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Biomedical subjects

R Benne

Publications and source records attributed to R Benne.

At least 19 recordsLinked to original sources
PubMed2000

MitBASE : a comprehensive and integrated mitochondrial DNA database. The present status.

MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects, under a single interface, databases for Plant, Vertebrate, Invertebrate, Human, Protist and Fungal mtDNA and a Pilot database on nuclear genes involved in mitochondrial biogenesis in Saccharomyces cerevisiae. MitBASE reports all available information from different organisms and from intraspecies variants and mutants. Data have been drawn from the primary databases and from the literature; value adding information has been structured, e.g., editing information on protist mtDNA genomes, pathological information for human mtDNA variants, etc. The different databases, some of which are structured using commercial packages (Microsoft Access, File Maker Pro) while others use a flat-file format, have been integrated under ORACLE. Ad hoc retrieval systems have been devised for some of the above listed databases keeping into account their peculiarities. The database is resident at the EBI and is available at the following site: http://www3.ebi.ac.uk/Research/Mitbase/mitbas e.pl. The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecies diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme.

Animals↗
PubMed2000

Mitochondrial minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks.

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85-90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.

Amino Acid Sequence↗
PubMed2000

Molecular misreading. A new type of transcript mutation in gerontology.

Molecular misreading is a novel process that causes mutations in neuronal transcripts. It is defined as the inaccurate conversion of genomic information from DNA into nonsense transcripts and the subsequent translation into mutant proteins. As a result of dinucleotide deletions (delta GA, delta GU, delta CU) in and around GAGAG motifs in mRNA the reading frame shifts to the +1 frame, and subsequently the so-called +1 proteins are synthetized. +1 Proteins have a wild-type NH2 terminus and from the site of the dinucleotide deletion onwards an aberrant, nonfunctional COOH terminus. Molecular misreading was found in the rat vasopressin gene associated with diabetes insipidus and in the human genes linked to Alzheimer's disease (AD), that is, beta-amyloid precursor protein (beta APP) and ubiquitin-B (UBB). Moreover, beta APP+1 and UBB+1 proteins accumulate in the neuropathological hallmarks of AD. Inasmuch as these +1 proteins were also found in elderly, nondemented control patients, but not in younger ones (< 72 years), molecular misreading may act as a factor that becomes manifest in aged people. A hotspot for dinucleotide deletions is GAGAG motifs. Because statistically an average of 2.1 GAGAG motifs per gene can be expected, other genes expressed in other tissues may undergo molecular misreading as well. Indeed, we recently detected +1 proteins in proliferating cells present in tissues such as the liver, epididymis, parotid gland, and neuroblastoma cell lines. Therefore, molecular misreading can be regarded as a general biological source of transcript errors that may be involved in cellular derangements in numerous age-related pathologic conditions apart from Alzheimer's disease.

Aging↗
PubMed1999

MitBASE: a comprehensive and integrated mitochondrial DNA database.

MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects all available information from different organisms and from intraspecie variants and mutants. Research institutions from different countries are involved, each in charge of developing, collecting and annotating data for the organisms they are specialised in. The design of the actual structure of the database and its implementation in a user-friendly format are the care of the European Bioinformatics Institute. The database can be accessed on the Web at the following address: http://www.ebi.ac. uk/htbin/Mitbase/mitbase.pl. The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecie diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme.

Animals↗
PubMed1998

Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity.

We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem. 227, 780-786).

Alternative Splicing↗
PubMed1998

RNA editing in the free-living bodonid Bodo saltans.

