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1.
Summary For each of eleven different types of nuclear genes, comparisons of the protein coding sequences were made between human, mouse and rat pairwisely, and the evolutionary rate of silent substitution, v S nucl. , was estimated. It is shown that the v S nucl. is not only very high (=5.37×10–9/site/yr), but also approximately uniform for different genes regardless of the types, which confirms our previous results (Miyata et al. 1980b). This is in sharp contrast to the rate of protein evolution which differes greatly from protein to protein. Furthermore the v S nucl. is shown to be approximately constant with respect to different divergence times, at least within a short time period (75 Myr). Based on these observations, we propose a new molecular clock which has several advantages over a protein clock. Using this clock, we show that the rate of amino acid replacement in the immunoglobulin Ck gene of b4 rabbit is unexpectedly high, almost comparable to the rate of silent changes. This rate may be the highest one for protein evolution that we know so far. We further examine the rate of silent substitutions in mitochondrial genes comparing mouse and rat. Surprisingly the rate is extremely high (35×10–9/site/yr), at least 6-times as high as the corresponding rate of nuclear genes. Based on the estimate, we discuss a possible origin of the rapid rate found in mitochondrial DNA.  相似文献   

2.
Summary In analyzing the silent nucleotide substitutions in some mammalian mitochondrial mRNA coding genes, we had found that the frequency of each of the four nucleotides in rat, mouse, and cow, but not in humans, is the same in the silent third codon position (Lanave C, Preparata G, Saccone C, Serio G (1984) J Mol Evol 20:86-93). Because our findings for these three species were compatible with a stationary Markov process for the evolution of nucleotide sequences, we applied such a model to calculate the effective evolutionary silent substitution rate (vs) and the divergence times among the species. In this paper we have analyzed the first and second codon positions in the same mammalian mitochondrial genes. We found that in the first and second codon positions the human mitochondrial genes satisfy the stationarity conditions. This has allowed us to use the stochastic model mentioned above to calculate the divergence times among mouse, rat, cow, and human. Furthermore, we have analyzed the silent substitution rate in one nuclear gene for these four mammals. We found that in this gene the effective silent substitution rate is about 3 times lower than in mitochondrial genes, and that humans are in this case stationary with respect to the other three mammals in the third codon position as well. Application of our Markov model to this latter gene yields divergence times consistent with our previous determinations.  相似文献   

3.
We obtained 16 nucleotide sequences (∼1400 bp each) of the first intron of the mitochondrial (mt) gene for NADH subunit 4 (nad4) from 10 species of Brassicaceae. Using these new sequences and five published sequences from GenBank, we constructed a phylogenetic tree of the Brassicaceae species under study and showed that the rate of nucleotide substitution in the first intron of nad4 is very low, about 0.16–0.23 × 10−9 substitution per site per year, which is about half of the silent rate in exons of nad4. The ratios of substitution rates in this intron, ITS, and IGS are approximately 1:23:73, where ITS is the nuclear intergenic spacer between 18S and 25S rRNA genes and IGS is the intergenic spacer of 5S rRNA genes. A segment (335 bp) in the first intron of nad4 in Brassicaceae species that is absent in wheat was considered as a nonfunctional sequence and used to estimate the neutral rate (the rate of mutation) in mtDNA to be 0.5–0.7 × 10−9 substitution per site per year, which is about three times higher than the substitution rate in the rest of the first intron of nad4. We estimated that the dates of divergence are 170–235 million years (Myr) for the monocot–dicot split, 112–156 Myr for the Brassicaceae–Lettuce split, 14.5–20.4 Myr for the Brassica–Arabidopsis split, and 14.5–20.4 Myr for the Arabidopsis–Arabideae split. Received: 14 July 1998 / Accepted: 1 October 1998  相似文献   

