33 results on '"Simon LD"'
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2. MurF inhibitors with antibacterial activity: effect on muropeptide levels.
- Author
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Baum EZ, Crespo-Carbone SM, Foleno BD, Simon LD, Guillemont J, Macielag M, and Bush K
- Subjects
- Anti-Bacterial Agents chemistry, Enterococcus faecalis drug effects, Enterococcus faecium drug effects, Enzyme Inhibitors chemistry, Escherichia coli drug effects, Microbial Sensitivity Tests, Molecular Structure, Peptide Synthases genetics, Peptide Synthases physiology, Quinolines chemistry, Staphylococcus aureus drug effects, Anti-Bacterial Agents pharmacology, Bacteria drug effects, Enzyme Inhibitors pharmacology, Peptide Synthases antagonists & inhibitors, Quinolines pharmacology
- Abstract
MurF catalyzes the last cytoplasmic step of bacterial cell wall synthesis and is essential for bacterial survival. Our previous studies used a pharmacophore model of a MurF inhibitor to identify additional inhibitors with improved properties. We now present the characterization of two such inhibitors, the diarylquinolines DQ1 and DQ2. DQ1 inhibited Escherichia coli MurF (50% inhibitory concentration, 24 microM) and had modest activity (MICs, 8 to 16 microg/ml) against lipopolysaccharide (LPS)-defective E. coli and wild-type E. coli rendered permeable with polymyxin B nonapeptide. DQ2 additionally displayed activity against gram-positive bacteria (MICs, 8 to 16 microg/ml), including methicillin (meticillin)-susceptible and -resistant Staphylococcus aureus isolates and vancomycin-susceptible and -resistant Enterococcus faecalis and Enterococcus faecium isolates. Treatment of LPS-defective E. coli cells with >or=2x MIC of DQ1 resulted in a 75-fold-greater accumulation of the MurF substrate compared to the control, a 70% decline in the amount of the MurF product, and eventual cell lysis, consistent with the inhibition of MurF within bacteria. DQ2 treatment of S. aureus resulted in similar effects on the MurF substrate and product quantities. At lower levels of DQ1 (
- Published
- 2009
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3. Expression of different-size transcripts from the clpP-clpX operon of Escherichia coli during carbon deprivation.
- Author
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Li C, Tao YP, and Simon LD
- Subjects
- ATPases Associated with Diverse Cellular Activities, Carbon metabolism, Culture Media, DNA-Directed RNA Polymerases genetics, DNA-Directed RNA Polymerases metabolism, Endopeptidase Clp, Escherichia coli genetics, Escherichia coli Proteins, Heat-Shock Proteins genetics, Heat-Shock Proteins metabolism, Molecular Chaperones, Promoter Regions, Genetic, Transcription Factors genetics, Transcription Factors metabolism, Transcription, Genetic, Transcriptional Activation, Adenosine Triphosphatases genetics, Escherichia coli enzymology, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Operon, RNA, Bacterial biosynthesis, Serine Endopeptidases genetics, Sigma Factor
- Abstract
Transcription of the clpP-clpX operon of Escherichia coli leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entire clpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpX coding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the sigma(32)-dependent promoter of the clpP-clpX operon within 15 min after the start of carbon starvation. Expression of the clpP-clpX operon from the promoters upstream of the clpP gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation. E. coli rpoB mutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript. The E. coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpX transcript when E. coli is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.
- Published
- 2000
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4. A motif shared by TFIIF and TFIIB mediates their interaction with the RNA polymerase II carboxy-terminal domain phosphatase Fcp1p in Saccharomyces cerevisiae.
- Author
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Kobor MS, Simon LD, Omichinski J, Zhong G, Archambault J, and Greenblatt J
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Binding Sites, Gene Expression Regulation, Fungal, Holoenzymes chemistry, Holoenzymes genetics, Holoenzymes metabolism, Magnetic Resonance Spectroscopy, Methyl Methanesulfonate pharmacology, Mutation, Phenotype, Phosphoprotein Phosphatases genetics, Phosphorylation, Protein Binding, Protein Structure, Tertiary genetics, RNA Polymerase II chemistry, RNA Polymerase II genetics, RNA Polymerase II metabolism, RNA, Messenger biosynthesis, RNA, Messenger genetics, RNA, Messenger metabolism, Recombinant Fusion Proteins metabolism, Repetitive Sequences, Nucleic Acid, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Transcription Factor TFIIB, Transcription Factors genetics, Transcriptional Activation, Phosphoprotein Phosphatases metabolism, Saccharomyces cerevisiae enzymology, Transcription Factors chemistry, Transcription Factors metabolism, Transcription Factors, TFII
- Abstract
Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo.
- Published
- 2000
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5. An amino acid change in the exodomain of the E2 protein of Sindbis virus, which impairs the release of virus from chicken cells but not from mosquito cells.
