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G Attardi

Publications and source records attributed to G Attardi.

At least 19 recordsLinked to original sources
PubMed1996

Evidence for aminoacylation-induced conformational changes in human mitochondrial tRNAs.

Analysis by acid polyacrylamide/urea gel electrophoresis of 14 individual mitochondrial tRNAs (mt-tRNAs) from human cells has revealed a variable decrease in mobility of the aminoacylated relative to the nonacylated form, with the degree of separation of the two forms not being correlated with the mass, polar character, or charge of the amino acid. Separation of the charged and uncharged species has been found to be independent of tRNA denaturation, being observed also in the absence of urea. In another approach, electrophoresis through a perpendicular denaturing gradient gel of several individual mt-tRNAs has shown a progressive unfolding of the tRNA with increasing denaturant concentration, which is consistent with an initial disruption of tertiary interactions, followed by the sequential melting of the four stems of the cloverleaf structure. A detailed analysis of the unfolding process of charged and uncharged tRNALys and tRNALeu(UUR) has revealed that the separation of the two forms of these tRNAs persisted throughout the almost entire range of denaturant concentrations used and was lost upon denaturation of the last helical domain(s), which most likely included the amino acid acceptor stem. These observations strongly suggest that the electrophoretic retardation of the charged species reflects an aminoacylation-induced conformational change of the 3'-end of these mt-tRNAs, with possible significant implications in connection with the known role of the acceptor end in tRNA interactions with the ribosomal peptidyl transferase center and the elongation factor Tu.

Base Sequence↗
PubMed1996

Aging-dependent functional alterations of mitochondrial DNA (mtDNA) from human fibroblasts transferred into mtDNA-less cells.

To investigate the role that aging-dependent accumulation of mitochondrial DNA (mtDNA) mutations plays in the senescence processes, mitochondria from fibroblasts of 21 normal human individuals between 20 weeks (fetal) and 103 years of age were introduced into human mtDNA-less (rhoo) 206 cells by cytoplast x rhoo cell fusion, and 7-31 transformant clones were isolated from each fusion. A slight cell donor age-dependent decrease in growth rate was detected in the transformants. Using an O2 consumption rate of 1 fmol/min/cell, which was not observed in any transformant among 158 derived from individuals 20 weeks (fetal) to 37 years of age, as a cut-off to identify respiratory-deficient clones, 11 such clones were found among 198 transformants derived from individuals 39-103 years of age. Furthermore, conventional and nonparametric analysis of the respiratory rates of 356 clones revealed a very significant decrease with donor age. In other analyses, a very significant age-dependent decline in the mtDNA content of the clones was observed, without, however, any significant correlation with the decrease in O2 consumption rate in the defective transformants. These observations clearly indicate the occurrence in the fibroblast-derived transformants of two independent, age-related functional alterations of mtDNA, presumably resulting from structural damage to this genome.

Adolescent↗
PubMed1996

Respiration and growth defects in transmitochondrial cell lines carrying the 11778 mutation associated with Leber's hereditary optic neuropathy.

Mitochondrial DNA from two genetically unrelated patients carrying the mutation at position 11778 that causes Leber's hereditary optic neuropathy has been transferred with mitochondria into human mtDNA-less rho0206 cells. As analyzed in several transmitochondrial cell lines thus obtained, the mutation, which is in the gene encoding subunit ND4 of the respiratory chain NADH dehydrogenase (ND), did not affect the synthesis, size, or stability of ND4, nor its incorporation into the enzyme complex. However, NADH dehydrogenase-dependent respiration, as measured in digitonin-permeabilized cells, was specifically decreased by approximately 40% in cells carrying the mutation. This decrease, which was significant at the 99.99% confidence level, was correlated with a significantly reduced ability of the mutant cells to grow in a medium containing galactose instead of glucose, indicating a clear impairment in their oxidative phosphorylation capacity. On the contrary, no decrease in rotenone-sensitive NADH dehydrogenase activity, using a water-soluble ubiquinone analogue as electron acceptor, was detected in disrupted mitochondrial membranes. This is the first cellular model exhibiting in a foreign nuclear background mitochondrial DNA-linked biochemical defects underlying the optic neuropathy phenotype.

