scholarly journals In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.

1996 ◽  
Vol 16 (11) ◽  
pp. 6468-6476 ◽  
Author(s):  
S A Shaaban ◽  
E V Bobkova ◽  
D M Chudzik ◽  
B D Hall
Keyword(s):  
Rna Polymerase Iii ◽  
Multiple Roles ◽  
Wild Type ◽  
Protein Motifs ◽  
Study Support ◽  
Vitro Analysis ◽  
Pol Iii ◽  
In Vitro Analysis

We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467-1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA(Tyr) gene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.

scholarly journals Aggravated Lyme Carditis in CD11a−/− and CD11c−/− Mice

2005 ◽  
Vol 73 (11) ◽  
pp. 7637-7643 ◽  
Author(s):  
Mireia Guerau-de-Arellano ◽  
Joseph Alroy ◽  
Daniel Bullard ◽  
Brigitte T. Huber
Keyword(s):  
Wild Type ◽  
Lyme Carditis ◽  
Disease Induction ◽  
In Vivo Analysis ◽  
The Common ◽  
Vitro Analysis ◽  
Β2 Integrins ◽  
In Vitro Analysis

ABSTRACT CD18 hypomorph mice expressing reduced levels of the common β2 integrin chain develop aggravated Lyme carditis, compared to that developed by wild-type (WT) mice, upon infection with the spirochete Borrelia burgdorferi. The enhancement of Lyme carditis in these mice is characterized by increased macrophage infiltration, correlating with augmented expression of the monocyte/macrophage chemoattractant protein 1 (MCP-1). The lack of CD18 results in the deficiency of all β2 integrins, i.e., CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1/CR3), CD11c/CD18 (p150,95/CR4), and CD11d/CD18. To determine the roles of the various β2 integrins in controlling the development of aggravated Lyme carditis, disease induction was analyzed in CD11a−/−, CD11b−/−, and CD11c−/− mice. CD11a−/− and CD11c−/− mice, but not CD11b−/− mice, developed aggravated Lyme carditis after exposure to B. burgdorferi. Similarly to CD18 hypomorph mice, CD11c−/− mice expressed higher levels of MCP-1, compared to both WT and CD11a−/− mice, as determined by in vitro analysis of MCP-1 secretion by bone marrow-derived dendritic cells and in vivo analysis of MCP-1 mRNA expression in B. burgdorferi-infected hearts. On the other hand, CD11a deficiency was associated with heightened heart B. burgdorferi burden relative to that of WT mice. Overall, our results suggest that the increased severity of Lyme carditis in CD18 hypomorph mice is caused by deficiency in CD11a or CD11c, possibly via different mechanisms.

scholarly journals Changes in the Morphology and Cation Content of a Bambusa edulis Xylem Mutant, vse, Derived from Somaclonal Variation

2006 ◽  
Vol 131 (4) ◽  
pp. 445-451 ◽  
Author(s):  
Choun-Sea Lin ◽  
Huey-Ling Lin ◽  
Wann-Neng Jane ◽  
Han-Wen Hsiao ◽  
Chung-Chih Lin ◽  
...  
Keyword(s):  
Potassium Deficiency ◽  
Naphthaleneacetic Acid ◽  
Wild Type ◽  
Blade Angle ◽  
Young Leaves ◽  
Growth Habits ◽  
Vitro Analysis ◽  
In Vitro Analysis

A xylem mutant (vse) was isolated from a Bambusa edulis (Odashima) Keng plantlet following vegetative micropropagation and subculture for 7 consecutive years and induced to proliferate in medium supplemented with 0.1 mg·L-1 (0.5 μm) thidiazuron (TDZ) and to develop roots in medium supplemented with 5 mg·L-1 (26.9 μm) α-naphthaleneacetic acid (NAA). Subsequent investigations comparing the growth habits of mutant plantlets with those of the wild type indicated that the growth of the former was retarded in a greenhouse. Several morphological abnormalities were observed in the vse mutant: it had thinner stems with fewer trichromes on the surface; the xylem vessels were smaller in diameter and contained crystal-like structures in the pith; the leaves were shorter and narrower with a sharp leaf blade angle; the roots were thinner and contained fewer xylem cells. The cation concentrations of both the mutant and wild type were similar in the in vitro analysis, except for those of iron and potassium, which were lower in mutant leaves in vivo. In 2-month-old mutant plants, iron chlorosis was observed on young leaves and a potassium deficiency was observed on older leaves. After 1 year of growth in the greenhouse, all of the wild-type plants had survived, but only 27% (16/60) of the mutant vse plants were alive.