In parasitic kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g) RNAs. In order to further explore the role and evolutionary history of this process, we searched for editing in mt RNAs of the free-living bodonid Bodo saltans. We found extensive editing in the transcript for NADH dehydrogenase (ND) subunit 5, which is unedited in trypanosomatids. In contrast, B.saltans cytochrome c oxidase (cox) subunit 2 and maxicircle unidentified reading frame (MURF) 2 RNAs display limited editing in the same regions as their trypanosomatid counterparts. A putative intramolecular cox2 gRNA and the gene for gMURF2-I directing the insertion of only one U in the 5' editing domain of MURF2 RNA, are conserved in B.saltans. This lends (further) evolutionary support to the proposed role of these sequences as gRNAs. Phylogenetic analysis showed that B.saltans is more closely related to trypanosomatids than the cryptobiids Trypanoplasma borreli and Cryptobia helicis, in line with the trypanosomatid-like cox2 and MURF2 RNA editing patterns. Nevertheless, other features like the apparent absence of a catenated mtDNA network, are shared with bodonid and cryptobiid species. ND5 RNA editing may represent yet another example of editing 'on the way out' during kinetoplastid evolution, but in view of the fact that cox2 RNA is unedited in T. borreli and C.helicis, we infer that the editing of this RNA may have arisen relatively recently. Our results provide the first examples of RNA editing in a free-living kinetoplastid, indicating that there is no direct link between U-insertion/deletion editing and a parasitic lifestyle.

Amino Acid Sequence↗
PubMed1997

Characterization of the respiratory chain from cultured Crithidia fasciculata.

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F0F1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F0F1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F0 subunits (b and c) and all of the F1 subunits. The F0 b subunit has a low degree of similarity to subunit b from other organisms. The F1 alpha subunit is extremely small making the beta subunit the largest F1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.

Adenosine Triphosphate↗
PubMed1997

RNA editing in kinetoplastid parasites: what to do with U.

The editing of the mitochondrial RNAs of kinetoplastid protozoa is a bizarre form of transcript maturation that involves insertion and deletion of uridylate residues. Editing leads to the formation of translational initiation and termination codons, the correction of gene-encoded reading frame shifts and the creation of complete reading frames in mRNAs. It is therefore an essential step in mitochondrial gene expression.

Animals↗
PubMed1997

Further evidence for the presence of mitochondrially encoded subunits in cytochrome c oxidase of the trypanosomatid Crithidia fasciculata.

Mitochondrial mRNAs in trypanosomatids are edited by uridylate insertion and deletion. The respiratory chain complexes cytochrome c reductase, cytochrome c oxidase and F0F1-ATPase of the insect trypanosomatid Crithidia fasciculata have been isolated and analysed by peptide microsequencing, but so far, proteins encoded by edited (and unedited) mitochondrial mRNAs have not been found. In this paper, we provide evidence that the mitochondrial mRNAs encoding the three large subunits of cytochrome c oxidase are indeed translated. First, purified holo cytochrome c oxidase turned out to be cysteine-rich, in agreement with the high cysteine codon-content of the sequence of mitochondrial cox subunit mRNAs. Second, in mass spectrometry measurements of cytochrome c oxidase, a protein was detected with the predicted molecular weight of cytochrome c oxidase subunit 2. Finally, an antibody generated against a fusion protein produced in Escherichia coli from constructs containing a segment of cytochrome c oxidase subunit 2 cDNA, specifically recognised protein bands present in cytochrome c oxidase following SDS PAGE. However, these proteins were present in the high molecular weight region of the gel, suggesting that cytochrome c oxidase subunit 2 aggregates in the presence of SDS.

Animals↗
PubMed1996

An intronic mutation in a lariat branchpoint sequence is a direct cause of an inherited human disorder (fish-eye disease).