Adult↗
PubMed1996

Relationship of genotype to phenotype in fibroblast-derived transmitochondrial cell lines carrying the 3243 mutation associated with the MELAS encephalomyopathy: shift towards mutant genotype and role of mtDNA copy number.

Transmitochondrial cell lines were isolated by fusing mtDNA-less rho degrees 206 cells with enucleated fibroblasts derived from four members of a pedigree carrying in their muscle varying proportions of the mutation at position 3243 in the tRNA(Leu(UUR)) gene associated with the MELAS encephalomyopathy. The mitochondrial transformants derived from an asymptomatic individual were all homoplasmic for wild-type mtDNA. The proportion of wild-type transformants derived from clinically affected members of the pedigree appeared to decrease in correspondence with an increase in severity of the clinical symptoms of the cell donor. Furthermore, the average proportion of wild-type mtDNA in the transformants derived from each member of the pedigree was very similar to that found in mtDNA from the fibroblasts of that individual, suggesting that the distribution of genotypes in the transformants reflected fairly closely that in the fibroblasts. The genotype and phenotype of ten transformants derived from one severely affected individual were investigated during continuous culture up to 17-24 weeks after the transformation step. Six heteroplasmic clones showed a progressive increase in the proportion of mutant mtDNA, whereas the mitochondrial genotype remained constant in four clones apparently homoplasmic for wild-type mtDNA or nearly homoplasmic for mutant mtDNA. An analysis of the rate of repopulation of rho degrees 206 cells with fibroblast-derived mtDNA revealed a large variability among different transformants, with the full re-establishment of the control ratio of mtDNA to nuclear DNA being observed between approximately 6 weeks and more than 22 weeks after the transformation step. An increase in rate of O2 consumption generally accompanied the increase in mtDNA copy number of the transformants, pointing to the important role of the mtDNA copy number in determining the phenotype of a cell. The observation that a very small amount of wild-type mtDNA (2 to 5% of the control level), coexisting with strongly predominant mutant mtDNA, conferred upon the transformants a substantial respiratory capacity (50% or more) and the evidence of proportionality between O2 consumption rate and mtDNA copy number, which occurred at widely different mutant to wild-type mtDNA ratios, strongly suggest a contribution of the mutant mtDNA to the cell respiratory competence.

Cell Line↗
PubMed1996

Biochemical evidence for nuclear gene involvement in phenotype of non-syndromic deafness associated with mitochondrial 12S rRNA mutation.

The phenotypic effects of the human mitochondrial 12S rRNA gene mutation at position 1555 associated with maternally inherited non-syndromic deafness and sensitivity to aminoglycoside-induced deafness have been analyzed in 25 lymphoblastoid cell lines derived from members of a large family carrying this mutation in homoplasmic form and from control individuals. A clear decrease in the rates of growth in galactose medium, mitochondrial protein synthesis, total oxygen consumption, and complex I-, complex III- and complex IV-dependent respiration was observed in two groups of nine and 10 mutant cell lines derived, respectively, from symptomatic and asymptomatic members of the family, as compared with six control cell lines. The severity of mitochondrial dysfunction in the mutant cell lines was correlated with the presence or absence of hearing loss in the donor individuals. These observations strongly suggest a role of a nuclear factor(s) in the phenotypic manifestation of the mutation. The approach used here provides a paradigm for the analysis of the nuclear background involvement in other mtDNA-linked disorders, including the putative ones associated with neurodegenerative diseases. Exposure of the cell lines derived from several symptomatic or asymptomatic individuals from the same family to high concentrations of neomycin or paromomycin decreased to a significant, nearly identical extent their rate of growth in glucose-containing medium, as contrasted with the unchanged growth rate of control cell lines or of mtDNA-less cells. These results support the hypothesis that the main target of the antibiotics is the mitochondrial 12S rRNA carrying the 1555 mutation, without any apparent role of the nuclear background.

