Biomedical subjects

A Brennicke

Publications and source records attributed to A Brennicke.

At least 19 recordsLinked to original sources

PubMed2001

The Y chromosome in the liverwort Marchantia polymorpha has accumulated unique repeat sequences harboring a male-specific gene.

The haploid liverwort Marchantia polymorpha has heteromorphic sex chromosomes, an X chromosome in the female and a Y chromosome in the male. We here report on the repetitive structure of the liverwort Y chromosome through the analysis of male-specific P1-derived artificial chromosome (PAC) clones, pMM4G7 and pMM23-130F12. Several chromosome-specific sequence elements of approximately 70 to 400 nt are combined into larger arrangements, which in turn are assembled into extensive Y chromosome-specific stretches. These repeat sequences contribute 2-3 Mb to the Y chromosome based on the observations of three different approaches: fluorescence in situ hybridization, dot blot hybridization, and the frequency of clones containing the repeat sequences in the genomic library. A novel Y chromosome-specific gene family was found embedded among these repeat sequences. This gene family encodes a putative protein with a RING finger motif and is expressed specifically in male sexual organs. To our knowledge, there have been no other reports for an active Y chromosome-specific gene in plants. The chromosome-specific repeat sequences possibly contribute to determining the identity of the Y chromosome in M. polymorpha as well as to maintaining genes required for male functions, as in mammals such as human.

Amino Acid Sequence↗

PubMed2001

The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis.

Two-component signal systems regulate a variety of cellular activities. They involve at least two common signalling molecules: a signal-sensing kinase and a response regulator that mediates the output response. Multistep systems also require proteins containing phosphotransfer domains. Here we report that the response regulator ARR2 from Arabidopsis is predominantly expressed in pollen and is localized in the nuclear compartment of the plant cell. Furthermore, ARR2 is transcriptionally active in yeast and binds to the promoters of nuclear genes for several components of mitochondrial respiratory chain complex I (nCI) from Arabidopsis. The nuclear nCI genes are up-regulated in pollen during spermatogenesis. The transcription factor functions of ARR2 are mediated by its C-terminal output domain. Our data identify ARR2 as the first eukaryotic response regulator which functions as a transcription factor at a known promoter sequence. Yeast two-hybrid analysis and in vitro interaction studies suggest that ARR2 very probably forms part of a multistep two-component signalling mechanism that includes HPt proteins like AHP1 or AHP2. These findings point to an as yet unidentified signal transduction system that may regulate aspects of floral and mitochondrial gene expression.

Arabidopsis↗

PubMed2001

From gene to protein in higher plant mitochondria.

Higher plant mitochondria contain a genetic system with a genome, transcription and translation processes, which have to be logistically integrated with the two other genomes in the nucleus and the plastid. In plant mitochondria, after transcripts have been synthesised, at least in some cases by a phage-type RNA polymerase, they have to go through a complex processing apparatus, which depends on protein factors imported from the cytosol. Processing involves cis- and trans-splicing, internal RNA editing and maturation at the transcript termini, these steps often occurring in parallel. Transcript life is terminated by RNA degradation mechanisms, one of which involves polyadenylation. RNA metabolism seems to be a key element of the regulation of gene expression in higher plant mitochondria.

Genome, Plant↗

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

Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha.

Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male-specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.

Base Sequence↗

PubMed2000

A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant.

