Abstract: Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar and pat. The use of these screening assays allows covering many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and inter-laboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.
Abstract: Since their first commercialization, the diversity of taxa and the genetic composition of transgene sequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs and derived products is commonly performed by PCR-based methods targeting specific DNA sequences introduced into the host genome. Information available regarding the GMOs' molecular characterization is dispersed and not appropriately organized. For this reason, GMO testing is very challenging and requires more complex screening strategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying on reliable information.
Abstract: In routine analysis, screening methods based on real-time PCR are most commonly used for the detection of genetically modified (GM) plant material in food and feed. In this paper, it is shown that the combination of five DNA target sequences can be used as a universal screening approach for at least 81 GM plant events authorised or unauthorised for placing on the market and described in publicly available databases. Except for maize event LY038, soybean events DP-305423 and BPS-CV127-9 and cotton event 281-24-236 x 3006-210-23, at least one of the five genetic elements has been inserted in these GM plants and is targeted by this screening approach. For the detection of these sequences, fully validated real-time PCR methods have been selected. A screening table is presented that describes the presence or absence of the target sequences for most of the listed GM plants. These data have been verified either theoretically according to available databases or experimentally using available reference materials. The screening table will be updated regularly by a network of German enforcement laboratories.
Abstract: Polymerase Chain Reaction (PCR)-based screening methods targeting genetic elements commonly used in genetically modified (GM) plants are important tools for the detection of GM materials in food, feed, and seed samples. To expand and harmonize the screening capability of enforcement laboratories, the German Federal Office of Consumer Protection and Food Safety conducted collaborative trials for interlaboratory validation of real-time PCR methods for detection of the phosphinothricin acetyltransferase (bar) gene from Streptomyces hygroscopicus and a construct containing the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens sp. strain CP4 (ctp2-cp4epsps), respectively. To assess the limit of detection, precision, and accuracy of the methods, laboratories had to analyze two sets of 18 coded genomic DNA samples of events LLRice62 and MS8 with the bar method and NK603 and GT73 with the ctp2-cp4epsps method at analyte levels of 0, 0.02, and 0.1% GM content, respectively. In addition, standard DNAs were provided to the laboratories to generate calibration curves for copy number quantification of the bar and ctp2-cp4epsps target sequences present in the test samples. The study design and the results obtained are discussed with respect to the difficult issue of developing general guidelines and concepts for the collaborative trial validation of qualitative PCR screening methods.
Abstract: Insect resistant Bt 176 maize has been developed by genetic modification to resist European borer infection. In the present investigation, the experiment was conducted to determine the effect of feeding a new hybrid of Bt 176 maize (NX 6262- Bt 176) on general health condition and performance of broiler chickens. Maize grains and diets were subjected to proximate analysis. Amino and fatty acids investigation were applied for both maize grains before used. To evaluate the degradation of NX 6262- Bt 176 maize DNA and its metabolic fate in broiler blood, muscles and organs. One-day-old male broilers were fed ad libitum on either an experimental diet containing NX 6262- Bt 176 or a control diet containing the non-modified maize grains for 35 days. Feed consumption and body weight were recorded weekly during the experimental period. All chickens were subjected to nutritional evaluation period at day 20 of age for 5 successive days, to calculate the percentage of apparent digestible nutrients in both diets. At day 35 samples were collected at several intervals after feed withdrawal. Prior to slaughter blood samples were collected from all birds by heart puncture to prevent DNA cross contamination. Samples from pectoral and thigh muscles, liver, spleen, kidney, heart muscle, bursa and thymus glands were collected. Digesta from different sections of the gastrointestinal tract (GIT) were collected as well. Packed cell volume (PCV) and some serum parameters were investigated. There were no significant differences between control and experimental group concerning chemical composition of feeds, apparent digestible nutrients, and all performance parameters measured (P > 0.05). Furthermore, there were no differences in the PCV and the analysed serum parameters between the control and experimental group. The results of maize DNA digestibility showed that the new variety takes the normal physiological passage along broiler GIT similar to the conventional line. In addition, Bt 176 maize DNA appears to be partially degraded in different parts of GIT comparable to the DNA of the control maize line. Results of the metabolic fate of maize DNA in broiler blood, muscles and organs indicated that only short DNA fragments (199 bp) derived from the plant chloroplast gene could be detected in the blood, skeletal muscles, liver, spleen and kidney, which disappeared after prolongation the fasting time. In heart muscle, bursa of Fabricius and thymus, no plant chloroplast DNA was found. Bt gene specific constructs from Bt 176 maize were not detected in any investigated blood or tissue samples.