Adult↗
PubMed1996

The mutation rate of the human mtDNA deletion mtDNA4977.

The human mitochondrial mutation mtDNA4977 is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation fate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA4977 in cultured human cells is 5.95 x 10(-8) per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations.

Adult↗
PubMed1995

Complementation and segregation behavior of disease-causing mitochondrial DNA mutations in cellular model systems.

The recent development of cellular models of mitochondrial DNA-linked diseases by transfer of patient-derived mitochondria into human mtDNA-less (rho o) cells has provided a valuable tool for investigating the complementation and segregation of mtDNA mutations. In transformants carrying in heteroplasmic form the mitochondrial tRNA(Lys) gene 8344 mutation or tRNA(Leu(UUR)) gene 3243 mutation associated, respectively, with the MERRF or the MELAS encephalomyopathy, full protection of the cells against the protein synthesis and respiration defects caused by the mutations was observed when the wild-type mtDNA exceeded 10% of the total complement. In the MERRF transformants, the protective effect of wild-type mtDNA was shown to involve interactions of the mutant and wild-type gene products, probably coexisting within the same organelle from the time of the mutation event. In striking contrast, in experiments in which two mtDNAs carrying either the MERRF or the MELAS mutation were sequentially introduced within distinct organelles into the same rho o cells, no evidence of cooperation between their products was observed. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which a chloramphenicol (CAP) resistance-conferring mtDNA mutation was introduced into CAP-sensitive cells. In the area of segregation of mtDNA mutations, in unstable heteroplasmic MELAS transformants, observations were made which pointed to a replicative advantage of mutant molecules, leading to a rapid shift of the genome towards the mutant type. These results are consistent with a model in which the mitochondrion, rather than the mtDNA molecule, is the segregating unit.

DNA, Mitochondrial↗
PubMed1995

Multiple deficiencies of mitochondrial DNA- and nuclear-encoded subunits of respiratory NADH dehydrogenase detected with peptide- and subunit-specific antibodies in mitochondrial myopathies.

Antibodies have been raised against synthetic peptides corresponding to several computer-predicted epitopes of three mtDNA-encoded subunits, ND4, ND5 and ND6, of the human respiratory chain NADH dehydrogenase (Complex I). Antibodies were characterized by a sensitive immunoblotting assay using proteins from human skeletal muscle mitochondria and by immunoprecipitation of radio-labeled HeLa cell mitochondrial translation products. Only antibodies against two of six selected peptides of the ND4 subunit, i.e., the C-terminal peptide and an internal peptide close to the C-terminus, reacted in both assays with the subunit. Antibodies raised against an internal peptide close to the N-terminus of the ND5 subunit and antibodies raised against an internal epitope of the ND6 subunit also reacted in both the immunoblotting and immunoprecipitation assays. The antibodies described above and other Complex I subunit- or holoenzyme-specific antibodies were used to investigate the subunit deficiencies of the respiratory NADH dehydrogenase in the skeletal muscle of patients affected by mitochondrial myopathies associated with Complex I defects. The reduction in enzyme activity correlated in an immunoblot assay with a decrease of four mtDNA-encoded subunits of the enzyme, as well as with a decrease of other subunits of Complex I encoded in the nDNA. The present work provides the first evidence of a decrease in NADH dehydrogenase subunits encoded in the mitochondrial genome in myopathy patients.

Adolescent↗
PubMed1995

Heteroplasmic mitochondrial tRNA(Lys) mutation and its complementation in MERRF patient-derived mitochondrial transformants.

