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PubMed1997

The predominance of hepatitis delta virus genotype I among chronically infected Italian patients.

The sera of 46 Italian patients with chronic hepatitis delta virus (HDV) infection were analyzed for HDV RNA by polymerase chain reaction (PCR) amplification. Genetic analysis of sequences amplified from two regions of the HDV genome indicated that all HDV RNA-positive patients (98%) were infected with HDV genotype I. In Italy, infection with this genotype appeared to be associated with a broad spectrum of chronic disease. No subtypes of HDV genotype I were identified, nor were genetic variations clearly associated with different disease patterns; however, clustering of some sequences suggested correlations with geography and transmission route. Italian HDV genotype I sequences were more diverse than those from east Asia and North America, suggesting that HDV genotype I was likely introduced to Italy earlier and/or from multiple sources as compared to those areas. All sequences analyzed were predicted to fold into the unbranched rod structure typical of HDV RNA. Within this structure, three conserved features were identified, including sequences around the RNA editing site and the polyadenylation signal site. We conclude that in Italy, where HDV infection has been endemic, the overwhelmingly predominant genotype of HDV is genotype I.

Adult↗

PubMed2002

Phylogenetic analysis of 18S rRNA and the mitochondrial genomes of the wombat, Vombatus ursinus, and the spiny anteater, Tachyglossus aculeatus: increased support for the Marsupionta hypothesis.

The monotremes, the duck-billed platypus and the echidnas, are characterized by a number of unique morphological characteristics, which have led to the common belief that they represent the living survivors of an ancestral stock of mammals. Analysis of new data from the complete mitochondrial (mt) genomes of a second monotreme, the spiny anteater, and another marsupial, the wombat, yielded clear support for the Marsupionta hypothesis. According to this hypothesis marsupials are more closely related to monotremes than to eutherians, consistent with a basal split between eutherians and marsupials/monotremes among extant mammals. This finding was also supported by analysis of new sequences from a nuclear gene--18S rRNA. The mt genome of the wombat shares some unique features with previously described marsupial mtDNAs (tRNA rearrangement, a missing tRNA(Lys), and evidence for RNA editing of the tRNA(Asp)). Molecular estimates of genetic divergence suggest that the divergence between the platypus and the spiny anteater took place approximately 34 million years before present (MYBP), and that between South American and Australian marsupials approximately 72 MYBP.

Animals↗

PubMed2000

Sequence analysis of the genes encoding the phosphoprotein of recent isolates of canine distemper virus in Japan.

The nucleotide sequences of the phosphoprotein (P) of canine distemper virus (CDV) strains isolated between 1992 and 1996 in Japan were determined. This is the first report of the complete sequences of the P genes of recently prevalent CDV strains. The deduced amino acid sequences of the P, C and V proteins showed that in the new Japanese isolates, these proteins have approximately 93%, 90-91% and 92% identities with those of the Onderstepoort vaccine strain, respectively. The predicted functional regions were conserved. RNA editing resulting in a shift to the open reading frame (ORF) of the V protein was shown to occur with the same efficiency in both the field isolates and vaccine strain.

Amino Acid Sequence↗

PubMed1993

Diversity of glutamate receptor subunit mRNA expression within live hippocampal CA1 neurons.

Glutamate-mediated neurotransmission occurs through the activation of multimeric postsynaptic receptors. One mechanism by which functional diversity of glutamate responsiveness may occur is by a single cell expressing multiple receptors containing different subunits. In a direct test of this hypothesis, we examined the glutamate receptor subunit mRNA composition of several individual CA1 neurons in hippocampal slices. Experiments used amplified antisense RNA coupled with expression profiling and polymerase chain reaction amplification to identify and determine the relative amounts of subunit mRNAs co-localized in single cells. The results demonstrate that each CA1 neuron contains varying amounts of most glutamate receptor mRNAs. In addition to relative mRNA levels, the single-cell approach also highlighted other possible sources of receptor diversity. This included the existence of novel, alternatively spliced forms of the N-methyl-D-aspartate receptor type 1 and glutamate-kainate receptor type 2 subunits. Surprisingly, levels of N-methyl-D-aspartate receptor type 1 mRNA were relatively low, compared with those of other glutamate receptor mRNAs. One postulated source of potential heterogeneity, RNA editing, was not a general cellular mechanism. There was no evidence that glutamate receptor type 5 mRNA was edited in any of the cells that were examined. These data show that individual CA1 neurons, in the intact synaptic network of hippocampal slices, generate glutamate receptor mRNA diversity in several ways, which together contribute to the diversity of functional receptors observed electrophysiologically.

