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1.
Abstract— The binding capacity for amino acids of low molecular wt. RNAs isolated from mitochondrial and cytoplasmic fractions from brain was studied in the presence of partially purified aminoacyl-tRNA synthetases obtained from both subcellular fractions. The ability of mitochondrial tRNAs to bind amino acids was greater by about three times in the presence of mitochondrial aminoacyl-tRNA synthetases than in the presence of cytoplasmic enzymes. In contrast, the amino acid-binding ability of cytoplasmic tRNA was the same in the presence of mitochondrial enzymes as in the presence of those from the cytoplasm. When homologous (rabbit) and heterologous (calf) tRNAs were tested in the presence of mitochondrial or cytoplasmic enzymes obtained from rabbit brain and a mixture of amino acids, a significant species specificity was seen: in both heterologous systems the highest amount of tRNA binding was only 44-66 per cent of that obtained with the homologous enzyme system.  相似文献   

2.
—Total tRNA fractions were isolated from pure mitochondrial and cytoplasmic calf brain preparations. After incubation with homologous crude preparations of aminoacyl-tRNA ligases in the presence of [14C]-glutamic acid, tRNAs were separated chromatographically on BD-cellulose columns and in reversed phase chromatography systems. In both of the methods used, cytoplasmic tRNA preparations revealed a larger number of radioactivity peaks. In experiments with double labelling, five radioactivity peaks for cytoplasmic glutamyl-tRNAs corresponded to only three mitochondrial glutamyl-tRNA fractions. The results imply the presence of isoaccepting species of tRNA in brain.  相似文献   

3.
The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase.  相似文献   

4.
1. Mitochondrial and cytoplasmic ribosomes of Euglena gracilis differ in their total RNA and protein content. 2. Mitochondrial ribosomes dissociate to subunits at higher Mg(2+) concentrations than do cytoplasmic ribosomes. 3. A separable 5S RNA is obtained from cytoplasmic and chloroplast ribosomes, but not from mitochondrial ribosomes. 4. For protein-synthesizing activity with a natural mRNA, mitochondrial ribosomes use tRNA from any cell compartment and are partly active with supernatant enzymes from cytoplasm. Cytoplasmic ribosomes are partly active with enzymes and tRNA from mitochondria or chloroplasts. 5. Both mitochondrial and cytoplasmic ribosomes show high specificity for the homologous salt-extractable ribosomal fraction for protein-synthesizing activity.  相似文献   

5.
Initiator methionine tRNA from the mitochondria of Neurospora crassa has been purified and sequenced. This mitochondrial tRNA can be aminoacylated and formylated by E. coli enzymes, and is capable of initiating protein synthesis in E. coli extracts. The nucleotide composition of the mitochondrial initiator tRNA (the first mitochondrial tRNA subjected to sequence analysis) is very rich in A + U, like that reported for total mitochondrial tRNA. In two of the unique features which differentiate procaryotic from eucaryotic cytoplasmic initiator tRNAs, the mitochondrial tRNA appears to resemble the eucaryotic initiator tRNAs. Thus unlike procaryotic initiator tRNAs in which the 5′ terminal nucleotide cannot form a Watson-Crick base pair to the fifth nucleotide from the 3′ end, the mitochondrial tRNA can form such a base pair; and like the eucaryotic cytoplasmic initiator tRNAs, the mitochondrial initiator tRNA lacks the sequence -TΨCG(or A) in loop IV. The corresponding sequence in the mitochondrial tRNA, however, is -UGCA- and not -AU(or Ψ)CG-as found in all eucaryotic cytoplasmic initiator tRNAs. In spite of some similarity of the mitochondrial initiator tRNA to both eucaryotic and procaryotic initiator tRNAs, the mitochondrial initiator tRNA is basically different from both these tRNAs. Between these two classes of initiator tRNAs, however, it is more homologous in sequence to procaryotic (56–60%) than to eucaryotic cytoplasmic initiator tRNAs (45–51%).  相似文献   

