scholarly journals Ni2+ Transport and Accumulation inRhodospirillum rubrum

1999 ◽  
Vol 181 (15) ◽  
pp. 4554-4560 ◽  
Author(s):  
Richard K. Watt ◽  
Paul W. Ludden
Keyword(s):  
Gene Products ◽  
Wild Type ◽  
Divalent Metal Ions ◽  
Co Dehydrogenase ◽  
Reading Frame ◽  
Carbonyl Cyanide ◽  
Transport Assay ◽  
Fold Excess ◽  
Ni Accumulation

ABSTRACT The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni2+ transport assay was used to monitor uptake and accumulation of 63Ni2+ into R. rubrum and to observe the effect of mutations in thecooC, cooT, and cooJ genes on63Ni2+ transport and accumulation. Cells grown either in the presence or absence of CO transported Ni2+with a Km of 19 ± 4 μM and aV max of 310 ± 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni2+ the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn2+, Mg2+, Ca2+, and Zn2+ showed no inhibition of63Ni2+ transport but Co2+, Cd2+, and Cu2+ inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4°C), by protonophores (carbonyl cyanidem-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N,N′-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni2+ transport. 63Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated 63Ni2+ accumulation coincided with the appearance of CODH activity in the culture, suggesting that the 63Ni2+ was accumulating in CODH. The cooC, cooT, and cooJgenes are required for the increased 63Ni2+accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated63Ni2+ like cells unexposed to CO.

scholarly journals The Mof2/Sui1 Protein Is a General Monitor of Translational Accuracy

1998 ◽  
Vol 18 (3) ◽  
pp. 1506-1516 ◽  
Author(s):  
Ying Cui ◽  
Jonathan D. Dinman ◽  
Terri Goss Kinzy ◽  
Stuart W. Peltz
Keyword(s):  
Site Selection ◽  
Start Site ◽  
Wild Type ◽  
Virus Propagation ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Translational Accuracy ◽  
Its Gene ◽  
Killer Virus

ABSTRACT Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed −1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene.mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed −1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.

scholarly journals Altered RNA Editing in Mice Lacking ADAR2 Autoregulation

2006 ◽  
Vol 26 (2) ◽  
pp. 480-488 ◽  
Author(s):  
Yi Feng ◽  
Christopher L. Sansam ◽  
Minati Singh ◽  
Ronald B. Emeson
Keyword(s):  
Alternative Splicing ◽  
Protein Expression ◽  
Translation Termination ◽  
Editing Site ◽  
Wild Type ◽  
Complementary Sequence ◽  
Double Stranded Rna ◽  
Reading Frame ◽  
Site Selective

ABSTRACT ADAR2 is a double-stranded-RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-selective conversion of adenosine to inosine. Previous studies from our laboratory have demonstrated that ADAR2 can modify its own pre-mRNA to create a proximal 3′ splice site containing a noncanonical adenosine-inosine dinucleotide. Alternative splicing to this proximal acceptor adds 47 nucleotides to the mature ADAR2 transcript, thereby resulting in the loss of functional ADAR2 protein expression due to premature translation termination in an alternate reading frame. To examine whether the editing of ADAR2 transcripts represents a negative autoregulatory strategy to modulate ADAR2 protein expression, we have generated genetically modified mice in which the ability of ADAR2 to edit its own pre-mRNA has been selectively ablated by deletion of a critical sequence (editing site complementary sequence [ECS]) required for adenosine-to-inosine conversion. Here we demonstrate that ADAR2 autoediting and subsequent alternative splicing are abolished in homozygous ΔECS mice and that ADAR2 protein expression is increased in numerous tissues compared to wild-type animals. The observed increases in ADAR2 protein expression correlate with the extent of ADAR2 autoediting observed with wild-type tissues and correspond to increases in the editing of ADAR2 substrates, indicating that ADAR2 autoediting is a key regulator of ADAR2 protein expression and activity in vivo.

scholarly journals Regulation of Adenovirus-Mediated Transgene Expression by the Viral E4 Gene Products: Requirement for E4 ORF3

1999 ◽  
Vol 73 (10) ◽  
pp. 8308-8319 ◽  
Author(s):  
M. Lusky ◽  
L. Grave ◽  
A. Dieterlé ◽  
D. Dreyer ◽  
M. Christ ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Transgene Expression ◽  
Cystic Fibrosis Gene ◽  
Gene Products ◽  
In Vivo Evaluation ◽  
Cmv Promoter ◽  
Reading Frame ◽  
E4 Region