Animals↗

PubMed1999

The interaction between Z-DNA and the Zab domain of double-stranded RNA adenosine deaminase characterized using fusion nucleases.

Zab is a structurally defined protein domain that binds specifically to DNA in the Z conformation. It consists of amino acids 133-368 from the N terminus of human double-stranded RNA adenosine deaminase, which is implicated in RNA editing. Zab contains two motifs with related sequence, Zalpha and Zbeta. Zalpha alone is capable of binding Z-DNA with high affinity, whereas Zbeta alone has little DNA binding activity. Instead, Zbeta modulates Zalpha binding, resulting in increased sequence specificity for alternating (dCdG)n as compared with (dCdA/dTdG)n. This relative specificity has previously been demonstrated with short oligonucleotides. Here we demonstrate that Zab can also bind tightly to (dCdG)n stabilized in the Z form in supercoiled plasmids. Binding was assayed by monitoring cleavage of the plasmids using fusion nucleases, in which Z-DNA-binding peptides from the N terminus of double-stranded RNA adenosine deaminase are linked to the nuclease domain of FokI. A fusion nuclease containing Zalpha shows less sequence specificity, as well as less conformation specificity, than one containing Zab. Further, a construct in which Zbeta has been replaced in Zab with Zalpha, cleaves Z-DNA regions in supercoiled plasmids more efficiently than the wild type but with little sequence specificity. We conclude that in the Zab domain, both Zalpha and Zbeta contact DNA. Zalpha contributes contacts that produce conformation specificity but not sequence specificity. In contrast, Zbeta contributes weakly to binding affinity but discriminates between sequences of Z-DNAs.

Adenosine Deaminase↗

PubMed2001

The RNA world of plant mitochondria.

Mitochondria are well known as the cellular power factory. Much less is known about these organelles as a genetic system. This is particularly true for mitochondria of plants, which subsist with respect to attention by the scientific community in the shadow of the chloroplasts. Nevertheless the mitochondrial genetic system is essential for the function of mitochondria and thus for the survival of the plant. In plant mitochondria the pathway from the genetic information encoded in the DNA to the functional protein leads through a very diverse RNA world. How the RNA is generated and what kinds of regulation and control mechanisms are operative in transcription are current topics in research. Furthermore, the modes of posttranscriptional alterations and their consequences for RNA stability and thus for gene expression in plant mitochondria are currently objects of intensive investigations. In this article current results obtained in the examination of plant mitochondrial transcription, RNA processing, and RNA stability are illustrated. Recent developments in the characterization of promoter structure and the respective transcription apparatus as well as new aspects of RNA processing steps including mRNA 3' processing and stability, mRNA polyadenylation, RNA editing, and tRNA maturation are presented. We also consider new suggestions concerning the endosymbiont hypothesis and evolution of mitochondria. These novel considerations may yield important clues for the further analysis of the plant mitochondrial genetic system. Conversely, an increasing knowledge about the mechanisms and components of the organellar genetic system might reveal new aspects of the evolutionary history of mitochondria.

Base Sequence↗

PubMed1995

Complete sequence and gene organization of the mitochondrial genome of the land snail Albinaria coerulea.