- Author
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Li ML, Liao HJ, Simon LD, and Stollar V
- Subjects
- Aedes virology, Amino Acid Substitution, Animals, Chick Embryo, Clone Cells, Fibroblasts cytology, Fibroblasts virology, Mutagenesis, Site-Directed, Phenotype, Recombinant Proteins metabolism, Sindbis Virus genetics, Viral Envelope Proteins genetics, DNA, Viral genetics, Sindbis Virus physiology, Transcription, Genetic, Viral Envelope Proteins physiology
- Abstract
In order to obtain a mutant of Sindbis virus (SV) with a low methionine-resistant (LMR) phenotype, i.e., able to replicate in methionine-deprived Aedes albopictus mosquito cells, standard SV (SV(STD)) was passaged 17 times in mosquito cells maintained in a low methionine medium and then plaque-purified, also in mosquito cells. Although the virus obtained by this procedure, SV(LM17), did have the desired LMR phenotype, it also appeared to have acquired a host-range phenotype. We have now characterized the host-range phenotype of SV(LM17) in greater detail. In yield assays, the titer of SV(LM17) produced by chick embryo fibroblasts (CEF) was 100- to 1000-fold lower than that from mosquito cells. SV(STD), in contrast, produced a similar titer of virus from the two cell types. On the other hand, when SV(LM17) was assayed directly by plaque formation on CEF and on mosquito cell monolayers, no host restriction in CEF was observed. When CEF were infected with SV(LM17), viral proteins were synthesized normally, pE2 was processed to E2, and E2 was demonstrated by the fluorescent antibody method to reach the cell surface. However, electron microscopy of SV(LM17)-infected cells revealed an absence of extracellular virions and of budding particles; also, nucleocapsids were not aligned beneath the plasma membrane. By sequence determination and by site-directed mutagenesis, it was determined that the host restriction of SV(LM17) was due to a change from Ala to Val at position 251 of the E2 protein. Substitution of Gly or Leu at this position also resulted in the same host range phenotype., (Copyright 1999 Academic Press.)
- Published
- 1999
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6. Mechanisms controlling diffusion and release of model proteins through and from partially esterified hyaluronic acid membranes.
- Author
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Simon LD, Stella VJ, Charman WN, and Charman SA
- Subjects
- Delayed-Action Preparations, Diffusion, Enzyme Stability, Esters chemistry, Kinetics, Molecular Weight, Permeability, Temperature, Thymidine chemistry, Deoxyribonucleases chemistry, Hyaluronic Acid chemistry, Membranes, Artificial, Ribonuclease, Pancreatic chemistry
- Abstract
The effects of polymer percent esterification and protein molecular weight on the diffusion of two model proteins, deoxyribonuclease (DNase) and ribonuclease A (RNase A), through and from partially esterified hyaluronic acid membranes were compared. The permeability of the polymer membranes was inversely related to the degree of polymer esterification and the molecular weight of the protein. Transport rates of proteins through the membranes decreased dramatically over narrow ranges of polymer esterification. As expected, the apparent diffusivity of the larger protein in the polymer matrix was more sensitive to changes in membrane hydration than that of the smaller protein. These observations demonstrated the dependence of the mobility of large molecular weight proteins on polymer hydration and chain relaxation. The relationship between protein diffusion through and release from the modified hyaluronate matrices was also investigated using RNase A as a model. The release profiles from fully esterified membranes showed lag behavior and varied with protein load and hyaluronate hydrolysis rates, while release from less esterified membranes was rapid and independent of polymer esterification or hydrolysis. Potential applications of modified hyaluronate matrices in the controlled delivery of proteins are discussed.
- Published
- 1999
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7. Variation in the diffusion and release of ribonuclease through and from esterified hyaluronic acid membranes: effect of changes in matrix characteristics.
- Author
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Simon LD, Stella VJ, Charman WN, and Charman SA
- Subjects
- Diffusion, Ribonuclease, Pancreatic administration & dosage, Spectroscopy, Fourier Transform Infrared, Drug Delivery Systems, Hyaluronic Acid administration & dosage, Ribonuclease, Pancreatic chemistry
- Abstract
50 h) were detected for the transport and release of a model protein (ribonuclease A) compared with that for the translucent region which showed no lag time. The results highlight the importance of carefully controlling matrix formation to ensure reproducible transport and release characteristics from polymer matrices.
- Published
- 1999
- Full Text
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8. Differences in hypothalamic serotonin between estrous phases and gender: an in vivo microdialysis study.
