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1.
2.
D A Hartman S R Kuo T R Broker L T Chow R D Wells 《The Journal of biological chemistry》1992,267(8):5488-5494
A conformational distortion in the DNA duplex at the regulatory region of human papillomavirus type-11 next to an intermolecular triplex, formed with a synthetic oligonucleotide, was investigated with several chemical probes. The sequence targeted for triplex formation borders on the binding sites for the regulatory proteins encoded by the viral E2 open reading frame. Dimethyl sulfate, diethyl pyrocarbonate, and OsO4 all react to a greater extent with nucleotides in the duplex that are immediately adjacent to the triplex as compared to other bases throughout the duplex. This hypermodification was observed on both the polypurine and polypyrimidine strands of the duplex DNA. Similar hyperreactivity of bases flanking a triplex also was seen when the contiguous target polypurine tract was effectively extended by mutating interrupting pyrimidines in the human papillomavirus type-11 sequence to purines. We propose that this hyperreactivity is due to a structural distortion caused by the junction between the triplex and the duplex tracts. 相似文献
3.
Salil K. Niyogi Thomas S. Soper Robert S. Foote Frank W. Larimer Richard J. Mural Sankar Mitra Eva H. Lee Richard Machanoff Fred C. Hartman 《Journal of biosciences》1987,11(1-4):203-214
Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted
to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested
are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as
active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of
the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains
largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier
postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway. 相似文献
4.
Rates of oxalate degradation by mixed bacterial populations in cecal contents from wild rats ranged from 2.5 to 20.6 mumol/g (dry weight) per h. The oxalate-degrading activity in cecal contents from three strains of laboratory rats (Long-Evans, Wistar, and Sprague-Dawley) from four commercial breeders was generally lower, ranging from 1.8 to 3.5 mumol/g (dry weight) of cecal contents per h. This activity did not increase when diets were supplemented with oxalate. When Sprague-Dawley rats from a fifth commercial breeder were fed an oxalate diet, rates of oxalate degradation in cecal contents increased from 2.0 to 23.1 mumol/g (dry weight) per h. Obligately anaerobic, oxalate-degrading bacteria, similar to ruminal strains of Oxalobacter formigenes, were isolated from the latter group of laboratory rats and from wild rats. Viable counts of these bacteria were as high as 10(8)/g (dry weight) of cecal contents, which was less than 0.1% of the total viable population. This report presents the first evidence for the presence of anaerobic oxalate-degrading bacteria in the cecal contents of rats and represents the first direct measurement of the concentration of these bacteria in the large bowel of monogastric animals. We propose that methods used for the maintenance of most commercial rat colonies often preclude the intestinal colonization of laboratory rats with anaerobic oxalate-degrading bacteria. 相似文献
5.
Improved membrane filtration media for enumeration of total coliforms and Escherichia coli from sewage and surface waters. 总被引:6,自引:5,他引:1
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Two media were developed that allowed both a total coliform count and an Escherichia coli count to be determined on the same medium after 24 h of incubation at 35 degrees C. The new media were tested along with two standard media on 10 surface water and 7 sewage samples. The experimental media yielded equivalent or higher counts relative to the standard media and recovered more specifically the desired indicator groups as determined by colony identification. 相似文献
6.
Excision Repair of Uv Radiation-Induced DNA Damage in Caenorhabditis Elegans 总被引:1,自引:0,他引:1
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Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts [cyclobutane dimers and (6-4) photoproducts]. Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed. 相似文献
7.
8.
Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16 总被引:1,自引:0,他引:1
The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK
d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [14C]reagent per mole of enzyme subunit. Amino acid analysis of the [14C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site. 相似文献
9.
Isolation and identification of a morpholine-degrading bacterium 总被引:2,自引:0,他引:2
A gram-positive, slowly growing rod effectively utilizing morpholine as the sole source of organic carbon, nitrogen, and energy was isolated from a mixed culture in a laboratory reactor. The strain was tentatively identified as Mycobacterium aurum. Its growth characteristics at 20 degrees C and pH 6.5 were as follows: maximum specific growth rate, 0.052 h-1; half-velocity constant, 1.3 mg/liter; and yield, 0.37 g/g. The optimum temperature and pH were 31 degrees C and 6.0, respectively. 相似文献
10.
Catalytic nonessentiality of an active-site cysteinyl residue of phosphoribulokinase 总被引:1,自引:0,他引:1
The activity of the Calvin cycle enzyme phosphoribulokinase is coupled to photosynthetic electron transport by reversible oxidation/reduction mediated by thioredoxin-f. Previous studies have shown that one of the regulatory sulfhydryl groups, that of Cys-16, is positioned at the nucleotide-binding domain of the active site. To determine if oxidative deactivation of the kinase reflects catalytic essentiality of Cys-16, the methylation of spinach phosphoribulokinase by methyl-4-nitrobenzenesulfonate has been examined. Methylation of the kinase results in a 50% loss of the initial activity relative to controls. The suppression of kcat is accompanied by a 6-fold increase in the Km for ATP, without change in the Km for ribulose 5-phosphate. The insensitivity of the modified enzyme, in contrast to the native, to iodoacetate and 5,5'-dithiobis(2-nitrobenzoate) indicates that Cys-16 is a site of methylation. This supposition is verified independently by peptide mapping and Edman degradation subsequent to S-carboxymethylation with [14C]iodoacetate of the methylated kinase. Retention of significant enzymatic activity after complete modification of Cys-16 with the small, uncharged methyl moiety demonstrates that this active-site residue is not essential for catalysis. 相似文献