6.
Yeast mitochondrial tRNA synthetase has been partially purified and chromatographic, catalytic and antigenic properties have been compared to the cytoplasmic homologous enzyme from yeast. No significant differences could be observed between the two enzymes with respect to their behaviour during ammonium sulfate precipitation or in chromatographic separation on DEAE cellulose, hydroxylapatite and Sephadex G 200. The Km of the two enzymes toward tRNAs from yeast mitochondria, yeast cytoplasm or E. coli are pratically identical. The antigenic properties of the two enzymes are very similar; antisera against either the mitochondria or the cytoplasmic enzyme lead to the inhibition of their catalytic properties. The mitochondrial ValRS is formed by a single polypeptide chain whose molecular weight is 125,000 daltons, a value very close to that of the yeast cytoplasmic enzyme.  相似文献   

7.
Differential rates of nucleotide substitution among different gene segments and between distinct evolutionary lineages is well documented among mitochondrial genes and is likely a consequence of locus-specific selective constraints that delimit mutational divergence over evolutionary time. We compared sequence variation of 18 homologous loci (15 coding genes and 3 parts of the control region) among 10 mammalian mitochondrial DNA genomes which allowed us to describe different mitochondrial evolutionary patterns and to produce an estimation of the relative order of gene divergence. The relative rates of divergence of mitochondrial DNA genes in the family Felidae were estimated by comparing their divergence from homologous counterpart genes included in nuclear mitochondrial DNA (Numt, pronounced "new might"), a genomic fossil that represents an ancient transfer of 7.9 kb of mitochondrial DNA to the nuclear genome of an ancestral species of the domestic cat (Felis catus). Phylogenetic analyses of mitochondrial (mtDNA) sequences with multiple outgroup species were conducted to date the ancestral node common to the Numt and the cytoplasmic (Cymt) mtDNA genes and to calibrate the rate of sequence divergence of mitochondrial genes relative to nuclear homologous counterparts. By setting the fastest substitution rate as strictly mutational, an empirical "selective retardation index" is computed to quantify the sum of all constraints, selective and otherwise, that limit sequence divergence of mitochondrial gene sequences over time.   相似文献   

8.
9.
We present here the complete 16,338 nucleotide DNA sequence of the bovine mitochondrial genome. This sequence is homologous to that of the human mitochondrial genome (Anderson et al., 1981) and the genes are organized in virtually identical fashion. The bovine mitochondrial protein genes are 63 to 79% homologous to their human counterparts, and most of the nucleotide differences occur in the third positions of codons. The minimum rate of base substitution that accounts for the nucleotide differences in the codon third positions is very high: at least 6 × 10?9 changes per position per year. The bovine and human mitochondrial transfer RNA genes exhibit more interspecies variation than do their cytoplasmic counterparts, with the “TΨC” loop being the most variable part of the molecule. The bovine 12 S and 16 S ribosomal RNA genes, when compared with those from human mitochondrial DNA, show conserved features that are consistent with proposed secondary structure models for the ribosomal RNAs. Unlike the pattern of moderate-to-high homology between the bovine and human mitochondrial DNAs found over most of the genome, the DNA sequence in the bovine D-loop region is only slightly homologous to the corresponding region in the human mitochondrial genome. This region is also quite variable in length, and accounts for the bulk of the size difference between the human and bovine mitochondrial DNAs.  相似文献   

10.
Mapping of mitochondrial structural genes in Neurospora crassa   总被引:5,自引:0,他引:5  
A hybridization method has been employed to study the organization of the mitochondrial genome of Neurospora crassa. The method involves the use of 5' end-labeled single-stranded restriction fragments obtained from cytoplasmic "petite" strains of Saccharomyces cerevisiae known to contain single mitochondrial genes. The presence and localization of genes homologous to Subunits 1, 2, and 3 of cytochrome oxidase, cytochrome b and Subunit 6 of the ATPase is thus established for the mitochondrial genome of N. crassa.  相似文献   

11.
Mitochondrial porin, the major protein of the outer mitochondrial membrane is synthesized by free cytoplasmic polysomes. The apparent molecular weight of the porin synthesized in homologous or heterologous cell-free systems is the same as that of the mature porin. Transfer in vitro of mitochondrial porin from the cytosolic fraction into the outer membrane of mitochondria could be demonstrated. Before membrane insertion, mitochondrial porin is highly sensitive to added proteinase; afterwards it is strongly protected. Binding of the precursor form to mitochondria occurs at 4 degrees C and appears to precede insertion into the membrane. Unlike transfer of many precursor proteins into or across the inner mitochondrial membrane, assembly of the porin is not dependent on an electrical potential across the inner membrane.  相似文献   