The heteroplasmic tRNA(Lys) mutation in the mitochondrial DNA (mtDNA) is responsible for the phenotypic expression and the transmission of MERRF syndrome. However, the genetic behaviors of the mutant and wild-type mtDNA molecules within a cell are still unknown. We demonstrated a clear genetic complementation of the mutant and wild-type mtDNAs, with a sharp threshold around 10% in the wild-type, in the MERRF transformants, and in their subclones by a cytoplast transfer of the mitochondria into an mtDNA-less cell line, rho o cell. By contrast, no interaction was observed between the two functionally complementary mtDNAs that were originally located in distinct organelles and sequentially introduced into a rho o cell line (genetic independence). These results imply that the sorting of the mtDNA molecules among mitochondria plays a crucial role in the phenotypic expression and transmission of the disease.

Chloramphenicol↗
PubMed1995

MtDNA mutation in MERRF syndrome causes defective aminoacylation of tRNA(Lys) and premature translation termination.

We have investigated the pathogenetic mechanism of the mitochondrial tRNA(Lys) gene mutation (position 8344) associated with MERRF encephalomyopathy in several mitochondrial DNA (mtDNA)-less cell transformants carrying the mutation and in control cells. A decrease of 50-60% in the specific tRNA(Lys) aminoacylation capacity per cell was found in mutant cells. Furthermore, several lines of evidence reveal that the severe protein synthesis impairment in MERRF mutation-carrying cells is due to premature termination of translation at each or near each lysine codon, with the deficiency of aminoacylated tRNA(Lys) being the most likely cause of this phenomenon.

Cell Line↗
PubMed1995

Efficient selection and characterization of mutants of a human cell line which are defective in mitochondrial DNA-encoded subunits of respiratory NADH dehydrogenase.

The mitochondrial NADH dehydrogenase (complex I) in mammalian cells is a multimeric enzyme consisting of approximately 40 subunits, 7 of which are encoded in mitochondrial DNA (mtDNA). Very little is known about the function of these mtDNA-encoded subunits. In this paper, we describe the efficient isolation from a human cell line of mutants affected in any of these subunits. In the course of analysis of eight mutants of the human cell line VA2B selected for their resistance to high concentrations of the complex I inhibitor rotenone, seven were found to be respiration deficient, and among these, six exhibited a specific defect of complex I. Transfer of mitochondria from these six mutants into human mtDNA-less cells revealed, surprisingly, in all cases a cotransfer of the complex I defect but not of the rotenone resistance. This result indicated that the rotenone resistance resulted from a nuclear mutation, while the respiration defect was produced by an mtDNA mutation. A detailed molecular analysis of the six complex I-deficient mutants revealed that two of them exhibited a frameshift mutation in the ND4 gene, in homoplasmic or in heteroplasmic form, resulting in the complete or partial loss, respectively, of the ND4 subunit; two other mutants exhibited a frameshift mutation in the ND5 gene, in near-homoplasmic or heteroplasmic form, resulting in the ND5 subunit being undetectable or strongly decreased, respectively. It was previously reported (G. Hofhaus and G. Attardi, EMBO J. 12:3043-3048, 1993) that the mutant completely lacking the ND4 subunit exhibited a total loss of NADH:Q1 oxidoreductase activity and a lack of assembly of the mtDNA-encoded subunits of complex I. By contrast, in the mutant characterized in this study in which the ND5 subunit was not detectable and which was nearly totally deficient in complex I activity, the capacity to assemble the mtDNA-encoded subunits of the enzyme was preserved, although with a decreased efficiency or a reduced stability of the assembled complex. The two remaining complex I-deficient mutants exhibited a normal rate of synthesis and assembly of the mtDNA-encoded subunits of the enzyme, and the mtDNA mutation(s) responsible for their NADH dehydrogenase defect remains to be identified. The selection scheme used in this work has proven to be very valuable for the isolation of mutants from the VA2B cell line which are affected in different mtDNA-encoded subunits of complex I and may be applicable to other cell lines.