The complete sequence (14,130 bp) of the mitochondrial DNA (mtDNA) of the land snail Albinaria coerulea was determined. It contains 13 protein, two rRNA and 22 tRNA genes. Twenty-four of these genes are encoded by one and 13 genes by the other strand. The gene arrangement shares almost no similarities with that of two other molluscs for which the complete gene content and arrangement are known, the bivalve Mytilus edulis and the chiton Katharina tunicata; the protein and rRNA gene order is similar to that of another terrestrial gastropod, Cepaea nemoralis. Unusual features include the following: (1) the absence of lengthy noncoding regions (there are only 141 intergenic nucleotides interspersed at different gene borders, the longest intergenic sequence being 42 nucleotides) (2) the presence of several overlapping genes (mostly tRNAs), (3) the presence of tRNA-like structures and other stem and loop structures within genes. An RNA editing system acting on tRNAs must necessarily be invoked for posttranscriptional extension of the overlapping tRNAs. Due to these features, and also because of the small size of its genes (e.g., it contains the smallest rRNA genes among the known coelomates), it is one of the most compact mitochondrial genomes known to date.

Animals↗

PubMed1990

RNA sequence and the nature of the CuA-binding site in cytochrome c oxidase.

For cytochrome c oxidase subunit II (COXII), DNA and protein sequences suggest that Met-207 (bovine numbering) is conserved in all species except plants. Sequencing of plant mitochondrial COXII mRNAs now indicates that Met-207 is also conserved among plants as a result of a C-to-U type of RNA editing. Considering the strict evolutionary conservation of Met-207 and the homology of COXII to type I (blue) copper proteins and nitrous oxide reductase, we propose a model in which Met-207 is associated with the CuA-binding site (along with Cys-196, Cys-200 and His-204) and plays a role in determining its reduction potential and stability.

Base Sequence↗

PubMed2006

Translational inhibition and increased interferon induction in cells infected with C protein-deficient measles virus.

In addition to the phosphoprotein, the P gene of measles virus (MV) also encodes the V and C proteins by an RNA editing process and by alternative initiation of translation in a different reading frame, respectively. Although the MV C protein is required for efficient MV replication in vivo and in some cultured cells, its exact functions in virus infection are currently unclear. Here, we report that a recombinant MV lacking the C protein (MVDeltaC) grew poorly in a human cell line possessing the intact interferon (IFN) pathway and that this growth defect was associated with reduced viral translation and genome replication. The translational inhibition was correlated with phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. Moreover, increased IFN induction was observed in MVDeltaC-infected cells. The NS1 protein of influenza virus, which binds to double-stranded RNA (dsRNA) and consequently inhibits IFN induction and dsRNA-dependent protein kinase activation, complemented the growth defect of MVDeltaC. These results indicate that the MV C protein inhibits IFN induction and modulates host antiviral responses, thereby ensuring MV growth in host cells.

Animals↗

PubMed2000

Complete sequence of the mitochondrial DNA of the primitive opisthobranch gastropod Pupa strigosa: systematic implication of the genome organization.

The complete sequence (14,189 bp) of the mitochondrial DNA of the opisthobranch gastropod Pupa strigosa was determined. The genome contains 13 protein, 2 rRNA, and 22 tRNA genes typical of metazoan mtDNA. The Pupa mitochondrial genome is highly compact and shows the following unusual features, like pulmonate land snails: (1) extremely small genome size, (2) absence of lengthy noncoding regions (with the largest intergenic spacer being only 46 nt), (3) size reduction of encoded genes, and (4) many overlapping genes. Several tRNA genes exhibit bizarre secondary structures with reduced T or D stems, and many tRNA genes have unstable acceptor stems that might be corrected by posttranscriptional RNA editing. The Pupa mitochondrial gene arrangement is almost identical to those of pulmonate land snails but is radically divergent from those of the prosobranch gastropod Littorina saxatilis and other molluscs. Our finding that the unique gene arrangement and highly compact genome organization are shared between opisthobranch and pulmonate gastropods strongly suggests their close phylogenetic affinity.

Amino Acid Sequence↗

PubMed1991

The two ATPase 6 mRNAs of Leishmania tarentolae differ at their 3' ends.

We have determined the complete nucleotide sequence of ATPase 6 mRNA from Leishmania tarentolae. RNA editing occurs only in the 5' one-third of the mRNA and is the most extensive observed to date in this species. We have identified a potential gRNA sequence, encoded in a minicircle, for a portion of the edited sequence. The predicted amino acid sequence is about 85% homologous to that predicted from the more extensively edited Trypanosoma brucei ATPase 6 mRNA. The edited L. tarentolae mRNA exists as two distinct size classes which differ in the size of their 3' ends. Although variation in the length of the 3' untranslated region cannot be excluded, the size difference is probably to be due to variation in the length of the poly(A) tail.