Autocatalytic activity of some group II introns has been demonstrated in vitro, but helper functions such as the yeast MRS2 protein are essential for splicing in vivo. In our search for such helper factors in plants, we pursued the cloning of two Arabidopsis thaliana homologues, atmrs2-1 and atmrs2-2. Atmrs2-1, but not atmrs2-2, complements the yeast deletion mutant of mrs2, and this is congruent with the prediction of two adjacent transmembrane stretches in AtMRS2-1 and yeast MRS2 but not in AtMRS2-2. This complementation depends on fusion of the native yeast mitochondrial import sequence to atmrs2-1. A differing, non-mitochondrial, cellular targeting in Arabidopsis is supported by the analysis of green fluorescent protein fusion constructs after transient transformation into plant protoplasts. Further members of what now appears to be a family of 10 mrs2 homologues are identified in the Arabidopsis genome. Similarity searches with the PSI-BLAST algorithm in the protein database fail to identify homologues of this novel gene family in any eukaryotes other than yeasts, but do identify its distant relatedness to the corA group of bacterial magnesium transporters. In line with this observation, intramitochondrial magnesium concentrations are indeed restored to wild-type levels in the yeast mutant on complementation with atmrs2-1.

Amino Acid Sequence↗

PubMed2000

RNA degradation buffers asymmetries of transcription in Arabidopsis mitochondria.

To understand better the relative contributions of transcriptional and post-transcriptional processes towards the regulation of gene expression in plant mitochondria, we compared the steady state levels of RNAs with the respective transcriptional activities. All of the protein and rRNA coding genes of the Arabidopsis mitochondrial genome and several orfs were analyzed by run-on and northern experiments. rRNAs constitute the bulk of the steady state RNA in Arabidopsis mitochondria, but are (different from maize mitochondria) not equally prominent among the run-on transcripts. Their relatively low rate of active transcription is apparently compensated by their high stability. Run-on transcription values differ significantly between genes coding for different subunits of the same protein complex. The steady state RNA levels are considerably more homogeneous, indicating that high variations of transcription rates are counterbalanced by post-transcriptional processes. The relative amounts of the steady state transcripts for the different subunits in a given protein complex reflect the relative stoichiometries of the protein subunits much more closely than the respective transcriptional activities. Post-transcriptional RNA processing and stability thus contribute significantly to the regulation of gene expression in Arabidopsis mitochondria.

Arabidopsis↗

PubMed1999

RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs.

On the basis of the sequence of the mitochondrial genome in the flowering plant Arabidopsis thaliana, RNA editing events were systematically investigated in the respective RNA population. A total of 456 C to U, but no U to C, conversions were identified exclusively in mRNAs, 441 in ORFs, 8 in introns, and 7 in leader and trailer sequences. No RNA editing was seen in any of the rRNAs or in several tRNAs investigated for potential mismatch corrections. RNA editing affects individual coding regions with frequencies varying between 0 and 18.9% of the codons. The predominance of RNA editing events in the first two codon positions is not related to translational decoding, because it is not correlated with codon usage. As a general effect, RNA editing increases the hydrophobicity of the coded mitochondrial proteins. Concerning the selection of RNA editing sites, little significant nucleotide preference is observed in their vicinity in comparison to unedited C residues. This sequence bias is, per se, not sufficient to specify individual C nucleotides in the total RNA population in Arabidopsis mitochondria.

Arabidopsis↗

PubMed1999

An RNA helicase (AtSUV3) is present in Arabidopsis thaliana mitochondria.

The proteins involved in mitochondrial mRNA processing and degradation in higher plants have yet to be identified. As a first step towards this aim, we report here the characterisation of a nuclear-encoded DExH box RNA helicase (AtSUV3) localised in Arabidopsis thaliana mitochondria. The AtSUV3 mRNA is assembled from the 16 exons of a weakly expressed unique gene and the predicted protein has a calculated molecular weight of 63.6 kDa. Subcellular fractionation of transgenic plants expressing AtSUV3/GUS fusion proteins localises this protein in mitochondria. The N-terminal domain of AtSUV3 containing the motifs characteristic of DExH box RNA helicases exhibits a low endogenous ATPase activity in vitro which can be stimulated by the presence of mitochondrial RNA, confirming that AtSUV3 is an RNA helicase.

Adenosine Triphosphatases↗

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↗

PubMed1999

The mitochondrial genome of Arabidopsis is composed of both native and immigrant information.