4.
Summary In S. cerevisiae four isoacceptor mitochondrial tRNAs for serine have been separated by reversed phase chromatography. At least two of these species are products of different genes. In this work the deletion mapping technique has been used to locate two genes for tRNAser. The gene for tRNAser previously localized in the oli I region of the mitochondrial genome has been found to code for tRNA ser 2 , and another gene coding for tRNA ser 1 has been detected in the region where most of other tRNA genes are found. Results of fine mapping experiments allowed to localize this gene in the proximity of the gene for tRNAarg.  相似文献   

5.
We previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg2+] reported by an Mg2+-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg2+. In the current article, we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity, and myosin ATPase activity. Here we report that the addition of BeF3 and sodium orthovanadate (Na3VO4) to medium containing digitonin-permeabilized cells inhibits all ADP-ATP-using reactions except the adenine nucleotide translocase (ANT)-mediated mitochondrial ADP-ATP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F1Fo-ATPase due to its sensitivity to BeF3 and Na3VO4. With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler and expressed as a function of citrate synthase activity per total amount of protein.  相似文献   

6.
All tRNAHis possess an essential extra G–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G–1 is added by a tRNAHis guanylyl transferase. In prokaryotes, G–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G–1 we find here that both maturation pathways can be used. Indeed, tRNAHis with or without a G–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNAHis precursors at both positions G+1 and G–1. The G–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNAHis without G–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNAHis with a G–1 are recovered. This shows that a previously unreported tRNAHis guanylyltransferase activity is present in plant mitochondria.  相似文献   

7.
Loss of mitochondrial function is a fundamental determinant of cell injury and death. In heart cells under metabolic stress, we have previously described how the abrupt collapse or oscillation of the mitochondrial energy state is synchronized across the mitochondrial network by local interactions dependent upon reactive oxygen species (ROS). Here, we develop a mathematical model of ROS-induced ROS release (RIRR) based on reaction-diffusion (RD-RIRR) in one- and two-dimensional mitochondrial networks. The nodes of the RD-RIRR network are comprised of models of individual mitochondria that include a mechanism of ROS-dependent oscillation based on the interplay between ROS production, transport, and scavenging; and incorporating the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and Ca2+ handling. Local mitochondrial interaction is mediated by superoxide (O2 .−) diffusion and the O2 .−-dependent activation of an inner membrane anion channel (IMAC). In a 2D network composed of 500 mitochondria, model simulations reveal ΔΨm depolarization waves similar to those observed when isolated guinea pig cardiomyocytes are subjected to a localized laser-flash or antioxidant depletion. The sensitivity of the propagation rate of the depolarization wave to O2.− diffusion, production, and scavenging in the reaction-diffusion model is similar to that observed experimentally. In addition, we present novel experimental evidence, obtained in permeabilized cardiomyocytes, confirming that ΔΨm depolarization is mediated specifically by O2 .−. The present work demonstrates that the observed emergent macroscopic properties of the mitochondrial network can be reproduced in a reaction-diffusion model of RIRR. Moreover, the findings have uncovered a novel aspect of the synchronization mechanism, which is that clusters of mitochondria that are oscillating can entrain mitochondria that would otherwise display stable dynamics. The work identifies the fundamental mechanisms leading from the failure of individual organelles to the whole cell, thus it has important implications for understanding cell death during the progression of heart disease.  相似文献   