Adenosine Triphosphatases↗

PubMed1991

The cytochrome oxidase II gene in mitochondria of the sugar-beet Beta vulgaris L.

We have cloned and analyzed the sugar-beet mitochondrial gene for cytochrome oxidase subunit II (coxII). The sugar-beet and its deduced amino acid sequence were compared to its homologous coxII gene sequences from both monocot and dicot plants. It was found to be highly conserved (89-95%) compared to homologue in other plant species. The 780 bp coding sequence of the sugar beet coxII gene is interrupted at position 383 by a 1463 bp intron. This intron contains an additional 107 bp sequence that is not found in any of the plant coxII genes studied thus far. The structure of the intron suggests that a large intron existed in an ancestral coxII gene before monocots and dicots diverged in evolution. Three CGG codons in the sugar-beet coxII coding sequence align with conserved tryptophan residues in the homologous gene of other species, suggesting that RNA editing takes place also in sugar-beet mitochondria. In 13 out of 24 codons of coxII mRNA that were found to be edited in four other plants, the sugar-beet gene already utilizes the edited codons. This phenomenon may indicate that the mitochondrial genome in sugar-beet is phylogenetically more archaic relative to these plants. An additional sequence of 279 bp that is identical to the first exon of coxII was identified in the mtDNA of the sugar-beet. This 'pseudo-gene' is transcribed and its existence in the mitochondrial genome is unexplained.

Amino Acid Sequence↗

PubMed1992

Transcript-specific developmental regulation of polyadenylation in Trypanosoma brucei mitochondria.

Transcripts from many mitochondrial genes in kinetoplastids are heterogeneous in size, often occurring as 2 distinct size classes, but this cannot be accounted for by RNA editing alone. Analyses of transcripts from 6 mitochondrial genes of Trypanosoma brucei indicates that the size variation is due to poly(A) tail length. A larger fraction of CYb, COI and COII transcripts have longer poly(A) tails in procyclic than in bloodstream forms. These transcripts are also more abundant in the procyclic forms. In contrast, a more substantial fraction of CR1 transcripts have longer poly(A) tails in bloodstream than in procyclic forms and these transcripts tend to be more abundant in bloodstream forms. Both ND4 and MURF1 transcripts show a similar size distribution of poly(A) tail lengths in these life cycle states although both transcripts are more abundant in bloodstream forms. Furthermore, genes with edited transcripts tend to have longer poly(A) tails than unedited transcripts. Transcript abundance is not strictly correlated with longer poly(A) tails. Thus, poly(A) length variation appears to be developmentally regulated in a transcript-specific fashion in T. brucei. This regulation of polyadenylation may influence mitochondrial gene expression as polyadenylation can regulate cytoplasmic gene expression in eukaryotes.

Animals↗

PubMed1997

Genes encoding subunit 6 of NADH dehydrogenase and subunit 6 of ATP synthase are co-transcribed in maize mitochondria.

The mitochondrial nad6 gene of maize was identified and mapped 1 kb downstream from the atp6 gene. It encodes a 220 amino-acid polypeptide. Using Northern hybridization experiments and RT-PCR analysis, we showed that both nad6 and atp6 are co-transcribed in maize mitochondria. RNA editing of the mitochondrial nad6 transcript was studied by cDNA sequencing. Twelve edited sites were identified at the same positions as those already identified in the wheat mitochondria nad6 transcript. Alignments of nucleotide and amino-acid sequences of the mitochondrial nad6 genes of maize, wheat, and Brassica campestris, show that the wheat gene encodes a shorter polypeptide (229 amino acids) than was previously thought.

Amino Acid Sequence↗

PubMed1994

Structure and organization of mouse GlcNAc-1-phosphate transferase gene.