The first step in the splicing of an intron from nuclear precursors of mRNA results in the formation of a lariat structure. A distinct intronic nucleotide sequence, known as the branchpoint region, plays a central role in this process. We here describe a point mutation in such a sequence. Three sisters were shown to suffer from fish-eye disease (FED), a disorder which is caused by mutations in the gene coding for lecithin:cholesterol acyltransferase (LCAT). Sequencing of the LCAT gene of all three probands revealed compound heterozygosity for a missense mutation in exon 4 which is reported to underlie the FED phenotype, and a point mutation located in intron 4 (IVS4:T-22C). By performing in vitro expression of LCAT minigenes and reverse transcriptase PCR on mRNA isolated from leukocytes of the patient, this gene defect was shown to cause a null allele as the result of complete intron retention. In conclusion, we demonstrated that a point mutation in a lariat branchpoint consensus sequence causes a null allele in a patient with FED. In addition, our finding illustrates the importance of this sequence for normal human mRNA processing. Finally, this report provides a widely applicable strategy which ensures fast and effective screening for intronic defects that underlie differential gene expression.

Amino Acid Sequence↗
PubMed1996

The sequence of a small subunit of cytochrome c oxidase from Crithidia fasciculata which is homologous to mammalian subunit IV.

The sequence of subunit 8 of cytochrome c oxidase from Crithidia fasciculata was determined by sequencing cDNA and N-terminus of the mature protein (Mr = 15.7 kDA). The (inferred) protein is homologous to mammalian cox IV and the corresponding cox subunits from yeast, Neurospora crassa and Dictyostelium discoideum, which is reflected in a very similar hydropathy profile. Elements that are conserved in the C. fasciculata sequence include (i) an N-terminal (D/E)-(K/R)-X-K-(X2)-W-(X2)-(I/L) motif, (ii) a putative membrane-spanning region in the middle portion of the protein, and (iii) a C-terminal W-(X13)-(N/D)-P motif. The C. fasciculata protein is synthesized with a cleavable presequence.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of cytochrome c oxidase from the insect trypanosomatid Crithidia fasciculata.

Cytochrome c oxidase was purified from the mitochondrial lysate of the insect trypanosomatid Crithidia fasciculata with the aid of a methyl hydrophobic interaction column in a rapid one-step procedure. The purified complex displayed all characteristics expected from a eukaryotic cytochrome c oxidase: the presence of CuA in electron paramagnetic resonance analysis, a characteristic 605 nm peak in reduced-minus-oxidized optical spectroscopy, and the capacity to efficiently oxidize homologous, but not heterologous, cytochrome c. Two-dimensional PAGE showed that C. fasciculata cytochrome c oxidase consists of at least 10 different subunits. N-terminal sequences were obtained from the six smallest subunits of the complex, one of them showing significant similarity to Neurospora crassa cytochrome c oxidase subunit V. The N-terminus of each of the four largest subunits was found to be blocked.

Amino Acid Sequence↗
PubMed1996

RNA editing: how a message is changed.

Considerable progress has been made in unraveling the mechanistic features of RNA editing processes in a number of genetic systems. Recent highlights include the identification of the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme as a zinc-dependent cytidine deaminase that binds to RNA, the demonstration that adenosines in brain glutamate receptor pre-mRNAs are converted into inosines and that double-stranded RNA A deaminase (dsRAD), the candidate enzyme, is another zinc-dependent RNA nucleotide deaminase, and a mounting body of evidence for a cleavage-ligation mechanism for U insertion/deletion editing in kinetoplastid protozoa.

Animals↗
PubMed1995

Identification by UV cross-linking of oligo(U)-binding proteins in mitochondria of the insect trypanosomatid Crithidia fasciculata.