- Author
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Gundlah C, Simon LD, and Auerbach SB
- Subjects
- Animals, Diestrus drug effects, Diestrus metabolism, Dose-Response Relationship, Drug, Estrus drug effects, Extracellular Space, Female, Fenfluramine pharmacology, Hypothalamus drug effects, Male, Microdialysis methods, Paroxetine pharmacology, Rats, Rats, Sprague-Dawley, Sex Characteristics, Estrus metabolism, Hypothalamus metabolism, Serotonin metabolism
- Abstract
The aim of the present study was to assess whether there are gender differences in (1) levels of extracellular serotonin (5-HT) in the forebrain, and (2) the effect on 5-HT of a reuptake inhibitor, paroxetine, or a releasing drug, fenfluramine. In vivo microdialysis was used to measure 5-HT in the hypothalamus of male and regularly cycling female rats. Hypothalamic 5-HT was significantly lower in estrous females (0.83 +/- 0.05 pg/sample, n=33) than in male rats (1.04 +/- 0.06 pg, n=38). Levels in diestrous females (0.98 +/- 0.09 pg, n=38) were not significantly different from males. Paroxetine (1 mg/kg) increased hypothalamic 5-HT in males, and diestrous and estrous females to approximately 2 pg/sample. However, the increase in hypothalamic 5-HT produced by a maximally effective dose of paroxetine (10 mg/kg) was significantly greater in male rats and during diestrous than during estrous. d,l-Fenfluramine (10 mg/kg) evoked an increase in extracellular 5-HT to approximately 15 pg/sample in all groups. A higher dose of d,l-fenfluramine (20 mg/kg) produced a significantly greater increase in hypothalamic 5-HT in males than in females during estrous or diestrous. These results are consistent with other evidence that during estrous, when rats are responding to peak levels of estrogen and progesterone, 5-HT release is decreased., (Copyright 1998 Elsevier Science B.V.)
- Published
- 1998
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9. Isolation and characterization of the phage T4 PinA protein, an inhibitor of the ATP-dependent lon protease of Escherichia coli.
- Author
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Hilliard JJ, Maurizi MR, and Simon LD
- Subjects
- ATP-Dependent Proteases, Adenosine Triphosphatases antagonists & inhibitors, Adenosine Triphosphatases metabolism, Amino Acid Sequence, Biopolymers, Hydrolysis, Molecular Sequence Data, Protein Binding, Protein Conformation, Viral Proteins chemistry, Viral Proteins metabolism, Adenosine Triphosphate metabolism, Bacteriophage T4 metabolism, Endopeptidase Clp, Escherichia coli enzymology, Escherichia coli Proteins, Heat-Shock Proteins metabolism, Protease Inhibitors metabolism, Protease La, Serine Endopeptidases metabolism, Viral Proteins isolation & purification
- Abstract
The bacteriophage T4 PinA protein, expression of which leads to inhibition of protein degradation in Escherichia coli cells, has been purified from cells carrying multiple copies of the pinA gene. PinA is a heat-stable protein with a subunit Mr of 18,800 and an isoelectric point of 4.6. Under nondenaturing conditions on a gel filtration column, PinA migrated in two peaks corresponding to a dimer and a tetramer. Purified PinA inhibited ATP-dependent protein degradation by Lon protease in vitro; it did not inhibit the activity of other E. coli ATP-dependent proteases, ClpAP or ClpYQ. Furthermore, PinA did not inhibit ATP-independent proteolysis in E. coli cell extracts. PinA binds with high affinity to Lon protease (Kd approximately 10 nM for dimer binding), and a complex with approximately 1 dimer of PinA per tetramer of Lon protease could be isolated by gel filtration. Lon activity was partially restored upon dilution of the PinA-Lon complex to subnanomolar concentrations, indicating that inhibition was reversible and that PinA did not covalently modify Lon protease. PinA was not cleaved by Lon protease, and heating the Lon-PinA complex at 65 degrees C denatured Lon protease and released active PinA. The properties of PinA in vitro suggest that PinA inhibits protein degradation in vivo by forming a tight, reversible complex with Lon protease.
- Published
- 1998
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10. PinA inhibits ATP hydrolysis and energy-dependent protein degradation by Lon protease.
- Author
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Hilliard JJ, Simon LD, Van Melderen L, and Maurizi MR
- Subjects
- ATP-Dependent Proteases, Bacteriophage T4 metabolism, Hydrolysis, Protein Denaturation, Adenosine Triphosphate metabolism, Escherichia coli Proteins, Heat-Shock Proteins metabolism, Protease Inhibitors metabolism, Protease La, Serine Endopeptidases metabolism, Viral Proteins metabolism
- Abstract
The bacteriophage T4 PinA protein inhibited degradation of [3H]alpha-methyl casein by purified Lon protease from Escherichia coli, but inhibition was noncompetitive with respect to casein. PinA did not inhibit cleavage of the fluorogenic peptide, N-glutaryl-alanylalanylphenylalanyl-3-methoxynaphthylamide and, moreover, did not block the ability of protein substrates, such as casein, to activate cleavage of fluorogenic peptides by Lon. Thus, PinA does not block the proteolytic active site or the allosteric protein-binding site on Lon. Inhibition of basal ATPase activity was variable (50-90%), whereas inhibition of protein-activated ATPase activity was usually 80-95%. Inhibition was noncompetitive with respect to ATP. PinA did not block activation of peptide cleavage by nonhydrolyzable analogs of ATP. These data suggest that PinA does not bind at the ATPase active site of Lon and does not interfere with nucleotide binding to the enzyme. PinA inhibited cleavage of the 72-amino acid protein, CcdA, degradation of which requires ATP hydrolysis, but did not inhibit cleavage of the carboxyl-terminal 41-amino acid fragment of CcdA, degradation of which does not require ATP hydrolysis. PinA thus appears to interact at a novel regulatory or enzymatic site involved in the coupling between ATP hydrolysis and proteolysis, possibly blocking the protein unfolding or remodeling step essential for degradation of high molecular weight protein substrates by Lon.