8.
O-linked β-N-acetylglucosamine (O-GlcNAc) is an inducible, dynamically cycling and reversible post-translational modification of Ser/Thr residues of nucleocytoplasmic and mitochondrial proteins. We recently discovered that O-GlcNAcylation confers cytoprotection in the heart via attenuating the formation of mitochondrial permeability transition pore (mPTP) and the subsequent loss of mitochondrial membrane potential. Because Ca2+ overload and reactive oxygen species (ROS) generation are prominent features of post-ischemic injury and favor mPTP formation, we ascertained whether O-GlcNAcylation mitigates mPTP formation via its effects on Ca2+ overload and ROS generation. Subjecting neonatal rat cardiac myocytes (NRCMs, n ≥ 6 per group) to hypoxia, or mice (n ≥ 4 per group) to myocardial ischemia reduced O-GlcNAcylation, which later increased during reoxygenation/reperfusion. NRCMs (n ≥ 4 per group) infected with an adenovirus carrying nothing (control), adenoviral O-GlcNAc transferase (adds O-GlcNAc to proteins, AdOGT), adenoviral O-GlcNAcase (removes O-GlcNAc to proteins, AdOGA), vehicle or PUGNAc (blocks OGA; increases O-GlcNAc levels) were subjected to hypoxia–reoxygenation or H2O2, and changes in Ca2+ levels (via Fluo-4AM and Rhod-2AM), ROS (via DCF) and mPTP formation (via calcein-MitoTracker Red colocalization) were assessed using time-lapse fluorescence microscopy. Both OGT and OGA overexpression did not significantly (P > 0.05) alter baseline Ca2+ or ROS levels. However, AdOGT significantly (P < 0.05) attenuated both hypoxia and oxidative stress-induced Ca2+ overload and ROS generation. Additionally, OGA inhibition mitigated both H2O2-induced Ca2+ overload and ROS generation. Although AdOGA exacerbated both hypoxia and H2O2-induced ROS generation, it had no effect on H2O2-induced Ca2+ overload. We conclude that inhibition of Ca2+ overload and ROS generation (inducers of mPTP) might be one mechanism through which O-GlcNAcylation reduces ischemia/hypoxia-mediated mPTP formation.  相似文献   

9.
Mitochondrial DNA (mtDNA) variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA). We detected a total of 28 point mutations and eight insertion-deletion (indel) mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 × 10−8 per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G→A mutations on the major strand (the sense strand for the majority of mitochondrial genes). These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10× higher than the nuclear mutation rate, but the mitochondrial major strand G→A mutation rate is about 70× higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base composition of the mitochondrial genome of Drosophila.  相似文献   

10.
Rabbit spermatozoa from the cauda epididymis produced 0.7–0.8nmol of H2O2/min per 108 cells at cell concentrations below 107 cells/ml with linear dependence on cell concentration. Above 2 × 107 cells/ml, the rate again became linear with cell concentration but decreased to 0.1–0.2nmol/min per 108 cells. Spermatozoa treated with amphotericin B, which makes the plasma membrane highly permeable to low-molecular-weight compounds, showed a similar dependence of H2O2 production rate on cell concentration; below 107 cells/ml the rate was 0.3–0.4nmol/min per 108 cells; above 2 × 107 cells/ml, the rate was 0.1–0.2nmol/min per 108 cells. Hypo-osmotically treated rabbit epididymal spermatozoa, a preparation useful for studying mitochondrial function in sperm [Keyhani & Storey (1973) Biochim. Biophys. Acta 305, 557–565] produced 0.1–0.2nmol/min per 108 cells in the absence of added substrates. The dependence of rate on cell concentration was linear from 107 to 2.2 × 108 cells/ml. This endogenous rate was unaffected by rotenone, but stimulated 4-fold by antimycin A. Addition of the mitochondrial substrates lactate plus malate increased the rate of H2O2 production to 0.3nmol/min per 108 cells. The decreased rate of H2O2 production observed with intact sperm at high cell concentrations is attributed to reaction of H2O2 with the cells, possibly with the plasma membrane, which is lost after hypo-osmotic treatment. Rabbit spermatozoa have glutathione peroxidase and glutathione reductase activities, but these seem to play little role in removal of H2O2 generated. The rate at low cell concentration is taken to be the unperturbed rate. The sources of H2O2 production in rabbit spermatozoa have been tentatively resolved into a low-molecular-weight component, lost after amphotericin treatment, a mitochondrial component and a rotenone-insensitive component that has not been identified.  相似文献   

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