RNA editing in trypanosomes is the process of insertion and deletion of U residues at specific sites of mitochondrial transcripts mediated by short guide RNAs (gRNAs) that have a 3' oligo(U) extension. Here we describe the identification by UV cross-linking of proteins present in mitochondrial extracts from Crithidia fasciculata with a high affinity for gRNAs, and the characterization of the binding specificity. A 65-kDa protein binds to gRNAs provided they are equipped with a U tail, to post-transcriptionally labelled mitoribosomal 9S and 12S RNAs that also possess a 3' terminal stretch of U residues, and to free oligo(U) sequences with a minimal length of 23-29 nucleotides. It does not bind to a number of control RNAs, one of which has an internal U stretch of 13 residues. Poly(U), but not poly(C) or total yeast RNA, efficiently competes for binding to gRNA. Proteins of 88 kDa and 30 kDa also bind to gRNAs with a U tail, to mitochondrial ribosomal RNAs and to oligo(U). These proteins, however, require longer oligo(U) for binding (> 39 nucleotides) and they also have an affinity for other U-rich RNAs and poly(C). For comparison, part of the analysis was also carried out with a mitochondrial extract from Trypanosoma brucei. In this organism, gRNA-binding proteins of 83 kDa and 64 kDa were found with the same preference for 3'-terminal oligomeric U stretches as the C. fasciculata 65-kDa protein, whereas the binding specificity of a 26-kDa protein resembled that of the C. fasciculata 88-kDa and 30-kDa proteins. The possible involvement of the proteins in the editing process is discussed.

Animals↗
PubMed1995

A possible role for the guide RNA U-tail as a specificity determinant in formation of guide RNA-messenger RNA chimeras in mitochondrial extracts of Crithidia fasciculata.

Chimeric g(uide) RNA:pre-mRNA molecules are potential intermediates of the RNA editing process in kinetoplastid mitochondria. We have studied the characteristics of chimeric molecules formed in mitochondrial extracts of the insect trypanosomatid Crithidia fasciculata which had been supplied with synthetic NADH dehydrogenase (ND) subunit-7 gRNA and pre-mRNA variants. The ability of a gRNA to participate in chimera formation in this system depends on the possibility of base pairing with the pre-mRNA via the anchor sequence, but not on the presence of a U-tail or a full-length informational part. Chimeras formed with a specific gRNA:pre-mRNA pair displayed a large variation in length, due to variably sized 3' end truncations of the gRNA moieties and variation in the sites in the pre-mRNA to which the gRNAs were attached. Surprisingly, the presence of a U-tail in the gRNA for a large part determined the specificity of the linkage. In 60% of the cases gRNAs possessing a U-tail of at least one residue were attached to an editing site, whereas 75% of the gRNAs without Us were attached to non-editing sites. Furthermore, the chimera forming activity was greatly stimulated by the addition of ATP but not by AMP-CPP, an ATP-analogue with a non-hydrolyzable alpha-beta phosphate bond. This suggests the involvement in the chimera formation of an RNA ligase.

Adenosine Triphosphate↗
PubMed1994

Novel pattern of editing regions in mitochondrial transcripts of the cryptobiid Trypanoplasma borreli.

In mitochondria of Kinetoplastida belonging to the suborder Trypanosomatina, the nucleotide sequence of transcripts is post-transcriptionally edited via insertion and deletion of uridylate residues. In order to shed more light on the evolutionary history of this process we have searched for editing in mitochondrial RNAs of Trypanoplasma borreli, an organism belonging to the suborder Bodonina. We have cloned and sequenced a 5.3 kb fragment derived from a 37 kb mitochondrial DNA molecule which does not appear to be a part of a network structure and have found genes encoding cytochrome c oxidase (cox) subunit 1, cox 2 and apocytochrome (cyt) b, and genes encoding the small and large subunit mitoribosomal RNAs. The order in which these genes occur is completely different from that of trypanosomatid maxicircle genes. The 5' and 3' termini of both the cytb and cox1 gene are cryptic, the protein coding sequences being created by extensive insertion/deletion of Us in the corresponding mRNA sections. Phylogenetic analyses of the protein and ribosomal RNA sequences demonstrated that the separation between T.borreli and Trypanosomatina was an early event, implying that U-insertion/deletion processes are ancient. Different patterns of editing have persisted in different lineages, however, since editing of cox1 RNA and of relatively small 3'-terminal RNA sections is not found in trypanosomatids. In contrast, cox2 RNA which is edited in trypanosomatids by the insertion of four Us, is unedited in T.borreli.

Amino Acid Sequence↗