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J A Enriquez

Publications and source records attributed to J A Enriquez.

10 recordsLinked to original sources
PubMed2001

Mechanism of mammalian mitochondrial DNA replication: import of mitochondrial transcription factor A into isolated mitochondria stimulates 7S DNA synthesis.

The light strand promoter of mammalian mitochondrial DNA gives rise to a primary transcript, but also to the RNA primer necessary for initiation of replication and 7S DNA synthesis as well as 7S RNA. Here we have studied the turnover of 7S DNA in isolated rat liver mitochondria and whether import of mitochondrial transcription factor A (mtTFA), which is necessary for transcription initiation, increases its rate of synthesis. 7S DNA was present as two species, probably due to two different sites of RNA-DNA transition. Time course and pulse-chase experiments showed that the half-life of this DNA is approximately 45 min. Import of mtTFA, produced in vitro, into the mitochondrial matrix in stoichiometric amounts significantly increased the rate of 7S DNA formation. We conclude that isolated rat liver mitochondria faithfully synthesize and degrade 7S DNA and that increased matrix levels of mtTFA are sufficient to increase its rate of synthesis, strongly supporting the hypothesis that this process is transcription primed.

Animals↗
PubMed2000

The mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode syndrome-associated human mitochondrial tRNALeu(UUR) mutation causes aminoacylation deficiency and concomitant reduced association of mRNA with ribosomes.

The pathogenetic mechanism of the mitochondrial tRNA(Leu(UUR)) A3243G transition associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondrial DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtDNA-less cells. An in vivo footprinting analysis of the mtDNA segment within the tRNA(Leu(UUR)) gene that binds the transcription termination factor failed to reveal any difference in occupancy of sites or qualitative interaction with the protein between mutant and wild-type mtDNAs. Cell lines nearly homoplasmic for the mutation exhibited a strong (70-75%) reduction in the level of aminoacylated tRNA(Leu(UUR)) and a decrease in mitochondrial protein synthesis rate. The latter, however, did not show any significant correlation between synthesis defect of the individual polypeptides and number or proportion of UUR codons in their mRNAs, suggesting that another step, other than elongation, may be affected. Sedimentation analysis in sucrose gradient showed a reduction in size of the mitochondrial polysomes, while the distribution of the two rRNA components and of the mRNAs revealed decreased association of mRNA with ribosomes and, in the most affected cell line, pronounced degradation of the mRNA associated with slowly sedimenting structures. Therefore, several lines of evidence indicate that the protein synthesis defect in A3243G MELAS mutation-carrying cells is mainly due to a reduced association of mRNA with ribosomes, possibly as a consequence of the tRNA(Leu(UUR)) aminoacylation defect.

Acylation↗
PubMed1998

Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency.

Leigh disease associated with cytochrome c oxidase deficiency (LD[COX-]) is one of the most common disorders of the mitochondrial respiratory chain, in infancy and childhood. No mutations in any of the genes encoding the COX-protein subunits have been identified in LD(COX-) patients. Using complementation assays based on the fusion of LD(COX-) cell lines with several rodent/human rho0 hybrids, we demonstrated that the COX phenotype was rescued by the presence of a normal human chromosome 9. Linkage analysis restricted the disease locus to the subtelomeric region of chromosome 9q, within the 7-cM interval between markers D9S1847 and D9S1826. Candidate genes within this region include SURF-1, the yeast homologue (SHY-1) of which encodes a mitochondrial protein necessary for the maintenance of COX activity and respiration. Sequence analysis of SURF-1 revealed mutations in numerous DNA samples from LD(COX-) patients, indicating that this gene is responsible for the major complementation group in this important mitochondrial disorder.

Animals↗
PubMed1998

The deafness-associated mitochondrial DNA mutation at position 7445, which affects tRNASer(UCN) precursor processing, has long-range effects on NADH dehydrogenase subunit ND6 gene expression.

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3' end of the tRNASer(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of approximately 70% in the tRNASer(UCN) level and a decrease of approximately 45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNASer(UCN) to support the wild-type protein synthesis rate, which corresponds to approximately 40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located approximately 7 kbp upstream and is cotranscribed with the tRNASer(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O2 consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells.

Base Sequence↗
PubMed1998

Familial multiple symmetric lipomatosis associated with the A8344G mutation of mitochondrial DNA.

We describe familial multiple symmetric lipomatosis in a pedigree harboring the 8344 mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA). The proband showed neuromuscular involvement but lacked the typical manifestations of myoclonic epilepsy and ragged-red fibers disease. The distribution of the mutation was unusual because the proportion of mutated genomes was higher in blood and lipomas than in muscle tissue.

Adult↗
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↗
PubMed1992

A simple procedure for recovering the denaturing effect of methylmercury in agarose gel electrophoresis.

A simple and rapid procedure for recovering the denaturing effect of methylmercuric hydroxide in agarose gel electrophoresis is described. The procedure consisted of the treatment of the commercial methylmercuric hydroxide solutions with Amberlite, a mixture of anion- and cation-exchange resins. This treatment greatly improved the resolution of RNA species when fractionated by electrophoresis through agarose-CH3HgOH slab gel.

Animals↗
PubMed1991

Saturation of the processing of newly synthesized rRNA in isolated brain mitochondria.

Isolated rat brain mitochondria, when incubated in the presence of [alpha-32P]UTP in an appropriate incubation buffer, in which the energy requirements are provided by exogenous ADP in the presence of an oxidizable substrate, are able to support mitochondrial DNA transcription and RNA processing in a way faithfully resembling the in vivo process. Furthermore, we have strikingly found a saturation of the synthesis of mature 16 S and 12 S rRNA under conditions in which their RNA precursor as well as the mature mRNAs continue being synthesized. This suggests that synthesis of mature rRNAs could be regulated at the level of processing of their precursor rather than at the level of transcription.

Adenosine Diphosphate↗
PubMed1990

Rat brain synaptosomes prepared by phase partition.

Synaptosomes from rat forebrain can easily be isolated by combining centrifugation with partition in an aqueous two-phase system composed of dextran T500 and polyethylene glycol 4000 in which synaptosomes have an extreme affinity for the upper phase. The fraction thus obtained has been characterized by electron microscopy and biochemical markers for synaptosomes and some other cell components. The contamination by microsomes, free mitochondria, and myelin was 4.4, 3.2, and 0.1%, respectively. The morphometric analysis of the electron micrographs shows that greater than 60% of the structures are synaptosomes. This preparation of the isolation procedure is remarkably short (less than 1 h), formance as assayed by their respiratory activities and ATP level in the absence and presence of depolarizing agents. Synaptosomes prepared by phase partition release the neurotransmitter glutamate in a Ca2(+)-dependent manner. The duration of the isolation procedure is remarkably short (less than 1 h), no ultracentrifuge is required, and the method can be applied for small- or large-scale preparations.

Adenosine Diphosphate↗