Autoradiography↗
PubMed1994

Complementation of mutant and wild-type human mitochondrial DNAs coexisting since the mutation event and lack of complementation of DNAs introduced separately into a cell within distinct organelles.

The rules that govern complementation of mutant and wild-type mitochondrial genomes in human cells were investigated under different experimental conditions. Among mitochondrial transformants derived from an individual affected by the MERRF (myoclonus epilepsy associated with ragged red fibers) encephalomyopathy and carrying in heteroplasmic form the mitochondrial tRNA(Lys) mutation associated with that syndrome, normal protein synthesis and respiration was observed when the wild-type mitochondrial DNA exceeded 10% of the total complement. In these transformants, the protective effect of wild-type mitochondrial DNA was shown to involve interactions of the mutant and wild-type gene products. Very different results were obtained in experiments in which two mitochondrial DNAs carrying nonallelic disease-causing mutations were sequentially introduced within distinct organelles into the same human mitochondrial DNA-less (rho 0) cell. In transformants exhibiting different ratios of the two genomes, no evidence of cooperation between their products was observed, even 3 months after the introduction of the second mutation. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which mitochondria carrying a chloramphenicol resistance-inducing mitochondrial DNA mutation were introduced into chloramphenicol-sensitive cells. A plausible interpretation of the different results obtained in the latter two sets of experiments, compared with the complementation behavior observed in the heteroplasmic MERRF transformants, is that in the latter, the mutant and wild-type genomes coexisted in the same organelles from the time of the mutation. This would imply that the way in which mitochondrial DNA is sorted among different organelles plays a fundamental role in determining the oxidative-phosphorylation phenotype in mammalian cells. These results have significant implications for mitochondrial genetics and for studies on the transmission and therapy of mitochondrial DNA-linked diseases.

Base Sequence↗
PubMed1994

Platelet-mediated transformation of mtDNA-less human cells: analysis of phenotypic variability among clones from normal individuals--and complementation behavior of the tRNALys mutation causing myoclonic epilepsy and ragged red fibers.

In the present work, we demonstrate the possibility of using human blood platelets as mitochondrial donors for the repopulation of mtDNA-less (rho 0) cells. The noninvasive nature of platelet isolation, combined with the prolonged viability of platelet mitochondria and the simplicity and efficiency of the mitochondria-transfer procedure, has substantially increased the applicability of the rho 0 cell transformation approach for mitochondrial genetic analysis and for the study of mtDNA-linked diseases. This approach has been applied to platelets from several normal human individuals and one individual affected by the myoclonic-epilepsy-and-ragged-red-fibers (MERRF) encephalomyopathy. A certain variability in respiratory capacity was observed among the platelet-derived rho 0 cell transformants from a given normal subject, and it was shown to be unrelated to their mtDNA content. The results of sequential transfer of mitochondria from selected transformants into a rho 0 cell line different from the first rho 0 acceptor strongly suggest that this variability reflected, at least in part, differences in nuclear gene content and/or activity among the original recipient cells. A much greater variability in respiratory capacity was observed among the transformants derived from the MERRF patient and was found to be related to the presence and amount of the mitochondrial tRNALys mutation associated with the MERRF syndrome. An analysis of the relationship between proportion of mtDNA carrying the MERRF mutation and degree of respiratory activity in various transformants derived from the MERRF patient revealed an unusual complementation behavior of the tRNALys mutation, possibly reflecting the distribution of mutant mtDNA among the platelet mitochondria.

Adult↗
PubMed1993

Post-transcriptional regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells.