Plants contain large mitochondrial genomes, which are several times as complex as those in animals, fungi or algae. However, genome size is not correlated with information content. The mitochondrial genome (mtDNA) of Arabidopsis specifies only 58 genes in 367 kb, whereas the 184 kb mtDNA in the liverwort Marchantia polymorpha codes for 66 genes, and the 58 kb genome in the green alga Prototheca wickerhamii encodes 63 genes. In Arabidopsis' mtDNA, genes for subunits of complex II, for several ribosomal proteins and for 16 tRNAs are missing, some of which have been transferred recently to the nuclear genome. Numerous integrated fragments originate from alien genomes, including 16 sequence stretches of plastid origin, 41 fragments of nuclear (retro)transposons and two fragments of fungal viruses. These immigrant sequences suggest that the large size of plant mitochondrial genomes is caused by secondary expansion as a result of integration and propagation, and is thus a derived trait established during the evolution of land plants.

Journal Article↗

PubMed1999

In plants a putative isovaleryl-CoA-dehydrogenase is located in mitochondria.

In plants the degradation pathways of branched-chain amino acids have remained somewhat unclear with respect to both their biochemistry and their intracellular location. While biochemical evidence has localized some of the catabolic enzymes in peroxisomes/glyoxysomes, others cofractionate with mitochondria. We have now identified a candidate protein and corresponding cDNA for an enzyme of the leucine catabolic pathway, the isovaleryl-CoA-dehydrogenase (IVD). This polypeptide is a member of the acyl-CoA-dehydrogenase (ACDH) family and is encoded in the nuclear genome of Arabidopsis thaliana. Expression of the putative IVD gene in pea seedlings is documented by western blot analyses with an antibody against the mammalian IVD. Subcellular fractionation identifies the putative IVD enzyme in the mitochondrion. This localization suggests that in plants mitochondria contain at least part of the branched-chain amino acid degradation pathway(s).

Amino Acid Sequence↗

PubMed1999

Homologues of yeast and bacterial rotenone-insensitive NADH dehydrogenases in higher eukaryotes: two enzymes are present in potato mitochondria.

Two different cDNAs, homologous to genes for rotenone-insensitive NADH dehydrogenases of bacteria and yeast, were isolated from potato. The encoded proteins, called NDA and NDB, have calculated molecular masses of 55 and 65 kDa, respectively. The N-terminal parts show similarity to mitochondrial targeting peptides and the polypeptides are in vitro imported into potato mitochondria. Import processing to a smaller polypeptide is seen for the NDA but not the NDB protein. After import, NDA is intramitochondrially sorted to the matrix side of the inner membrane, whereas NDB becomes exposed to the intermembrane space. Imported proteins are associated to membranes upon digitonin permeabilization. On expression in Escherichia coli, NDB is released from the bacterial membrane in the absence of divalent cations whereas detergents are necessary for solubilization of NDA. Both deduced amino-acid sequences contain the dual motifs for nucleotide binding with the characteristics of the core criteria, similar to the bacterial homologues. Unique among NADH dehydro- genases, the NDB amino-acid sequence contains a non-conserved insert, which is similar to EF-hand motifs for calcium binding. Phylogenetic analyses group the rotenone-insensitive NADH dehydrogenases largely by species, but suggest ancient gene duplications.

Amino Acid Motifs↗

PubMed1999

RNA editing.

The term RNA editing describes those molecular processes in which the information content is altered in an RNA molecule. To date such changes have been observed in tRNA. rRNA and mRNA molecules of eukaryotes, but not prokaryotes. The demonstration of RNA editing in prokaryotes may only be a matter of time, considering the range of species in which the various RNA editing processes have been found. RNA editing occurs in the nucleus, as well as in mitochondria and plastids, which are thought to have evolved from prokaryotic-like endosymbionts. Most of the RNA editing processes, however, appear to be evolutionarily recent acquisitions that arose independently. The diversity of RNA editing mechanisms includes nucleoside modifications such as C to U and A to I deaminations, as well as non-templated nucleotide additions and insertions. RNA editing in mRNAs effectively alters the amino acid sequence of the encoded protein so that it differs from that predicted by the genomic DNA sequence.

