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Biomedical subjects

A Araya

Publications and source records attributed to A Araya.

At least 19 recordsLinked to original sources
PubMed1992

An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts.

A posttranscriptional modification (C-to-U) at specific positions of plant mitochondrial mRNA leads to changes in the amino acid sequence as well as to the emergence of novel initiation or termination sites. This phenomenon, named RNA editing, has been described for several mitochondrial genes from different plant sources. We have found recently that RNA editing of the ATP synthase subunit 9 (atp9) mRNA involves eight changes including the creation of a new stop codon. In this article, we describe an in vitro system devised to follow the editing of wheat mitochondrial atp9 mRNA. Nonedited mRNA was obtained to serve as substrate for this reaction by in vitro transcription of the corresponding gene with T7 RNA polymerase. The source of conversion factor(s) was a soluble fraction obtained from purified wheat mitochondria lysed with salt and detergent. Edited RNA molecules were detected by hybridization with an end-labeled synthetic oligodeoxynucleotide probe complementary to a short region containing four editing events. Optimal conditions for the in vitro RNA editing reaction were determined. The reaction is sensitive to high temperature and protease digestion. Pretreatment with micrococcal nuclease decreased RNA editing activity in the mitochondrial extract, suggesting that a nucleic acid is necessary for the enzymatic reactions. Analysis of the edited mRNA showed that the in vitro reaction led to the same products as those observed in vivo.

Amino Acid Sequence↗
PubMed1990

Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants.

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.

Amino Acid Sequence↗
PubMed1990

RNA editing of wheat mitochondrial ATP synthase subunit 9: direct protein and cDNA sequencing.

RNA editing of subunit 9 of the wheat mitochondrial ATP synthase has been studied by cDNA and protein sequence analysis. Most of the cDNA clones sequenced (95%) showed that editing by C-to-U transitions occurred at eight positions in the coding region. Consequently, 5 amino acids were changed in the protein when compared with the sequence predicted from the gene. Two edited codons gave no changes (silent editing). One of the C-to-U transitions generated a stop codon by modifying the arginine codon CGA to UGA. Thus, the protein produced is 6 amino acids shorter than that deduced from the genomic sequence. Minor forms of cDNA with partial or overedited sequences were also found. Protein sequence and amino acid composition analyses confirmed the results obtained by cDNA sequencing and showed that the major form of edited atp9 mRNA is translated.

Adenosine Triphosphatases↗
PubMed1988

Characterization of the cDNA synthesized by avian retrovirus reverse transcriptase using 35 S avian myeloblastosis virus RNA and an exogenous bovine primer tRNA.

Bovine tRNA(Trp) can be partially hybridized to the avian myeloblastosis virus (AMV) 35 S RNA at 37 degrees C, in the presence of AMV RNA-dependent DNA polymerase (reverse transcriptase). This template-primer complex is active in the synthesis of viral cDNA. The size of the cDNA products synthesized in the in vitro reconstituted AMV system was determined by urea-polyacrylamide gel electrophoresis using a tRNA labelled at the 3'-end by yeast tRNA nucleotidyl transferase. The synthesized cDNA has a size of about 100 nucleotides and was shown by Southern blotting to be complementary to a specific sequence of the 5'-end of the retroviral genome. These results indicate that reverse transcriptase is able to anneal the exogenous primer tRNA at the 'primer-binding site' near the 5'-end of the long terminal repeat (LTR) of AMV RNA.

Avian Leukosis Virus↗
PubMed1986

Primary structure of adult hemoglobin of white-throated capuchin, Cebus capucinus.

The alpha and beta chains of White-Throated Capuchin (Cebus capucinus) hemoglobin were separated and digested by trypsin. The tryptic peptides were isolated and sequenced by conventional methods. The peptides in each chain were aligned by the homology of their sequences with those of human adult hemoglobin. The primary structures thus deduced are compared with those of other primate hemoglobins, and we discuss the molecular evolution of hemoglobins, in particular the rate of evolution in New World monkey hemoglobins.

Amino Acid Sequence↗
PubMed1984

Cloning, physical mapping and genome organization of mitochondrial DNA from Cyprinus carpio oocytes.