In human mitochondrial DNA (mtDNA), the tRNA genes are located in three different transcription units that are transcribed at three different rates. To analyze the regulation of tRNA formation by the three transcription units, we have examined the steady-state levels and metabolic properties of the tRNAs of HeLa cell mitochondria. DNA excess hybridization experiments utilizing separated strands of mtDNA and purified tRNA samples from exponential cells long term labeled with [32P]orthophosphate have revealed a steady-state level of 6 x 10(5) tRNA molecules/cell, with three-fourths being encoded in the H-strand and one-fourth in the L-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNA(Phe) and tRNA(Val) are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs isolated from cells labeled with very short pulses of [5-3H]uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady-state amounts. Therefore, the approximately 25-fold higher rate of transcription of the tRNA(Phe) and tRNA(Val) genes relative to the other H-strand tRNA genes and the 10-16-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes are not reflected in the steady-state levels or the rates of formation of the corresponding tRNAs. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters.

Cloning, Molecular↗
PubMed1993

Molecular characterization of the transcription termination factor from human mitochondria.

The transcription termination factor (mTERF), which plays a central role in the control of mitochondrial rRNA and mRNA synthesis in mammalian mitochondria, has been previously identified and purified by DNA affinity chromatography from a human mitochondrial lysate (Kruse, B., Narasimhan, N., and Attardi, G. (1989) Cell 58, 391-397). In the present work, this factor has been characterized as to its protein composition and the activities of the protein components. Three polypeptides, two of approximately 34-kDa molecular mass and one of approximately 31 kDa, were shown to be associated with the specific DNA binding and footprinting activity of the factor, with the 31-kDa component having a much lower affinity for the recognition sequence than the 34-kDa components. On the other hand, the transcription termination activity, as assayed in an in vitro system, was found to be associated exclusively with the two 34-kDa polypeptides. Mass spectroscopic analysis of tryptic peptides derived from highly purified polypeptides indicated that all three polypeptides share regions with common sequences. The evidence obtained suggests that differential phosphorylation is not responsible for the difference in electrophoretic mobility of the three polypeptides.

Base Sequence↗
PubMed1993

Lack of assembly of mitochondrial DNA-encoded subunits of respiratory NADH dehydrogenase and loss of enzyme activity in a human cell mutant lacking the mitochondrial ND4 gene product.

In most eukaryotic cells, the respiratory chain NADH dehydrogenase (Complex I) is a multimeric enzyme under dual (nuclear and mitochondrial) genetic control. Several genes encoding subunits of this enzyme have been identified in the mitochondrial genome from various organisms, but the functions of these subunits are in most part unknown. We describe here a human cell line in which the enzyme lacks the mtDNA-encoded subunit ND4 due to a frameshift mutation in the gene. In this cell line, the other mtDNA-encoded subunits fail to assemble, while at least some of the nuclear-encoded subunits involved in the redox reactions appear to be assembled normally. In fact, while there is a complete loss of NADH:Q1 oxidoreductase activity, the NADH:Fe(CN)6 oxidoreductase activity is normal. These observations provide the first clear evidence that the ND4 gene product is essential for Complex I activity and give some insights into the function and the structural relationship of this polypeptide to the rest of the enzyme. They are also significant for understanding the pathogenetic mechanism of the ND4 gene mutation associated with Leber's hereditary optic neuropathy.

Cell Line↗
PubMed1992

Marked replicative advantage of human mtDNA carrying a point mutation that causes the MELAS encephalomyopathy.

The segregation of mutant and wild-type mtDNA was investigated in transformants constructed by transferring human mitochondria from individuals belonging to four pedigrees with the MELAS encephalomyopathy-associated mtDNA mutation (MELAS is mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) into human mtDNA-less (rho 0) cells. Five of 13 clonal cell lines containing mixtures of wild-type and mutant mtDNAs were found to undergo a rapid shift of their genotype toward the pure mutant type. The other 8 cell lines, which included 6 exhibiting nearly homoplasmic mutant mtDNA, on the contrary, maintained a stable genotype. Subcloning experiments and growth rate measurements clearly indicated that an intracellular replicative advantage of mutant mtDNA was mainly responsible for the dramatic shift toward the mutant genotype observed in the unstable cell lines.

Cell Division↗