Abstract: The results of a survey study on food samples produced from genetically modified soybean and maize collected from the Egyptian market are presented. Forty samples of soybean and 40 samples of maize products have been gathered randomly from markets in Cairo and Giza. The genetic modification was detected by polymerase chain reaction (PCR) using official detection methods according to section 35 of the German Foodstuffs Act. Samples were investigated for the presence of material derived from the following genetically modified organisms (GMOs) all of which are approved for food use in Europe: Roundup Ready soybean (RRS) and maize lines Bt176, Bt11, T25 and MON810. In addition, samples were examined in qualitative and quantitative analysis for the presence of material derived from the transgenic maize line StarLink (Aventis) which was approved for animal feed use exclusively in the US. Twenty % of 40 investigated soy samples contained Roundup Ready soybean; 15% of 40 maize samples tested positive for Bt176 and 12.5% positive for Bt11 maize. Furthermore, the presence of StarLink maize could clearly be demonstrated in four samples mixed with Bt176 and Bt11. The percentage of StarLink was less than 1% in quantitative analysis. The maize lines T25 and MON810 were not detected.
Abstract: Fluorescence-coupled PCR technology was employed to quantify DNA segments specific for Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacteriaceae. The PCR procedure is put forward as an alternative method for detecting microbial contaminations in pharmaceutical preparations and is compared to the tests for specified microorganisms described in European Pharmacopoeia (EP) 2, 2.6.13 and the USP, chapter 61. Data presented here describe the validation of this analytical method when used for proof of absence of specified microorganisms. The detection systems were specific for the microorganisms analyzed, and led to linear results over a wide range (more than 6-7 log intervals). The correlation coefficients lay above 0.99. The precision of replicate determinations within a single test was observed to be high, the relative standard deviation being between 0.39% and 1.53%. The precision between different tests was also high, with a relative standard deviation between 0.76% and 1.91%. The sensitivity without pre-enrichment amounted to 1-10 CFU. Since determination of the specified bacteria was performed following pre-enrichment, the limit of detection amounted to 1 CFU. Equivalent results were obtained in a study on nine batches of a milky hydrophilic cream (SH-No. M 440 A) with the conventional test for microbial contamination and the PCR procedure. The data presented here strongly indicate that the use of fluorescence-coupled PCR techniques can prove the absence of specified bacteria faster and more efficiently than conventional methods.
Abstract: 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.
Abstract: Genes encoding subunits of complex I (EC 1.6.5.3) of the mitochondrial respiratory chain vary in their locations between the mitochondrial and nuclear genomes in different organisms, whereas genes for a homologous multisubunit complex in chloroplasts have to date only been found on the plastid genome. In potato (Solanum tuberosum L.), the gene coding for the mitochondrial 76 kDa iron-sulphur protein is identified in the nuclear genome. The gene is transcribed into polyadenylated mRNA which is most abundant in flowers, and more frequent in tubers than in leaves. The amino acid sequence is well conserved relative to the nuclear-encoded 75 kDa and 78 kDa subunits of Bos taurus and Neurospora crassa, respectively, and to the Paracoccus denitrificans homologue, most prominently in the region presumed to carry the iron-sulphur clusters. Polyclonal antibodies directed against the 78 kDa complex I subunit of N. crassa recognise the 76 kDa polypeptide in potato mitochondrial complex I, and additionally a polypeptide of 75 kDa in solubilised stroma thylakoids from spinach chloroplasts. The 32 amino acid residues long presequence of the potato mitochondrial 76 kDa complex I subunit targets the precursor polypeptide into isolated potato mitochondria but not into isolated chloroplasts. These results suggest that chloroplast stroma thylakoids contain a protein similar in size and antigenicity to, but genetically distinct from, the mitochondrial subunit.
Abstract: 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.