The mitochondrial genome from Cyprinus carpio oocytes is a 10.5 megadalton, circular DNA molecule. The carp mitochondrial DNA was cloned in pBR325. Three recombinant plasmids accounted for the entire genome. Mapping of this DNA using 11 different restriction endonucleases is reported here. Both the large and small rRNA genes were then localized using Southern blot analysis. The subunit I of the cytochrome oxidase, the cytochrome b, the tRNAGlu and the URF 4 genes were localized by nucleotide sequence analysis and homology studies with human mtDNA. Our results suggest that a similar gene order has been maintained in the mitochondrial genomes of Chordata and support the hypothesis of a common ancestor for all vertebrate organelle genomes. This study constitutes the first report on the genome organization of a fish mtDNA and provides information for further investigation in connection with sequence determination, replication, and gene expression in carp mitochondria.

Animals↗
PubMed1984

Analys. DNA: a computer program for nucleic acid sequence data processing.

A computer program written in BASIC language is described. The program allows processing and analysis of DNA data and has been designed to be used by persons with little or no computer experience. The operator using different options can search for direct homologies with varying degrees of matching, generate complementary strands, find restriction sites, invert the polarity of the sequence and edit a print-out.

Animals↗
PubMed1983

Involvement of tRNA in retrovirus expression: biological implications of reverse transcriptase-primer tRNA interactions.

We have previously studied the topographical and functional implications of the recognition of primer tRNATrp by avian retrovirus reverse transcriptase. Here we have presented evidence that the enzyme is able to deacylate beef liver Trp-tRNATrp, provided that 35-S viral RNA is present in the incubation mixture. No effect of dNTPs on this activity was observed. The extensive modification of tRNATrp with acrylonitrile led to a marked loss of priming activity by tRNATrp if the annealing between primer and template was performed at 37 degrees C, while the annealing of cyanoethylated tRNA with the viral genome at 75 degrees C gave almost normal levels of cDNA synthesis. We have also studied the priming behaviour of tRNATrp, modified by incorporation of various analogs of adenosine. Only tRNATrp-2'dA was active in cDNA initiation; 3'dA, 3'NH2-3'dA, and primer tRNA with formycin in the 3' end showed low or nonexistent priming activity.

Adenosine Triphosphate↗
PubMed1983

Characterization of the 42-S nucleoprotein particles of Cyprinus carpio oocytes.

Previtellogenic oocytes of the fish Cyprinus carpio contain 42S nucleoprotein particles that are composed of two proteins of molecular weights 48,500 and 39,300 (molar ratio 2:1), tRNA and 5S RNA (molar ratio 3:1). The tRNA population embodied in the 42S particle contains all amino acid acceptor species but their distribution differs from that found in tRNA from mature oocytes.

Amino Acids↗
PubMed1980

Study of the interactions between avian myeloblastosis virus reverse transcriptase and primer tRNA. Affinity labeling and inactivation of the enzyme by periodate-treated tRNATrp.

Reverse transcriptase from avian myeloblastosis virus can react with periodate-treated primer tRNATrp (beef) to form a Schiff's base between an epsilon-NH2 lysine group within the active center of the enzyme and the dialdehyde derivative of the 3' terminal ribose of tRNA. In the presence of cyanoborohydride the reversible imminium moiety of the Schiff's base is reduced to a more stable adduct. Non-primer tRNAs were not able to reduce the extent of primer fixation to the enzyme. Complete inactivation of the enzyme was attained when the ratio enzyme:tRNA in the complex was 1:1. When the 1:1 adduct was analyzed by polyacrylamide gel electrophoresis, radioactivity from the terminal adenosine of tRNA was found exclusively associated with the alpha subunit. At longer times of labeling the beta subunit was also found linked to the oxidized primer tRNA.

Animals↗
PubMed1979

Reverse transcriptase mediated binding of primer tRNA to the viral genome.

A complex between tRNATrp (beef) and 35 S RNA from avian myeloblastosis virus is obtained when the mixture is preincubated in the presence of reverse transcriptase at 35 degrees C. The tRNA-RNA complex is active in initiating DNA synthesis catalyzed by reverse transcriptase. The interaction of tRNA with reverse transcriptase involves the partial unwinding of the acceptor stem of tRNA, as evidenced by nuclease digestion with RNAase T1 and micrococcal nuclease. When tRNA2Glu (coli), having a high degree of similarity with primer tRNA at the level of the acceptor stem, was used as primer for DNA synthesis, a low but significant level of incorporation was obtained, if the reaction was performed at 35 degrees C, while a high incorporation, similar to the one obtained with tRNATrp was obtained when the annealing between tRNA2Glu and 35 S RNA was performed at 80 degrees C. Our evidences point out to an important role of the viral DNA polymerase in positioning the primer on the RNA genome.

Avian Leukosis Virus↗