- Published
- 1998
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11. Oxazole yellow dye interactions with short DNA oligomers of homogeneous base composition and their hybrids.
- Author
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Simon LD, Abramo KH, Sell JK, and McGown LB
- Subjects
- Base Composition, Binding, Competitive, Circular Dichroism, DNA, Single-Stranded chemistry, Fluorescence Polarization, Nucleic Acid Conformation, Poly A chemistry, Poly A metabolism, Poly C chemistry, Poly C metabolism, Poly G chemistry, Poly G metabolism, Poly T chemistry, Poly T metabolism, Poly dA-dT chemistry, Poly dA-dT metabolism, Polydeoxyribonucleotides chemistry, Repetitive Sequences, Nucleic Acid, Spectrometry, Fluorescence, Spectrophotometry, Ultraviolet, Structure-Activity Relationship, Benzoxazoles metabolism, DNA, Single-Stranded metabolism, Fluorescent Dyes metabolism, Polydeoxyribonucleotides metabolism, Quinolines metabolism
- Abstract
Interactions between short single-stranded DNA oligomers of homogeneous base composition and the fluorescent probes oxazole yellow (YO) and its homodimer YOYO are described. The oligomers included 15-mers and 30-mers of polydA, polydT, polydG, and polydC. Interactions between the dyes and DNA hybrids formed from complementary homogeneous strands of equal length were also investigated. No interactions were observed between the dyes and the monomeric monophosphate nucleosides A, G, T, or C. The dyes were found to interact much more strongly with the purine oligomers polydA and polydG than with the pyrimidine oligomers polydT and polydC. PolydA of both lengths has strong interactions with YOYO, whereas the polydG 30-mer interacts strongly with monomeric YO. The 15-mers of polydG and polydC of both lengths show little interaction with either dye. Interactions of the dyes with the polydA/polydT and polydG/polydC hybrids tend to be dominated by interactions with polydA and polydG, respectively. Although dye interactions generally were facilitated by hybridization, particularly for polydA/polydT, the interactions were similar to those with the single strands and different from those that have been observed in long double-stranded DNA.
- Published
- 1998
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12. Ho endonuclease cleaves MAT DNA in vitro by an inefficient stoichiometric reaction mechanism.
- Author
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Jin Y, Binkowski G, Simon LD, and Norris D
- Subjects
- DNA, Fungal chemistry, Deoxyribonucleases, Type II Site-Specific chemistry, Plasmids genetics, Saccharomyces cerevisiae Proteins, DNA, Fungal genetics, Deoxyribonucleases, Type II Site-Specific genetics, Saccharomyces cerevisiae genetics
- Abstract
Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a double-stranded DNA break at the yeast MAT locus. In this report, we characterize the fundamental biochemical properties of Ho. Using an assay that monitors cleavage of a MAT plasmid, we define an optimal in vitro reaction, showing in particular that the enzyme has a stringent requirement for zinc ions. This suggests that zinc finger motifs present in Ho are important for cleavage. The most unexpected feature of Ho, however, is its extreme inefficiency. Maximal cleavage occurs when Ho is present at a concentration of 1 molecule/3 base pairs of substrate DNA. Even under these conditions, complete digestion requires >2 h. This inefficiency results from two characteristics of Ho. First, Ho recycles slowly from cleaved product to new substrate, in part because the enzyme has an affinity for one end of its double strand break product. Second, high levels of cleavage in the in vitro reaction correlate with the appearance of large protein-DNA aggregates. At optimal Ho concentrations, these latter aggregates, referred to as "florettes," have an ordered structure consisting of a densely staining central region and loops of radiating DNA. These unusual properties may indicate that Ho plays a role in other aspects of mating type switching subsequent to double strand break formation.
- Published
- 1997
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13. Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants.
- Author
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Allison LA, Simon LD, and Maliga P
- Subjects
- Base Sequence, Molecular Sequence Data, Photosynthesis, Promoter Regions, Genetic, RNA, Ribosomal, 16S genetics, Sequence Deletion, Sigma Factor genetics, Transcription, Genetic, DNA-Directed RNA Polymerases genetics, Gene Expression Regulation, Plant, Plants, Toxic, Plastids enzymology, Nicotiana genetics
- Abstract
The plastid genome in higher plants encodes subunits of an Escherichia coli-like RNA polymerase which initiates transcription of plastid genes from sequences resembling E.coli sigma70-type promoters. By deleting the gene for the essential beta subunit of the tobacco E.coli-like RNA polymerase, we have established the existence of a second plastid transcription system which does not utilize E.coli-like promoters. In contrast to the E.coli-like RNA polymerase, the novel transcription machinery preferentially transcribes genetic system genes rather than photosynthetic genes. Although the mutant plants are photosynthetically defective, transcription by this polymerase is sufficient for plastid maintenance and plant development.
