the maturation of rRNA within ribosome precursors.

scholarly journals Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

1993 ◽  
Vol 4 (9) ◽  
pp. 931-939 ◽  
Author(s):  
D Feldheim ◽  
K Yoshimura ◽  
A Admon ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Functional Characterization ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Endoplasmic Reticulum Membrane ◽  
Restrictive Temperature ◽  
Permissive Temperature

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.

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.

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