The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, was isolated and characterized. Southern blot analyses demonstrated a single copy gene for GPT. The gene spans about 7.5 kilobase pairs of DNA and is divided into 9 exons by 8 introns. All the introns are found in the coding region, and most of them occur in segments separating the putative membrane-spanning domains. The exon/intron organization of the gene also correlates with the presence of several highly conserved regions of potential functional importance among yeast, leishmania, hamster, and mouse enzymes. Primer extension and reverse transcription-polymerase chain reaction analyses suggested the presence of several potential transcription start sites, with the closest one being approximately 200 base pairs upstream from the translation initiation codon. The 5'-flanking region lacks a typical TATA box, but is high in GC content and contains two putative Sp1 binding sites (GC boxes), consistent with promoters described for housekeeping genes. The 3'-end reverse transcription-polymerase chain reaction analysis indicated that the first of the two polyadenylation sites was used predominantly, in agreement with a approximately 2.0-kilobase pair GPT message seen on Northern blots of RNA from a wide variety of mouse tissues. This is the first report of cloning of a gene for an enzyme of the dolichol cycle in higher eukaryotes. A novel finding of this study is the observation of a G-->A change between the genomic sequence and nucleotide 280 in the cDNA. This could have important implications as an RNA editing mechanism for regulating the expression of the gene and therefore, protein N-glycosylation. A previous study (11) had shown that the activity of GPT was developmentally regulated in mouse mammary gland, with possible involvement by the hormone prolactin. The availability of the GPT gene with its promoter should facilitate future studies on delineating the mechanism for the hormonal regulation of GPT.

Amino Acid Sequence↗

PubMed2003

Functional cloning of genes encoding dsRNA binding proteins.

Over a dozen human genes code for proteins that specifically bind to double-stranded RNA. These proteins have been implicated in several important cellular processes such as transcriptional activation, inhibition of translational initiation, RNA editing, mRNA localization, signal transduction, and posttranscriptional gene silencing (PTGS or RNAi). The recent discovery that PTGS or RNAi is a dsRNA-mediated pathway has further added to the study of dsRNA binding proteins. A method that enables the cloning of genes encoding dsRNA binding proteins would greatly facilitate the identification and study of these molecules. Here we describe a method for isolating such genes from an expression library using radiolabeled poly(I):poly(C) as a binding substrate.

Cloning, Molecular↗

PubMed1999

Molecular tinkering of G protein-coupled receptors: an evolutionary success.

Among membrane-bound receptors, the G protein-coupled receptors (GPCRs) are certainly the most diverse. They have been very successful during evolution, being capable of transducing messages as different as photons, organic odorants, nucleotides, nucleosides, peptides, lipids and proteins. Indirect studies, as well as two-dimensional crystallization of rhodopsin, have led to a useful model of a common 'central core', composed of seven transmembrane helical domains, and its structural modifications during activation. There are at least six families of GPCRs showing no sequence similarity. They use an amazing number of different domains both to bind their ligands and to activate G proteins. The fine-tuning of their coupling to G proteins is regulated by splicing, RNA editing and phosphorylation. Some GPCRs have been found to form either homo- or heterodimers with a structurally different GPCR, but also with membrane-bound proteins having one transmembrane domain such as nina-A, odr-4 or RAMP, the latter being involved in their targeting, function and pharmacology. Finally, some GPCRs are unfaithful to G proteins and interact directly, via their C-terminal domain, with proteins containing PDZ and Enabled/VASP homology (EVH)-like domains.

Animals↗

PubMed1997

Evolution of trans-splicing plant mitochondrial introns in pre-Permian times.

Trans-splicing in angiosperm plant mitochondria connects exons from independent RNA molecules by means of group II intron fragments. Homologues of trans-splicing introns in the angiosperm mitochondrial nad2 and nad5 genes are now identified as uninterrupted group II introns in the ferns Asplenium nidus and Marsilea drummondii. These fern introns are correctly spliced from the pre-mRNA at the sites predicted from their well-conserved secondary structures. The flanking exon sequences of the nad2 and nad5 genes in the ferns require RNA editing, including the removal of in-frame stop codons by U-to-C changes for correct expression of the genetic information. We conclude that cis-splicing introns like the ones now identified in ferns are the ancestors of trans-splicing introns in angiosperm mitochondria. Intron disruption is apparently due to a size increase of the structurally variable group II intron domain IV followed by DNA recombination in the plant mitochondrial genome.

Amino Acid Sequence↗