- Published
- 1996
14. Increased ATP-dependent proteolytic activity in lon-deficient Escherichia coli strains lacking the DnaK protein.
- Author
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Kroh HE and Simon LD
- Subjects
- ATP-Dependent Proteases, DNA, Kinetoplast, Gene Expression Regulation, Bacterial, Heat-Shock Proteins physiology, Hydrolysis, Adenosine Triphosphate physiology, DNA, Circular physiology, Escherichia coli physiology, Escherichia coli Proteins, Protease La, Serine Endopeptidases deficiency
- Abstract
Extracts made from Escherichia coli null dnaK strains contained elevated levels of ATP-dependent proteolytic activity compared with levels in extracts made from dnaK+ strains. This ATP-dependent proteolytic activity was not due to Lon, Clp, or Alp-associated protease. Comparison of the levels of ATP-dependent proteolytic activity present in lon rpoH dnaK mutants and in lon rpoH dnaK+ mutants showed that the level of ATP-dependent proteolytic activity was elevated in the lon rpoH dnaK mutant strain. These findings suggest that DnaK negatively regulates a new ATP-dependent proteolytic activity, independently of sigma 32. Other results indicate that an ATP-dependent proteolytic activity was increased in a lon alp strain after heat shock. It is not yet known whether the same protease is associated with the increased ATP-dependent proteolytic activity in the dnaK mutants and in the heat-shocked lon alph strain.
- Published
- 1991
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15. The ClpP component of Clp protease is the sigma 32-dependent heat shock protein F21.5.
- Author
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Kroh HE and Simon LD
- Subjects
- ATP-Dependent Proteases, Electrophoresis, Gel, Two-Dimensional, Escherichia coli enzymology, Genotype, Heat-Shock Proteins biosynthesis, Heat-Shock Proteins isolation & purification, Macromolecular Substances, Promoter Regions, Genetic, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins isolation & purification, Serine Endopeptidases biosynthesis, Serine Endopeptidases isolation & purification, Escherichia coli genetics, Genes, Bacterial, Heat-Shock Proteins genetics, Serine Endopeptidases genetics, Sigma Factor metabolism
- Abstract
The genes that encode the subunits of the Clp protease of Escherichia coli, clpA and clpP, appear to be regulated differently from each other. The clpA gene does not seem to be under heat shock control (Y. S. Katayama, S. Gottesman, J. Pumphrey, S. Rudikoff, W. P. Clark, and M. R. Maurizi, J. Biol. Chem. 263:15226-15236, 1988). In contrast, the level of ClpP protein was increased in rpoH+ cells but not in null rpoH cells after an upshift in temperature from 17 to 43 degrees C. The level of ClpP protein in a null dnaK strain was also elevated relative to the level of ClpP protein in an otherwise isogenic dnaK+ strain. In two-dimensional gels, the ClpP protein was located in the position of the previously unidentified heat shock protein F21.5. No protein spot corresponding to F21.5 was present in two-dimensional gels of a null clpP strain. The clpP gene, therefore, appears to be a heat shock gene, expressed in a sigma 32-dependent manner and negatively regulated by DnaK; the product of clpP is the previously unidentified heat shock protein F21.5.
- Published
- 1990
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16. Bacteriophage T4 bypass31 mutations that make gene 31 nonessential for bacteriophage T4 replication: mapping bypass31 mutations by UV rescue experiments.
- Author
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Doermann AH and Simon LD
- Subjects
- Escherichia coli radiation effects, Gene Frequency, Phenotype, T-Phages radiation effects, Virus Replication radiation effects, DNA Replication radiation effects, Escherichia coli genetics, Genes, Viral, Mutation, T-Phages genetics, Ultraviolet Rays
- Abstract
The product of gene 31 is normally required for assembly of the T4 capsid. Two mutations that each bypass that requirement are shown to be located at separate sites in gene 23, which encodes the major structural protein of the capsid. A second phenotypic effect that characterizes both bypass31 mutant strains is the ability to multiply in host-defective strains, such as hdB3-1 and groEL mutants, on which wild-type T4 is unable to assemble capsids. The genetic data indicate that both phenotypic effects are due to the bypass31 mutation. Elimination of the requirement for both the phage protein, gp31, and the host protein, GroEL, by either of two single mutations in gene 23 indicates that GroEL and gp31 are normally needed to interact with gp23 in capsid assembly of wild-type T4.
- Published
- 1984
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17. Divergent effects of a dnaK mutation on abnormal protein degradation in Escherichia coli.
