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81.
The sequence selectivity of enzyme-DNA interactions was analyzed by comparing discrimination between synthetic oligonucleotides containing the canonical site GAATTC and altered DNA sequences with the EcoRI DNA methyltransferase. The specificities (kcat/KmDNA) are decreased from 5- to 23,000-fold relative to the unmodified site. For several substrates the decrease in kcat makes a disproportionate contribution to the specificity difference, suggesting that discrimination is mediated by the placement of critical catalytic residues rather than binding interactions. This is supported by our observation that specificity changes are generally not followed by changes in the stability of the methyltransferase-DNA complexes. Also, base pair substitutions near the site of methylation result in greater decreases in complex stability, suggesting that recognition and catalytic mechanisms overlap. 相似文献
82.
83.
Plasticity of the tobacco genome was studied by testing the DNAs of protoplast-derived regenerants with three different repetitive
DNA sequences by the method of quantitative DNA/DNA hybridizations. A large population of 91 regenerants belonging to 35 different
protoclones was analysed and a high degree of heterogeneity in the contents of the different DNA repeats was detected. The
contents of middle repetitive sequences of two types were more stable or changed in the same direction, while the highly repetitive
sequence varied independently and displayed a significant reduction in comparison with the two other sequences. Comparing
the variation within the subpopulations of plants of the same clonal origin and the variation among the protoclones led to
a conclusion that the pre-existing DNA variability in the starting plant material and/or thein vitro stress during the very early stages of protoclone regeneration played a decisive role in the formation of modified genomes
in regenerants. 相似文献
84.
Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) by two Alcaligenes eutrophus strains and one Pseudomonas cepacia strain containing the 2,4-D degrading plasmids pJP4 or pRO101 (=pJP4::Tn1721) was tested in 50 g (wet wt) samples of non-sterile soil. Mineralization was measured as 14C-CO2evolved during degradation of uniformly-ring-labelled 14C-2,4-D. When the strains were inoculated to a level of approximately 108 CFU/g soil, between 20 and 45% of the added 2,4-D (0.05 ppm, 10 ppm or 500 ppm) was mineralized within 72 h. Mineralization of 0.05 ppm and 10 ppm, 2,4-D by the two A. eutrophus strains was identical and rapid whereas mineralization by P. cepacia DBO1(pRO101) occurred more slowly. In contrast, mineralization of 500 ppm 2,4-D by the two A. eutrophus strains was very slow whereas mineralization by P. cepacia DBO1 was more rapid. Comparison of 2,4-D mineralization at different levels of inoculation with P. cepacia DBO1(pRO101) (6×104, 6×106 and 1×108 CFU/g soil) revealed that the maximum mineralization rate was reached earlier with the high inoculation levels than with the low level. The kinetics of mineralization were evaluated by nonlinear regression analysis using five different models. The linear or the logarithmic form of a three-half-order model were found to be the most appropriate models for describing 2,4-D mineralization in soil. In the cases in which the logarithmic form of the three-half-order model was the most appropriate model we found, in accordance with the assumptions of the model, a significant growth of the inoculated strains.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- CFU
colony forming units
- PTYG
peptone, tryptone, yeast & glucose
- DPM
disintegrations per minute 相似文献
85.
Inhibition of EcoRI DNA methylase with cofactor analogs 总被引:5,自引:0,他引:5
Four analogs of the natural cofactor S-adenosylmethionine (AdoMet) were tested for their ability to bind and inhibit the prokaryotic enzyme, EcoRI adenine DNA methylase. The EcoRI methylase transfers the methyl group from AdoMet to the second adenine in the double-stranded DNA sequence 5'GAATTC3'. Dissociation constants (KD) of the binary methylase-analog complexes obtained in the absence of DNA with S-adenosylhomocysteine (AdoHcy), sinefungin, N-methyl-AdoMet, and N-ethylAdoMet are 225, 43, greater than 1000, and greater than 1000 microM, respectively. In the presence of a DNA substrate, all four analogs show simple competitive inhibition with respect to AdoMet. The product of the enzymic reaction, AdoHcy, is a poor inhibitor of the enzyme (KI(AdoHcy) = 9 microM; KM(AdoMet) = 0.60 microM). Two synthetic analogs, N-methyl-AdoMet and N-ethyl-AdoMet, were also shown to be poor inhibitors with KI values of 50 and greater than 1000 microM, respectively. In contrast, the naturally occurring analog sinefungin was shown to be a highly potent inhibitor (KI = 10 nM). Gel retardation assays confirm that the methylase-DNA-sinefungin complex is sequence-specific. The ternary complex is the first sequence-specific complex detected for any DNA methylase. Potential applications to structural studies of methylase-DNA interactions are discussed. 相似文献
86.
Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements 总被引:1,自引:0,他引:1
P W Hager N O Reich J P Day T G Coche H W Boyer J M Rosenberg P J Greene 《The Journal of biological chemistry》1990,265(35):21520-21526
The x-ray structure of the EcoRI endonuclease-DNA complex (3) suggests that hydrogen bonds between amino acids, glutamic acid 144, arginine 145, and arginine 200, and major groove base moieties are the molecular determinants of specificity. We have investigated residue 144 using aspartate and glutamine substitutions introduced by site-directed mutagenesis. Substitution with glutamine results in a null phenotype (at least a 2000-fold reduction in activity). On the other hand, the aspartic acid mutant (ED144) retained in vivo activity. Substrate binding and catalytic studies were done with purified ED144 enzyme. The affinity of the ED144 enzyme for the canonical sequence 5'-GAATTC-3' is about 340-fold less than the wild-type (WT) enzyme, while its affinity for nonspecific DNA is about 50 times greater. The ED144 enzyme cleaves one strand in the EcoRI site in plasmid pBR322 with a kcat/Km similar to WT. In contrast to the WT enzyme, the ED144 enzyme dissociates after the first strand cleavage. Partitioning between cleavage and dissociation at the first and second cleavage steps for the ED144 enzyme is extremely salt-sensitive. The altered partitioning results largely from a destabilization of the enzyme-DNA complex, particularly the enzyme-nicked DNA complex, with only small changes in the respective cleavage rates. The hydrogen bonds of Glu-144 are critical, they appear to act cooperatively with other specificity contacts to stabilize the enzyme-DNA complex. 相似文献
87.