Abstract: The integrity of healthy mitochondria is supposed to depend largely on proper mitochondrial protein biosynthesis. Mitochondrial ribosomal proteins (MRPs) are directly involved in this process. To identify mammalian mitochondrial ribosomal proteins and their corresponding genes, we purified mature rat MRPs and determined 12 different N-terminal amino acid sequences. Using this peptide information, data banks were screened for corresponding DNA sequences to identify the genes or to establish consensus cDNAs and to characterize the deduced MRP open reading frames. Eight different groups of corresponding mammalian MRPs constituted from human, mouse, and rat origin were identified. Five of them show significant sequence similarities to bacterial and/or yeast mitochondrial ribosomal proteins. However, MRPs are much less conserved in respect to the amino acid sequence among species than cytoplasmic ribosomal proteins of eukaryotes and bacteria.
Abstract: Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCl) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5' upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the beta-glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the -200/-100 regions actively convey the pollen/anther-specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCl promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen-specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.
Abstract: The intrinsic 28.5-kDa iron-sulfur protein of complex I in the mitochondrial respiratory chain is encoded in the nucleus in animals and fungi, but specified by a mitochondrial gene in trypanosomes. In plants, the homologous protein is now found to be encoded by a single-copy nuclear gene in Arabidopsis thaliana and by two nuclear genes in potato. The cysteine motifs involved in binding two iron-sulfur clusters are conserved in the plant protein sequences. The locations of the seven introns, with sizes between 60 and 1700 nucleotides, are identical in the A. thaliana and the two potato genes, while their primary sequences diverge considerably. The A + T contents of the intron sequences range between 61% and 73%, as is characteristic for dicot plants, but are in some instances not higher than in the adjacent exons. Here, differences in T content may instead serve to discriminate exons and introns. In potato, both genes are expressed, with the highest levels found in flowers. Sequence similarities between the homologous nuclear and mitochondrial genes indicate that the nuclear forms in animals and plants originate from the endosymbiont genome.
Abstract: We have purified 13 large subunit proteins of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae and determined their partial amino acid sequences. To elucidate the structure and function of these proteins, we searched for their genes by comparing our sequence data with those deduced from the genomic nucleotide sequence data of S. cerevisiae and analyzed them. In addition, we searched for the genes encoding proteins whose N-terminal amino acid sequences we have reported previously [Grohmann, L., Graack, H.-R., Kruft, V., Choli, T., Goldschmidt-Reisin, S. & Kitakawa, M. (1991) FEBS Lett. 284, 51-56]. Thus, we were able to identify and characterize 12 new genes for large subunit proteins of the yeast mitochondrial ribosome. Furthermore, we determined the N-terminal amino acid sequences of seven small subunit proteins and subsequently identified the genes for five of them, three of which were found to be new.
Abstract: Genes for subunits of respiratory chain complex I are found in mitochondrial, plastid and/or nuclear genomes with varying distributions in the diverse eukaryotic species. The intrinsic PSST subunit of complex I is a mitochondrially encoded protein in Paramecium but is specified by a nuclear gene in animals. In plants to date only the homologous plastid encoded NDH-K gene product has been described. The analogous plant mitochondrial protein is now identified as the 22 kDa complex I subunit and found to be encoded in the nuclear genome of Arabidopsis and potato. The cDNA sequences of clones isolated from both plants are 79% identical in the conserved coding region, while the 5' parts of the reading frames specifying the N-terminal presequences for mitochondrial import differ significantly. The expression of the genes examined in different organs of both plants by Northern blot analysis shows elevated steady-state mRNA levels in flowers. Hence, expression of the gene appears to be organ-specifically regulated by its transcription rate and/or mRNA stability. A 1.6 kb long genomic DNA sequence of Arabidopsis upstream of the transcribed gene region encoding the PSST subunit in Arabidopsis contains several putative promoter sequence motifs. The results are discussed with regard to the appearance of a nuclearly integrated, former mitochondrial gene.