Amino Acid Sequence↗

PubMed1998

Physiological, biochemical and molecular aspects of mitochondrial complex I in plants

Respiratory complex I of plant mitochondria has to date been investigated with respect to physiological function, biochemical properties and molecular structure. In the respiratory chain complex I is the major entry gate for low potential electrons from matrix NADH, reducing ubiquinone and utilizing the released energy to pump protons across the inner membrane. Plant complex I is active against a background of several other NAD(P)H dehydrogenases, which do not contribute in proton pumping, but permit and establish several different routes of shuttling electrons from NAD(P)H to ubiquinone. Identification of the corresponding molecular structures, that is the proteins and genes of the different NADH dehydrogenases, will allow more detailed studies of this interactive regulatory network in plant mitochondria. Present knowledge of the structure of complex I and the respective mitochondrial and nuclear genes encoding various subunits of this complex in plants is summarized here. Copyright 1998 Elsevier Science B.V.

Journal Article↗

PubMed1998

The gene for ribosomal protein L7a-1 in Schizosaccharomyces pombe contains an intron after the initiation codon.

The gene encoding ribosomal protein L7a-1 in the fission yeast Schizosaccharomyces pombe is identified by the similarity of its open reading frame to the respective gene in Saccharomyces cerevisiae. The L7a gene is encoded in two different genomic environments as frequently found for ribosomal protein genes in this organism. One of these genes, L75a-1, is located on chromosome 2. The two consensus promoter elements homol D and homol E are both identified upstream of the start codon of this gene. The ATG start codon is separated from the main reading frame by an intron of 66 nucleotides.

Amino Acid Sequence↗

PubMed1998

Characterization of DNA-Binding Proteins from Pea Mitochondria

We studied transcription initiation in the mitochondria of higher plants, with particular respect to promoter structures. Conserved elements of these promoters have been successfully identified by in vitro transcription systems in different species, whereas the involved protein components are still unknown. Proteins binding to double-stranded oligonucleotides representing different parts of the pea (Pisum sativum) mitochondrial atp9 were analyzed by denaturation-renaturation chromatography and mobility-shift experiments. Two DNA-protein complexes were detected, which appeared to be sequence specific in competition experiments. Purification by hydroxyapatite, phosphocellulose, and reversed-phase high-pressure liquid chromatography separated two polypeptides with apparent molecular masses of 32 and 44 kD. Both proteins bound to conserved structures of the pea atp9 and the heterologous Oenothera berteriana atp1 promoters and to sequences just upstream. Possible functions of these proteins in mitochondrial promoter recognition are discussed.

Journal Article↗

PubMed1998

5' end maturation and RNA editing have to precede tRNA 3' processing in plant mitochondria.

We report the characterization and partial purification of potato mitochondrial RNase Z, an endonuclease that generates mature tRNA 3' ends. The enzyme consists of one (or more) protein(s) without RNA subunits. Products of the processing reaction are tRNA molecules with 3' terminal hydroxyl groups and 3' trailers with 5' terminal phosphates. The main processing sites are located immediately 3' to the discriminator and one nucleotide further downstream. This endonucleolytic processing at and close to the tRNA 3' end in potato mitochondria suggests a higher similarity to the eukaryotic than to the prokaryotic tRNA 3' processing pathway. Partial purification and separation of RNase Z from the 5' processing activity RNase P allowed us to determine biochemical characteristics of the enzyme. The activity is stable over broad pH and temperature ranges, with peak activity at pH 8 and 30 degrees C. Optimal concentrations for MgCl2 and KCl are 5 mM and 30 mM, respectively. The potato mitochondrial RNase Z accepts only tRNA precursors with mature 5' ends. The precursor for tRNAPhe requires RNA editing for efficient processing by RNase Z.

Base Sequence↗