Erik Steen Hansen Jens Knudsen 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,721(4):418-424
Heat production, free fatty acid and glycerol release from white adipose tissue fat pads from obese (ob/ob) mice and their lean littermates are determined. Heat production was significantly lower in obese mice compared to lean mice when expressed on wet weight basis but not when expressed on DNA basis. Noradrenaline significantly increased the heat production in fat pads from both groups of animals. However, the increase in heat production due to noradrenaline addition in fat pads from lean mice was significantly higher than in fat pads from obese mice. The release of free fatty acids and glycerol before incubation with noradrenaline was similar from fat pads from both groups of animals. Addition of noradrenaline to the fat pads increased the release of free fatty acids and glycerol in both groups of animals, but the increase was significantly larger from fat pads from lean mice. In the absence of noradrenaline the free fatty acid/glycerol ratio (mol/mol) in the effluent was 7.9:1 and 4.8:1 for lean mice and obese mice, respectively. In the presence of noradrenaline the ratio decreased to 3:1 for both groups of animals. 相似文献
88.
Regulation of the cellular p53 tumor antigen in teratocarcinoma cells and their differentiated progeny. 总被引:20,自引:10,他引:10
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F9 embryonal carcinoma cells express high levels of a 53,000-molecular-weight cellular tumor antigen called p53. When F9 cell cultures are treated with retinoic acid and dibutyryl adenosine 3',5'-phosphate, they differentiate, predominantly into endoderm-like cells. This differentiation is accompanied by a marked decrease in the levels of p53. The mechanism(s) responsible for this decline in the level of p53 in differentiated cells was investigated. The results demonstrate that the high levels of p53 in F9 cells relative to their differentiated progeny were not due to alterations in the stability or turnover of this protein. Rather, the regulation during differentiation involved a marked decrease in the amount of in vitro translatable p53 mRNA detected in the differentiated cell cultures. This mechanism is unlike the one operating during the simian virus 40 infection or transformation, where the increased levels of p53 are largely due to the increased stability of the p53 protein. 相似文献
89.
Bente Christiansen Jens Pete Lomholt Kjell Johansen 《Environmental Biology of Fishes》1982,7(3):291-296
Synopsis Oxygen uptake (Vo
2) was measured in carp of approximately 40 cm length swimming at controlled variable oxygen tensions (Po
2). At Po
2> 120 mm Hg Vo
2 increased with an increase in swimming speed from 5.6 to 11.3 cm · sec–1. Extrapolation of Vo
2 to zero activity at Po
2 = 140 mm Hg revealed a standard O2 uptake of 36.7 ml O2 · kg–1 · h–1 at 20° C. At the lowest swimming speed (5.6 cm · s–1) the oxygen uptake increased when the water Po
2 was reduced. A near doubling in Vo
2 was seen at Po
2 = 70 mm Hg compared to 140 mm Hg. At higher swimming speeds in hypoxic water Vo
2 decreased relative to the values at low swimming speeds. As a result the slope of the lines expressing log Vo
2 as a function of swimming speed decreased from positive to negative values with decreasing Po
2 of the water. pH of blood from the caudal vein drawn before and at termination of swimming at Po
2 = 70 mm Hg and 100 mm Hg did not show any decrease in relation to rest values at Po
2 = 140 mm Hg. Blood lactate concentration did not increase during swimming at these tensions. 相似文献
90.
Peter Sarnow Patrick Hearing Carl W. Anderson Nancy Reich Arnold J. Levine 《Journal of molecular biology》1982,162(3):565-583
Antisera from some hamsters bearing adenovirus-induced tumors contain antibodies to an 11,000 Mr adenovirus-induced protein. In adenovirus-infected HeLa cells, this early viral protein was specifically associated with the nuclear matrix fraction. After two-dimensional gel electrophoresis, two forms of the 11,000 Mr protein at pI 5.6 and pI 5.4 were found. Only the pI 5.4 form of this protein was associated with the nuclear matrix fraction. Adenoviruses from groups A, B, C, D and E all produced an early viral protein (10,000 to 12,000 Mr) that reacted with group C antibody to the 11,000 Mr protein. To date, this is the only known early viral protein that is immunologically conserved in all of the human adenovirus groups.The positions of two methionine and seven leucine residues were determined by sequencing the first 35 amino acids from the N terminus of the adenovirus serotype 2 group C 11,000 Mr protein. The positions of these amino acid residues were compared to the adenovirus serotype 2 nucleotide sequence, which uniquely localized the structural gene of the 11,000 Mr protein to region E4, subregion 3 in type 2 adenovirus. A frameshift mutant, which contained a deletion of one base-pair in the structural gene of the 11,000 Mr protein, was isolated and mapped by marker rescue and nucleotide sequence analysis. This mutant failed to produce immunologically detectable 11,000 Mr protein. The mutant had a viable phenotype, producing normal levels of infectious virus in both HeLa cells and WI38 cells in culture. These experiments identify the first adenovirus early region 4 protein detected in virus-infected cells. 相似文献