ABSTRACT In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persistence of the viral genome or affect the antiviral host immune response (Lusky et al., J. Virol. 72:2022–2032, 1998). In this study, the influence of the adenoviral E4 region on the strength and persistence of transgene expression was evaluated by using as a model system the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV) promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoter in vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4 ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 gene products but maintenance of stable transgene expression required E4 ORF3 together with either E4 ORF4 or E4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMV promoter was severely reduced in the absence of E4 functions but could be partially restored in the presence of either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of the E4-encoded proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6 mice are immunologically weakly responsive to the human CFTR protein. This observation implies that such mice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.

scholarly journals pbpB, a Gene Coding for a Putative Penicillin-Binding Protein, Is Required for Aerobic Nitrogen Fixation in the Cyanobacterium Anabaena sp. Strain PCC7120

2001 ◽  
Vol 183 (2) ◽  
pp. 628-636 ◽  
Author(s):  
Sara Lázaro ◽  
Francisca Fernández-Piñas ◽  
Eduardo Fernández-Valiente ◽  
Amaya Blanco-Rivero ◽  
Francisco Leganés
Keyword(s):  
Sequence Similarity ◽  
Shuttle Vector ◽  
Molecular Size ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Reading Frame ◽  
Cyanobacterium Anabaena ◽  
In The Wild ◽  
Peptidoglycan Layer

ABSTRACT Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequencedAnabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and theEscherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal aspbpB and its product as PBP2. The wild-type form ofpbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N2, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.

scholarly journals Atp10p Assists Assembly of Atp6p into the F0Unit of the Yeast Mitochondrial ATPase

2004 ◽  
Vol 279 (19) ◽  
pp. 19775-19780 ◽  
Author(s):  
Alexander Tzagoloff ◽  
Antoni Barrientos ◽  
Walter Neupert ◽  
Johannes M. Herrmann
Keyword(s):  
Nuclear Gene ◽  
Mitochondrial Atpase ◽  
Gene Products ◽  
Wild Type ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Inner Membrane ◽  
Atpase Subunits ◽  
Specific Factors

The F0F1-ATPase complex of yeast mitochondria contains three mitochondrial and at least 17 nuclear gene products. The coordinate assembly of mitochondrial and cytosolic translation products relies on chaperones and specific factors that stabilize the pools of some unassembled subunits. Atp10p was identified as a mitochondrial inner membrane component necessary for the biogenesis of the hydrophobic F0sector of the ATPase. Here we show that, following its synthesis on mitochondrial ribosomes, subunit 6 of the ATPase (Atp6p) can be cross-linked to Atp10p. This interaction is required for the integration of Atp6p into a partially assembled subcomplex of the ATPase. Pulse labeling and chase of mitochondrial translation productsin vivoindicate that Atp6p is less stable and more rapidly degraded in anatp10null mutant than in wild type. Based on these observations, we propose Atp10p to be an Atp6p-specific chaperone that facilitates the incorporation of Atp6p into an intermediate subcomplex of ATPase subunits.

scholarly journals A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth.

1988 ◽  
Vol 8 (8) ◽  
pp. 3094-3103 ◽  
Author(s):  
C K Shih ◽  
R Wagner ◽  
S Feinstein ◽  
C Kanik-Ennulat ◽  
N Neff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Atp Synthase ◽  
Resistance Mutation ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Reading Frame ◽  
F0f1 Atp Synthase ◽  
General Inhibitor

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

scholarly journals Vaccinia virus kelch protein A55 is a 64 kDa intracellular factor that affects virus-induced cytopathic effect and the outcome of infection in a murine intradermal model

2006 ◽  
Vol 87 (6) ◽  
pp. 1521-1529 ◽  
Author(s):  
Philippa M. Beard ◽  
Graham C. Froggatt ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Cytopathic Effect ◽  
Plaque Morphology ◽  
Wild Type ◽  
Intracellular Protein ◽  
Reading Frame ◽  
Btb Domain ◽  
Broad Complex ◽  
Vacv Strain