- Author
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Keller JA and Simon LD
- Subjects
- Escherichia coli metabolism, Genotype, Kinetics, beta-Galactosidase genetics, Bacterial Proteins genetics, Escherichia coli genetics, Escherichia coli Proteins, Genes, Genes, Bacterial, HSP70 Heat-Shock Proteins, Heat-Shock Proteins genetics, Mutation, Proteins metabolism
- Abstract
Escherichia coli bacteria produce at least one 70 kD stress protein, the product of the dnaK gene. We have compared the rates of degradation of different types of abnormal proteins in null Ion E. coli with a partial deletion of the dnaK gene with the rates observed in null Ion dnaK+ cells. We have found that both canavanyl proteins and puromycyl polypeptides are degraded more slowly in the null dnaK mutants than in the dnaK+ strain. However, a temperature-sensitive mutant LacI protein is degraded more rapidly in the null dnaK strain. The stability of this temperature-sensitive LacI protein was also examined in detail under various other conditions.
- Published
- 1988
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18. Hyperdegradation of proteins in Escherichia coli rho mutants.
- Author
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Simon LD, Gottesman M, Tomczak K, and Gottesman S
- Subjects
- Coliphages genetics, Escherichia coli genetics, Galactokinase metabolism, Kinetics, Mutation, Phenotype, Bacterial Proteins metabolism, Coliphages metabolism, Escherichia coli metabolism, Rho Factor metabolism, Transcription Factors metabolism
- Abstract
An Escherichia coli mutant, HDF026, defective for growth of phage T4, has been characterized biochemically and genetically. The mutant displays an elevated level of degradation of abnormal proteins, such as puromycyl polypeptides or canavanine-containing polypeptides. Genetically, HDF026 appears to be an allele of rho, which also encodes the transcription termination factor and RNA-dependent ATPase, Rho. The mutation contransduces by phage PI with ilv, weakly suppresses polar mutations in gal, and permits some growth of lambda N- phage. Temperature sensitive lambda mutants in gene O exhibit a reduced efficiency of plating at intermediate temperature on HDF026 mutants; presumably the lambda Ots protein is rapidly degraded in these strains. The ability of wild-type lambda to grow on HDF026 is also reduced, apparently the result of the lambda N product deficiency. gal escape synthesis, which reflects the level of lambda N activity, is decreased 50-66% in the HDF026 mutant. lambda r32, which requires more N function than wild-type phage, does not grow at all in HDF026. A lon mutation, which decreases protein degradation, partially reverses some of these phenotypes, suggesting that they are related to the protein hyperlability of HDF026.
- Published
- 1979
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19. Isolation and analysis of Escherichia coli mutants that allow increased replication of bacteriophage lambda.
- Author
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Keller JA and Simon LD
- Subjects
- Cytidine pharmacology, Escherichia coli physiology, Genes, Viral, Guanine pharmacology, Mutation, Phenotype, Rec A Recombinases genetics, Temperature, Viral Plaque Assay, Viral Proteins genetics, Virus Replication, Xanthine, Xanthines pharmacology, Bacteriophage lambda physiology, Escherichia coli genetics, Genes, Bacterial, Suppression, Genetic
- Abstract
Escherichia coli mutants were isolated that supported the growth of a lambda Ots and, in at least one case, a lambda Bts phage at the normally nonpermissive temperature of 39 degrees C. In one such strain, Ots and Bts suppression ability appeared to be a function of the guaB gene. Ots suppression by the mutant guaB strain was prevented if high levels of guanine or xanthine were present in the medium. No other base had any effect on Ots suppression in this strain. Other strains carrying spontaneous mutations resulting in guanine or xanthine auxotrophy (guaA or guaB lesions, respectively) all allowed lambda Ots replication at 39 degrees C; Ots suppression in these strains was also abolished by addition of guanine to the medium. Thus, reduced intracellular guanine levels resulting from guaA or guaB mutations appeared to suppress the inability of lambda Ots and, at least in some cases, Bts bacteriophage to form plaques at 39 degrees C. In burst size experiments, a guaB mutant produced a larger phage yield per infected cell of both lambda Ots and lambda O+ phage at 39 degrees C than did a similar guaB+ strain. It appeared that a lower-than-normal level of guanine (or a guanine derivative) in these cells may permit unusually efficient lambda replication. The fact that O+ and lambda Ots bursts in the guaB mutant were reduced significantly by addition of exogenous guanine to the medium is consistent with this suggestion. Another strain that suppresses the Ots allele has no known auxotrophic requirements, and suppression in this strain was unaffected by addition of guanine to the medium; however, addition of cytidine to the medium specifically eliminated Ots suppression in this strain. The mutation responsible for allowing Ots replication in this strain is unknown.
- Published
- 1987
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20. A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease.