Abstract: In higher plants, genes for subunits of respiratory chain complex I (NADH:ubiquinone oxidoreductase) have so far been identified solely in organellar genomes. At least nine subunits are encoded by the mitochondrial DNA and 11 homologues by the plastid DNA. One of the 'key' components of complex I is the subunit binding the substrate NADH. The corresponding gene for the mitochondrial subunit has now been cloned and identified in the nuclear genome from potato (Solanum tuberosum). The mature protein consists of 457 amino acids and is preceded by a mitochondrial targeting sequence of 30 amino acids. The protein is evolutionarily related to the NADH-binding subunits of complex I from other eukaryotes and is well conserved in the structural domains predicted for binding the substrate NADH, the FMN and one iron-sulphur cluster. Expression examined in different potato tissues by Northern blot analysis shows the highest steady-state mRNA levels in flowers. Precursor proteins translated in vitro from the cDNA are imported into isolated potato mitochondria in a delta psi-dependent manner. The processed translation product has an apparent molecular mass of 55 kDa, identical to the mature protein present in the purified plant mitochondrial complex I. However, the in-vitro translated protein is not imported into isolated chloroplasts. To further investigate whether the complex I-like enzyme in chloroplasts contains an analogous subunit for binding of NAD(P)H, different plastid protein fractions were tested with a polyclonal antiserum directed against the bovine 51 kDa NADH-binding subunit. In none of the different thylakoid or stroma protein fractions analysed were specific cross-reactive polypeptides detected. These results are discussed particularly with respect to the structure of a potential complex I in chloroplasts and the nature of its acceptor site.
Abstract: The mitochondrial complex I (NADH:ubiquinone oxidoreductase) isolated from potato (Solanum tuberosum) has been investigated for the presence of iron-sulfur clusters. EPR spectroscopic analysis detected signals arising from clusters N1, N2, N3 and N4. Quantitation of the content of iron and sulfur within the isolated complex I showed the preparation to contain 22.6 mol acid-labile sulfide and 30.4 mol iron/mol complex I. The iron-sulfur cluster composition of the plant complex I appears to be similar to the well-known composition found in Neurospora crassa.
Abstract: In order to characterize individual protein components of the mitochondrial (mt) ribosome for regulatory, functional and evolutionary studies, the yeast nuclear gene MRP-L4 (accession No. Z30582), coding for the mt ribosomal protein (MRP) YmL4, has been cloned using oligodeoxyribonucleotides (oligos) deduced from a partial amino acid (aa) sequence [Graack et al., FEBS Lett. 242 (1988) 4-8] as screening probes. MRP-L4 is located on chromosome XII and codes for a slightly basic protein of 319 aa. The first 14 aa have not been found in the mature protein, and putatively form a signal peptide that is cleaved off during or after mt import. YmL4 has an N terminus very rich in Pro residues, and at its C terminus contains four hydrophobic domains. YmL4 shows no significant sequence similarity to any other sequence from the databases. Gene disruption shows the MRP-L4 product to be indispensable for mt function in cells growing on non-fermentable carbon sources. In contrast to nearly all other MRPs investigated so far, gene disruption of MRP-L4 also affects growth of yeast cells on fermentable carbon sources, suggesting additional cytosolic and/or mt functions of YmL4 besides its involvement in mt protein biosynthesis.
Abstract: The plant NADH:ubiquinone oxidoreductase (or complex I) was isolated from potato (Solanum tuberosum) mitochondria. The multisubunit enzyme was solubilized with detergents, Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), out of the inner mitochondrial membranes and purified by hydroxylapatite and gel filtration chromatography. The preparation was found to be virtually free of any ATPase or transhydrogenase contamination. Complex I of potato is composed of at least 32 individual subunits as detected in silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a total molecular mass of about 900 kDa. The enzyme preparation showed an NADH:ubiquinone-2 reductase activity of 11.5 mumol x min-1 x mg-1 and is strongly inhibited by rotenone. Heterologous polyclonal antibodies against the 70- and 49-kDa subunits of the Neurospora crassa complex I and against the wheat NAD9 subunit cross-reacted specifically with the respective potato subunits. Four of the 10 NH2-terminal sequences determined show significant similarities to Neurospora or bovine complex I subunits and allow a tentative assignment of these subunits.
Abstract: The nuclear gene MRP-L13 of Saccharomyces cerevisiae, which codes for the mitochondrial ribosomal protein YmL13, has been cloned and characterized. It is a single-copy gene residing on chromosome XI. Its nucleotide sequence was found to be identical to that of the previously reported ORF YK105. A comparison of the predicted protein sequence of the MRP-L13 gene product and the actual N-terminal amino-acid sequence of the isolated YmL13 protein indicated that the mature protein is preceded by a mitochondrial signal peptide of 86 amino-acid residues, which is the longest among all known mitochondrial ribosomal proteins of S. cerevisiae. No sequence similarity was found to any other ribosomal protein in the current databases. The transcription of MRP-L13 was found to be repressed in the presence of glucose. Its protein product is not strictly essential for mitochondrial functions, but disruption of the gene by insertion of LEU2 noticeably affected cellular growth on non-fermentable carbon sources.