The vaccinia virus (VACV) protein A55 is a BTB/kelch protein with a broad-complex, tramtrack and bric-a-brac (BTB) domain in the N-terminal region and five kelch repeats in the C-terminal half. The BTB/kelch subgroup of the kelch superfamily of proteins has been associated with a wide variety of functions including regulation of the cytoskeleton. VACV contains three genes predicted to encode BTB/kelch proteins: A55R, F3L and C2L. The A55R gene product has been identified as an intracellular protein of 64 kDa that is expressed late in infection. A VACV strain lacking 93.6 % of the A55R open reading frame (vΔA55) was constructed and found to have an unaltered growth rate in vivo but a different plaque morphology and cytopathic effect, as well as reduced development of VACV-induced Ca2+-independent cell/extracellular matrix adhesion. In a murine intradermal model of VACV infection, a virus lacking the A55R gene induced larger lesions than wild-type and revertant control viruses.

scholarly journals Murine Cytomegalovirus Open Reading Frame M27 Plays an Important Role in Growth and Virulence in Mice

2001 ◽  
Vol 75 (4) ◽  
pp. 1697-1707 ◽  
Author(s):  
Gerardo Abenes ◽  
Manfred Lee ◽  
Erik Haghjoo ◽  
Tuong Tong ◽  
Xiaoyan Zhan ◽  
...  
Keyword(s):  
Scid Mice ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Viral Virulence ◽  
Carboxyl Terminal

ABSTRACT Using a Tn3-based transposon mutagenesis approach, we have generated a pool of murine cytomegalovirus (MCMV) mutants. In this study, one of the mutants, RvM27, which contained the transposon sequence at open reading frame M27, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. Our results suggest that the M27 carboxyl-terminal sequence is dispensable for viral replication in vitro. Compared to the wild-type strain and a rescued virus that restored the M27 region, RvM27 was attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. Specifically, the titers of RvM27 in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice at 21 days postinfection were 50- to 500-fold lower than those of the wild-type virus and the rescued virus. Moreover, the virulence of the mutant virus appeared to be attenuated, because no deaths occurred among SCID mice infected with RvM27 for up to 37 days postinfection, while all the animals infected with the wild-type and rescued viruses died within 27 days postinfection. Our observations provide the first direct evidence to suggest that a disruption of M27 expression results in reduced viral growth and attenuated viral virulence in vivo in infected animals. Moreover, these results suggest that M27 is a viral determinant required for optimal MCMV growth and virulence in vivo and provide insight into the functions of the M27 homologues found in other animal and human CMVs as well as in other betaherpesviruses.

scholarly journals Tropomyosin is essential in yeast, yet the TPM1 and TPM2 products perform distinct functions.

1995 ◽  
Vol 128 (3) ◽  
pp. 383-392 ◽  
Author(s):  
B Drees ◽  
C Brown ◽  
B G Barrell ◽  
A Bretscher
Keyword(s):  
Sequence Analysis ◽  
Wild Type ◽  
Purification And Characterization ◽  
Reading Frame ◽  
Over Expression ◽  
Sequence Identity ◽  
Bona Fide ◽  
Essential Function

Sequence analysis of chromosome IX of Saccharomyces cerevisiae revealed an open reading frame of 166 residues, designated TPM2, having 64.5% sequence identity to TPM1, that encodes the major form of tropomyosin in yeast. Purification and characterization of Tpm2p revealed a protein with the characteristics of a bona fide tropomyosin; it is present in vivo at about one sixth the abundance of Tpm1p. Biochemical and sequence analysis indicates that Tpm2p spans four actin monomers along a filament, whereas Tpmlp spans five. Despite its shorter length, Tpm2p can compete with Tpm1p for binding to F-actin. Over-expression of Tpm2p in vivo alters the axial budding of haploids to a bipolar pattern, and this can be partially suppressed by co-over-expression of Tpm1p. This suggests distinct functions for the two tropomyosins, and indicates that the ratio between them is important for correct morphogenesis. Loss of Tpm2p has no detectable phenotype in otherwise wild type cells, but is lethal in combination with tpm1 delta. Over-expression of Tpm2p does not suppress the growth or cell surface targeting defects associated with tpm1 delta, so the two tropomyosins must perform an essential function, yet are not functionally interchangeable. S. cerevisiae therefore provides a simple system for the study of two tropomyosins having distinct yet overlapping functions.

scholarly journals Role of rpoS in Stress Survival and Virulence of Vibrio cholerae

1998 ◽  
Vol 180 (4) ◽  
pp. 773-784 ◽  
Author(s):  
Fitnat H. Yildiz ◽  
Gary K. Schoolnik
Keyword(s):  
Vibrio Cholerae ◽  
Genomic Library ◽  
Bacterial Species ◽  
Wild Type ◽  
Reading Frame ◽  
Infant Mouse ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded byrpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivatedrpoS by homologous recombination. V. choleraestrains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoSmutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.

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