Multifactorial Regulation of Cardiac Gene Expression: an In Vivo and In Vitro Analysis

1995 ◽  
Vol 752 (1 Cardiac Growt) ◽  
pp. 370-386 ◽  
Author(s):  
J. L. SAMUEL ◽  
I. DUBUS ◽  
F. FARHADIAN ◽  
F. MAROTTE ◽  
P. OLIVIERO ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiac Gene Expression ◽  
Cardiac Gene ◽  
Vitro Analysis ◽  
In Vitro Analysis

scholarly journals Novel Small-Molecule Inhibitors of RNA Polymerase III

2003 ◽  
Vol 2 (2) ◽  
pp. 256-264 ◽  
Author(s):  
Liping Wu ◽  
Jing Pan ◽  
Vala Thoroddsen ◽  
Deborah R. Wysong ◽  
Ronald K. Blackman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Rna Polymerase Iii ◽  
Wild Type ◽  
Biochemical Assays ◽  
Transcription In Vitro ◽  
Pol Iii

ABSTRACT A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.

scholarly journals Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

2004 ◽  
Vol 384 (2) ◽  
pp. 337-348 ◽  
Author(s):  
Duane A. LEHTINEN ◽  
Fred W. PERRINO
Keyword(s):  
Dna Polymerase ◽  
Gel Filtration ◽  
Exonuclease Activity ◽  
Dna Polymerase Iii ◽  
Wild Type ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Pol Iii

The ε-subunit contains the catalytic site for the 3′→5′ proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the α-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the ε mutants were purified, and these proteins exhibited 3′→5′ exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type ε protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 ε mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified ε mutants with the α- and θ-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active ε511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The ε511 mutant associated tightly with the α-subunit, but the exonuclease activity of ε511 was not stimulated in the α–ε511 complex. Addition of the θ-subunit to generate the α–ε511–θ DNA pol III core partially restored stimulation of the ε511 exonuclease, indicating a role for the θ-subunit in co-ordinating the α–ε polymerase–exonuclease interaction. The α–ε511–θ DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the α–ε511–θ complex. Thus the ε511 mutant has wild-type 3′→5′ exonuclease activity and associates physically with the α- and θ-subunits to generate a proofreading-defective DNA pol III enzyme.

scholarly journals Comprehensive In Vitro Analysis of Voriconazole Inhibition of Eight Cytochrome P450 (CYP) Enzymes: Major Effect on CYPs 2B6, 2C9, 2C19, and 3A

2008 ◽  
Vol 53 (2) ◽  
pp. 541-551 ◽  
Author(s):  
Seongwook Jeong ◽  
Phuong D. Nguyen ◽  
Zeruesenay Desta
Keyword(s):  
Cytochrome P450 ◽  
Liver Microsomes ◽  
Competitive Inhibitor ◽  
Major Effect ◽  
Cyp Enzymes ◽  
Human Cytochrome ◽  
Vitro Analysis ◽  
In Vitro Analysis

ABSTRACT Voriconazole is an effective antifungal drug, but adverse drug-drug interactions associated with its use are of major clinical concern. To identify the mechanisms of these interactions, we tested the inhibitory potency of voriconazole with eight human cytochrome P450 (CYP) enzymes. Isoform-specific probes were incubated with human liver microsomes (HLMs) (or expressed CYPs) and cofactors in the absence and the presence of voriconazole. Preincubation experiments were performed to test mechanism-based inactivation. In pilot experiments, voriconazole showed inhibition of CYP2B6, CYP2C9, CYP2C19, and CYP3A (half-maximal [50%] inhibitory concentrations, <6 μM); its effect on CYP1A2, CYP2A6, CYP2C8, and CYP2D6 was marginal (<25% inhibition at 100 μM voriconazole). Further detailed experiments with HLMs showed that voriconazole is a potent competitive inhibitor of CYP2B6 (Ki < 0.5), CYP2C9 (Ki = 2.79 μM), and CYP2C19 (Ki = 5.1 μM). The inhibition of CYP3A by voriconazole was explained by noncompetitive (Ki = 2.97 μM) and competitive (Ki = 0.66 μM) modes of inhibition. Prediction of the in vivo interaction of voriconazole from these in vitro data suggests that voriconazole would substantially increase the exposure of drugs metabolized by CYP2B6, CYP2C9, CYP2C19, and CYP3A. Clinicians should be aware of these interactions and monitor patients for adverse effects or failure of therapy.

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