12.
O'Brien TW 《IUBMB life》2003,55(9):505-513
Mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S) and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial ribosomes. Typical of mammalian mitochondrial ribosomes, the bovine mitochondrial ribosome has been developed as a model system for the study of human mitochondrial ribosomes, to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from each subunit have been identified and characterized with respect to individuality and electrophoretic properties, amino acid sequence, topographic disposition, RNA binding properties, evolutionary relationships and reaction with affinity probes of ribosomal functional domains. Several distinctive properties of these ribosomes are being elucidated, including their antibiotic susceptibility and composition. Human mitochondrial ribosomes lack several of the major RNA stem structures of bacterial ribosomes but they contain a correspondingly higher protein content (as many as 80 proteins), suggesting a model where proteins have replaced RNA structural elements during the evolution of these ribosomes. Despite their lower RNA content they are physically larger than bacterial ribosomes, because of the 'extra' proteins they contain. The extra proteins in mitochondrial ribosomes are 'new' in the sense that they are not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in nuclear genes (a separate set from those encoding cytoplasmic ribosomal proteins) which are evolving more rapidly than those encoding cytoplasmic ribosomal proteins. The MRPs are imported into mitochondria where they assemble coordinately with mitochondrially transcribed rRNAs into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system.  相似文献   

13.
Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.  相似文献   

14.
Mitochondrial genome organization and cytoplasmic male sterility in plants   总被引:2,自引:0,他引:2  
Plant mitochondrial genomes are much larger and more complex than those of other eukaryotic organisms. They contain a very active recombination system and have a multipartite genome organization with a master circle resolving into two or more subgenomic circles by recombination through repeated sequences. Their protein coding capacity is very low and is comparable to that of animal and fungal systems. Several subunits of mitochondrial functional complexes, a complete set of tRNAs and 26S, 18S and 5S rRNAs are coded by the plant mitochondrial genome. The protein coding genes contain group II introns. The organelle genome contains stretches of DNA sequences homologous to chloroplast DNA. It also contains actively transcribed DNA sequences having open reading frames. Plasmid like DNA molecules are found in mitochondria of some plants Cytoplasmic male sterility in plants, characterized by failure to produce functional pollen grains, is a maternally inherited trait. This phenomenon has been found in many species of plants and is conveniently used for hybrid plant production. The genetic determinants for cytoplasmic male sterility reside in the mitochondrial genome. Some species of plants exhibit more than one type of cytoplasmic male sterility. Several nuclear genes are known to control expression of cytoplasmic male sterility. Different cytoplasmic male sterility types are distinguished by their specific nuclear genes(rfs) which restore pollen fertility. Cytoplasmic male sterility types are also characterized by mitochondrial DNA restriction fragment length polymorphism patterns, variations in mitochondrial RNAs, differences in protein synthetic profiles, differences in sensitivity to fungal toxins and insecticides, presence of plasmid DNAs or RNAs and also presence of certain unique sequences in the genome. Recently nuclear male sterility systems based on (i) over expression of agrobacterialrol C gene and (ii) anther specific expression of an RNase gene have been developed in tobacco andBrassica by genetic engineering methods.  相似文献   

15.
Comparative hybridization analyses of total DNA from fertile and cytoplasmic male-sterile (CMS) triticale plants which had been regenerated from embryogenic callus cultures revealed the organization and variation of the mitochondrial atp6 gene region. In order to compare different developmental phases, we analysed mitochondrial DNA (mtDNA) from both the shoots and full-grown regenerants. Somaclonal variants were identified on the basis of differences in the mtDNA from fertile and CMS triticale. Several shoots as well as all of the full-grown plants analysed showed somaclonal variation. This phenomenon could be traced back to having primarily orginated from the influence of the nuclear background, which give rise to a stoichiometric increase in a rye-specific orf25 gene copy, and a tissue culture-induced combination of fertile and CMS-specific mtDNA organization of the atp6 gene area. The latter event is probably caused by the homologous recombination of repetitive sequences that may be accompanied by selective amplifications.  相似文献   