- Author
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Skorupski K, Tomaschewski J, Rüger W, and Simon LD
- Subjects
- ATP-Dependent Proteases, Bacteriophage lambda growth & development, Base Sequence, Cloning, Molecular, Culture Media, DNA Restriction Enzymes, DNA, Viral genetics, Electrophoresis, Agar Gel, Genes, Viral, Genetic Vectors, Lysogeny, Molecular Sequence Data, Puromycin analogs & derivatives, Puromycin metabolism, Viral Plaque Assay, Viral Proteins metabolism, Deoxyribonucleases, Type II Site-Specific, Escherichia coli enzymology, Escherichia coli Proteins, Heat-Shock Proteins, Protease Inhibitors pharmacology, Protease La, Serine Endopeptidases metabolism, T-Phages genetics, Viral Proteins genetics
- Abstract
A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease has been identified. This pin (proteolysis inhibition) gene was selected for its ability to support plaque formation by a lambda Ots vector at 40 degrees C. Southern blot experiments indicated that this T4 gene is included within the 4.9-kilobase XbaI fragment which contains gene 49. Subcloning experiments showed that T4 gene 49.1 (designated pinA) is responsible for the ability of the Ots vector to form plaques at 40 degrees C. Deficiencies in Lon protease activity are the only changes known in E. coli that permit lambda Ots phage to form plaques efficiently at 40 degrees C. lon+ lysogens of the lambda Ots vector containing pinA permitted a lambda Ots phage to form plaques efficiently at 40 degrees C. Furthermore, these lysogens, upon comparison with similar lysogens lacking any T4 DNA, showed reduced levels of degradation of puromycyl polypeptides and of canavanyl proteins. The lon+ lysogens that contained pinA exhibited other phenotypic characteristics common to lon strains, such as filamentation and production of mucoid colonies. Levels of degradation of canavanyl proteins were essentially the same, however, in null lon lysogens which either contained or lacked pinA. We infer from these data that the T4 pinA gene functions to block Lon protease activity; pinA does not, however, appear to block the activity of proteases other than Lon that are involved in the degradation of abnormal proteins.
- Published
- 1988
- Full Text
- View/download PDF
21. Size changes among lambda capsid precursors.
- Author
-
Zachary A and Simon LD
- Subjects
- Coliphages growth & development, Escherichia coli, Lysogeny, Models, Biological, Morphogenesis, Mutation, Virus Replication, Capsid analysis, Coliphages ultrastructure, Protein Precursors analysis, Viral Proteins analysis
- Published
- 1977
- Full Text
- View/download PDF
22. Stabilization of proteins by a bacteriophage T4 gene cloned in Escherichia coli.
- Author
-
Simon LD, Randolph B, Irwin N, and Binkowski G
- Subjects
- Bacterial Proteins metabolism, Interferon Type I genetics, Interferon Type I metabolism, Cloning, Molecular methods, Escherichia coli genetics, Genes, Viral, T-Phages genetics
- Abstract
The cloned bacteriophage T4 pin gene functions to stabilize several different kinds of proteins in Escherichia coli bacteria. Incomplete proteins such as puromycyl polypeptides, abnormal but complete proteins such as the lambda phage tsO protein, and labile eukaryotic proteins encoded by genes cloned in E. coli such as mature human fibroblast interferon are stabilized in cells in which the T4 pin gene is expressed. The cloned T4 pin gene does not seem to affect the turnover of normal E. coli proteins.
- Published
- 1983
- Full Text
- View/download PDF
23. Bacterial mutation affecting T4 phage DNA synthesis and tail production.
- Author
-
Simon LD, Snover D, and Doermann AH
- Subjects
- Alleles, Autoradiography, Cell Count, Coliphages ultrastructure, Culture Media, Edetic Acid pharmacology, Electrophoresis, Polyacrylamide Gel, Escherichia coli ultrastructure, Genotype, Microscopy, Electron, Mutation, Sodium Dodecyl Sulfate, Coliphages metabolism, DNA, Viral biosynthesis, Escherichia coli metabolism, Virus Replication
- Published
- 1974
- Full Text
- View/download PDF
24. Effect of the position of the phenolic group in morphinans on their affinity for opiate receptor binding.
- Author
-
Simon LD, Simon FR, Mohasci E, Berger L, and Simon EJ
- Subjects
- Animals, Biological Assay, Isomerism, Mice, Morphinans pharmacology, Rats, Structure-Activity Relationship, Morphinans metabolism, Receptors, Opioid metabolism
- Published
- 1981
- Full Text
- View/download PDF
25. E. coli membrane lipid alteration affecting T4 capsid morphogenesis.
- Author
-
Simon LD, McLaughlin TJ, Snover D, Ou J, Grisham C, and Loeb M
- Subjects
- Cell Membrane analysis, Cell Membrane metabolism, Chromosome Mapping, Coliphages ultrastructure, Electron Spin Resonance Spectroscopy, Electrophoresis, Polyacrylamide Gel, Escherichia coli ultrastructure, Fatty Acids analysis, Microscopy, Electron, Morphogenesis, Mutation, Phospholipids analysis, Temperature, Coliphages growth & development, Escherichia coli metabolism, Lipid Metabolism
- Published
- 1975
- Full Text
- View/download PDF
26. Bacteriophage T4 bypass31 mutations that make gene 31 nonessential for bacteriophage T4 replication: isolation and characterization.
