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ATPase-defective derivatives of Escherichia coli DnaK that behave differently with respect to ATP-induced conformational change and peptide release. 下载免费PDF全文
We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a DeltadnaK mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell. 相似文献
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Klaus-Günther Barthel 《Polar Biology》1990,10(5):343-350
Summary During MIZEX'83 and MIZEX'84 food composition and food uptake by three Calanus species were investigated in the northern Greenland Sea. Samples were obtained from open water, the marginal ice zone, the pack ice region and the East Greenland shelf polynya. Food uptake of Calanus spp. was determined in ship-board incubation experiments under in situ conditions and volume of particulate matter in the upper 300 m of the water column was measured. Principal components analysis was used for data evaluation. Under the pack ice strongly reduced amounts of particulate matter in the euphotic zone were found while concentrations were elevated in the marginal ice zone and the East Greenland shelf polynya. Food uptake of C. finmarchicus was correlated to food concentration. In the pack ice region ingestion was close to zero for all species investigated. Likewise body weights were significantly lower than in MIZ or polynya. Principal components analysis of physical data shows a clustering of MIZ station groups at high temperature and salinity, clearly separated from pack ice and polynya stations. Analysis of biological data results in the same grouping of stations demonstrating a strong influence of hydrographic conditions on plankton development. It is concluded that herbivorous copepods cannot sustain themselves in the pack ice region but only at places of high productivity like marginal ice zone and East Greenland shelf polynya. 相似文献
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A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera 总被引:14,自引:1,他引:13
Levels of mitochondrial DNA (mtDNA) sequence divergence between species
within each of several avian (Anas, Aythya, Dendroica, Melospiza, and
Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were
compared. An analysis of digestion profiles generated by 13-18 restriction
endonucleases indicates little overlap in magnitude of mtDNA divergence for
the avian versus nonavian taxa examined. In 55 interspecific comparisons
among the avian congeners, the fraction of identical fragment lengths (F)
ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these
translate into estimates of nucleotide sequence divergence (p) ranging from
0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F
values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater
than 0.070. The small mtDNA distances among avian congeners are associated
with protein-electrophoretic distances (D values) less than approximately
0.2, while the mtDNA distances among assayed fish and amphibian congeners
are associated with D values usually greater than 0.4. Since the
conservative pattern of protein differentiation previously reported for
many avian versus nonavian taxa now appears to be paralleled by a
conservative pattern of mtDNA divergence, it seems increasingly likely that
many avian species have shared more recent common ancestors than have their
nonavian taxonomic counterparts. However, estimates of avian divergence
times derived from mtDNA- and protein-calibrated clocks cannot readily be
reconciled with some published dates based on limited fossil remains. If
the earlier paleontological interpretations are valid, then protein and
mtDNA evolution must be somewhat decelerated in birds. The empirical and
conceptual issues raised by these findings are highly analogous to those in
the long-standing debate about rates of molecular evolution and times of
separation of ancestral hominids from African apes.
相似文献
7.
Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes 总被引:23,自引:2,他引:21
Statistical methods for computing the standard errors of the branching
points of an evolutionary tree are developed. These methods are for the
unweighted pair-group method-determined (UPGMA) trees reconstructed from
molecular data such as amino acid sequences, nucleotide sequences,
restriction-sites data, and electrophoretic distances. They were applied to
data for the human, chimpanzee, gorilla, orangutan, and gibbon species.
Among the four different sets of data used, DNA sequences for an
895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the
most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979)
gave the least reliable one. The DNA sequence data suggested that the
chimpanzee is the closest and that the gorilla is the next closest to the
human species. The orangutan and gibbon are more distantly related to man
than is the gorilla. This topology of the tree is in agreement with that
for the tree obtained from chromosomal studies and DNA-hybridization
experiments. However, the difference between the branching point for the
human and the chimpanzee species and that for the gorilla species and the
human-chimpanzee group is not statistically significant. In addition to
this analysis, various factors that affect the accuracy of an estimated
tree are discussed.
相似文献
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Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves 总被引:4,自引:0,他引:4
A puzzling population-genetic phenomenon widely reported in allozyme
surveys of marine bivalves is the occurrence of heterozygote deficits
relative to Hardy-Weinberg expectations. Possible explanations for this
pattern are categorized with respect to whether the effects should be
confined to protein-level assays or are genomically pervasive and expected
to be registered in both protein- and DNA-level assays. Anonymous nuclear
DNA markers from the American oyster were employed to reexamine the
phenomenon. In assays based on the polymerase chain reaction (PCR), two
DNA-level processes were encountered that can lead to artifactual genotypic
scorings: (a) differential amplification of alleles at a target locus and
(b) amplification from multiple paralogous loci. We describe symptoms of
these complications and prescribe methods that should generally help to
ameliorate them. When artifactual scorings at two anonymous DNA loci in the
American oyster were corrected, Hardy-Weinberg deviations registered in
preliminary population assays decreased to nonsignificant values.
Implications of these findings for the heterozygote-deficit phenomenon in
marine bivalves, and for the general development and use of PCR-based
assays, are discussed.
相似文献
10.
A rapid two-step procedure has been developed for the purification of Despro(2)-Val15-Leu17-aprotinin from the culture supernatant of a recombinant yeast by affinity and ion-exchange chromatography. DesPro(2)-Val15-Leu17-aprotinin was purified to homogeneity, as demonstrated by dodecylsulfate gel electrophoresis and analysis of the N-terminal amino acid sequence. (c) 1993 John Wiley & Sons, Inc. 相似文献