16.
Aminoglycosides are a well known antibiotic family used to treat bacterial infections in humans and animals, but which can be toxic. By binding to the decoding site of helix44 of the small subunit RNA of the bacterial ribosome, the aminoglycoside antibiotics inhibit protein synthesis, cause misreading, or obstruct peptidyl-tRNA translocation. Although aminoglycosides bind helix69 of the bacterial large subunit RNA as well, little is known about their interaction with the homologous human helix69. To probe the role this binding event plays in toxicity, changes to thermal stability, base stacking, and conformation upon aminoglycoside binding to the human cytoplasmic helix69 were compared with those of the human mitochondrial and Escherichia coli helix69. Surprisingly, binding of gentamicin and kanamycin A to the chemically synthesized terminal hairpins of the human cytoplasmic, human mitochondrial, and E. coli helix69 revealed similar dissociation constants (1.3–1.7 and 4.0–5.4 μm, respectively). In addition, aminoglycoside binding enhanced conformational stability of the human mitochondrial helix69 by increasing base stacking. Proton one-dimensional and two-dimensional NMR suggested significant and specific conformational changes of human mitochondrial and E. coli helix69 upon aminoglycoside binding, as compared with human cytoplasmic helix69. The conformational changes and similar aminoglycoside binding affinities observed for human mitochondrial helix69 and E. coli helix69, as well as the increase in structural stability shown for the former, suggest that this binding event is important to understanding aminoglycoside toxicity.  相似文献   

17.
The sequences of three transfer RNAs from mosquito cell mitochondria, tRNAUCGArg, tRNAGUCAsp, and tRNAGAUIle, determined using a combination of rapid ladder and fingerprinting procedures are reported. These were compared with hamster mitochondrial tRNAUCGArg and tRNAGUCAsp determined similarly, and a bovine mitochondrial tRNAGAUIle determined using a somewhat different approach. The primary sequences of the mosquito tRNAs were 35 to 65% homologous to the corresponding mammalian mitochondrial species, and bore little homology to “conventional” (bacterial or eucaryotic cytoplasmic) tRNA. The modification status of the mosquito mitochondrial tRNAs resembled that of mammalian mitochondrial tRNA. The results contribute to the generalization that metazoan mitochondrial tRNA constitutes a distinctive, albeit loosely structured, phylogenetic group.  相似文献   

18.
A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.  相似文献   

19.
Summary The mitochondrial genome organizations of a number of independent culture-derived fertile CMS-S revertants with the nuclear genotype W182BN were compared to spontaneous field revertants with the genotypes WF9, M825/Oh07 and 38-11. Regions of the genome around sequences homologous to the terminal repeats of the linear S1 and S2 episomes characteristic of CMS-S mitochondria were used as hybridization probes on Southern blots of BamHI and SalI digested mitochondrial DNA. The results obtained suggest that the nuclear, not the cytoplasmic, genotype of the parent plant affects the type of novel mitochondrial DNA organization found in the revertant. The DNA reorganization during reversion from CMS-S in tissue culture appears to be similar to that observed in spontaneous revertants obtained during the normal plant life-cycle. Unlike the situation for reversion from CMS-T, no common DNA sequence or reading frame appeared to be lost or disrupted in revertants.  相似文献   

20.
Bovine mitochondrial ribosomes. Elongation factor specificity   总被引:2,自引:0,他引:2  
The activity of bovine mitochondrial ribosomes with elongation factors from a variety of sources including the mitochondria of lower eukaryotes, chloroplasts, Gram-negative bacteria, Gram-positive bacteria, and the eukaryotic cell cytoplasm has been investigated. Bovine mitochondrial ribosomes are active with homologous mitochondrial elongation factor (EF)-G but display no activity with the mitochondrial or chloroplast translocases from the lower eukaryote Euglena gracilis, with Escherichia coli or Bacillus subtilis EF-G or with cytoplasmic EF-2. In contrast to the results obtained with the translocases, E. coli EF-Tu, B. subtilis EF-Tu, and Euglena chloroplast EF-Tu all function to a significant extent on the mitochondrial ribosomes. Cytoplasmic EF-1 has barely detectable activity on the animal mitochondrial ribosomes. The polymerization of phenylalanine by these ribosomes is dependent on poly(U), displays a rather broad Mg2+ optimum around 12 mM, and proceeds most rapidly at low monovalent ion concentrations.  相似文献   

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