- Author
-
Simon LD and Randolph B
- Subjects
- DNA Replication, DNA, Viral genetics, Species Specificity, Suppression, Genetic, Viral Proteins analysis, Virus Replication, DNA, Viral isolation & purification, Escherichia coli genetics, Genes, Genes, Viral, Mutation, T-Phages genetics, Viral Proteins genetics
- Abstract
T4 bacteriophage mutants called bypass31 (byp31) that specifically suppress gene 31 amber mutations have been isolated and characterized. The mechanism by which the byp31 mutation, byp31-1, suppresses gene 31 nonsense mutations does not involve synthesis of gp31 or of a particular gp31 fragment; furthermore, the byp31 allele suppresses all nonsense mutations in gene 31 that have been tested. We detect no unusual properties among the T4 particles made in su- cells by the T4amN54byp31-1 double mutant. These virions, made in the absence of gp31, show normal heat sensitivity, normal sensitivity to osmotic shock, and normal morphology. Specific different gene 31 missense mutants are able to form plaques with high efficiencies on the following two types of host defective cells: (i) Escherichia coli groEL (Tilly et al., Proc. Natl. Acad. Sci. U.S.A. 78:1629-1633, 1981) mutants that block T4 capsid assembly and (ii) E. coli rho mutants in which T4+ heads are assembled, but in which tail production and DNA synthesis are blocked. (Note that not all rho mutants block T4 production [G. Binkowski and L. D. Simon, p. 342-350, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget, ed., Bacteriophage T4, 1983]; T4 is able to replicate in rho mutants such as rho ts15, whose principal defect is that they fail to terminate transcription.) The byp31-1 allele permits production of T4 particles in E. coli groEL host-defective mutants, but not in E. coli rho host mutants.
- Published
- 1984
- Full Text
- View/download PDF
27. Lambda head morphogenesis as seen in the electron microscope.
- Author
-
Zachary A, Simon LD, and Litwin S
- Subjects
- Coliphages ultrastructure, DNA, Viral, Escherichia coli, Lysogeny, Models, Biological, Morphogenesis, Mutation, Virus Replication, Capsid, Coliphages growth & development, Viral Proteins
- Published
- 1976
- Full Text
- View/download PDF
28. The infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope. II. Structure and function of the baseplate.
- Author
-
Simon LD and Anderson TF
- Subjects
- Microscopy, Electron, Coliphages, Escherichia coli
- Published
- 1967
- Full Text
- View/download PDF
29. Some morphological properties of P4 bacteriophage and P4 DNA.
- Author
-
Inman RB, Schnös M, Simon LD, Six EW, and Walker DH Jr
- Subjects
- Genes, Genetics, Microbial, Helper Viruses analysis, Helper Viruses isolation & purification, Lysogeny, Microscopy, Electron, Staining and Labeling, Viral Proteins, Coliphages analysis, Coliphages growth & development, Coliphages isolation & purification, DNA, Viral analysis, DNA, Viral isolation & purification, Escherichia coli
- Published
- 1971
- Full Text
- View/download PDF
30. The infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope. 3. Membrane-associated intracellular bacteriophages.
- Author
-
Simon LD
- Subjects
- Binding Sites, Cell Membrane, Microscopy, Electron, Coliphages, Escherichia coli, Virus Replication
- Published
- 1969
- Full Text
- View/download PDF
31. Infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope: T4 head morphogenesis.
- Author
-
Simon LD
- Subjects
- Cell Membrane metabolism, Coliphages metabolism, DNA, Viral metabolism, Microscopy, Electron, Morphogenesis, Mutation, Coliphages growth & development, Escherichia coli metabolism
- Abstract
Bacteriophage T4 capsids seem to be assembled on E. coli protoplasmic membranes. This process seems to involve "lumps" of head protein, which convert to tau particles, which in turn give rise to empty heads. The empty heads leave the bacterial membrane and are then filled with DNA in the central region of the cell. T4 gene 16 and 17 products appear to be necessary for head filling.
- Published
- 1972
- Full Text
- View/download PDF
32. The infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope. I. Attachment and penetration.
- Author
-
Simon LD and Anderson TF
- Subjects
- Microscopy, Electron, Coliphages, Escherichia coli
- Published
- 1967
- Full Text
- View/download PDF
33. Functional defects in T4 bacteriophages lacking the gene 11 and gene 12 products.
- Author
-
Simon LD, Swan JG, and Flatgaard JE
- Subjects
- Adsorption, Carbon Isotopes, Cell Wall, Centrifugation, Centrifugation, Density Gradient, Coliphages growth & development, Genetics, Microbial, Lysogeny, Microscopy, Electron, Molecular Biology, Staining and Labeling, Thymidine, Tritium, Viral Proteins metabolism, Coliphages metabolism, Escherichia coli, Genes, Mutation
- Published
- 1970
- Full Text
- View/download PDF
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