Abstract: The N-terminal amino acid sequence of a 42.5 kDa subunit of the NADH: ubiquinone oxidoreductase (complex I) from potato has been determined by direct protein sequencing. The sequence was found to be homologous to that of the nuclear-encoded 49 kDa complex I subunit of bovine and Neurospora mitochondria and to the sequence deduced from the mitochondrial nad7 gene identified in the mitochondrial (mt) DNA of tryp anosomes and the moss Marchantia. An oligonucleotide probe derived from the potato N-terminal protein sequence hybridized only to the plant mtDNA. Immunoprecipitation of in-organello 35S-labelled potato and wheat mitochondrial translation products with an antibody directed against the Neurospora 49 kDa complex I subunit indicates that at least in these plants the NAD7 protein is synthesized within the organelle. Comparisons of genomic, cDNA and protein sequences of the 5' coding region reveal three codons that are changed by RNA-editing and confirm translation of the edited transcripts in plant mitochondria. The NAD7 protein appears to undergo post-translational processing since the N-terminal methionine residue is absent from the mature mitochondrial protein.
Abstract: The pool of partially and completely edited mRNAs present in plant mitochondria could potentially be translated into a mixture of divergent proteins. This possibility was investigated for the nad9 gene in potato by characterization of the mRNA population and the corresponding protein sequence. The deduced amino acid sequence of the nad9 gene product has significant similarity to the nuclear-encoded 30 kDa subunit of the bovine and Neurospora NADH:ubiquinone oxidoreductase (complex I) and to the chloroplast ndhJ gene product. Immunoprecipitation of a 27 kDa in-organello 35S labelled mitochondrial translation product with an antibody directed against the wheat nad9 gene product demonstrates its functional expression in potato and wheat. Comparison of the nad9 genomic DNA and cDNA sequences reveals seven codons to be changed by a C to U RNA-editing. Direct sequencing of RT-PCR products derived from cDNAs of different tissues of potato plants shows the presence of a significant portion of only partially edited nad9 transcripts in the various tissues. Amino acid sequencing of internal peptides of the isolated 27 kDa protein from potato tubers demonstrates homogenous translation products of only completely edited nad9 mRNAs even in the presence of partially edited mRNAs. This result suggests a pretranslational selection between edited and incompletely edited mRNAs in plant mitochondria.
Abstract: The vast majority of mitochondrial proteins are in all eukaryotes encoded in the nuclear genomes by genes which have been transferred from the original endosymbiont. DNA as well as RNA was and is exchanged between organelles. A functionally successful information transfer, however, requires complex structural and regulatory alterations of the concerned gene. The recently identified variations of the information content in mitochondrial genomes of different plant species represent different stages of the transfer process. These evolutionary intermediates allow a definition of requirements and chances of successful gene transfers.
Abstract: The nuclear gene for mitochondrial ribosomal protein YmL9 (MRP-L9) of yeast has been cloned and sequenced. The deduced amino acid sequence characterizes YmL9 as a basic (net charge + 30) protein of 27.5 kDa with a putative signal peptide for mitochondrial import of 19 amino acid residues. The intact MRP-L9 gene is essential for mitochondrial function and is located on chromosome XV or VII. YmL9 shows significant sequence similarities to Escherichia coli ribosomal protein L3 and related proteins from various organisms of all three natural kingdoms as well as photosynthetic organelles (cyanelles). The observed structural conservation is located mostly in the C-terminal half and is independent of the intracellular location of the corresponding genes [Graack, H.-R., Grohmann, L. & Kitakawa, M. (1990) Biol. Chem. Hoppe Seyler 371, 787-788]. YmL9 shows the highest degree of sequence similarity to its eubacterial and cyanelle homologues and is less related to the archaebacterial or eukaryotic cytoplasmic ribosomal proteins. Due to their high sequence similarity to the YmL9 protein two mammalian cytoplasmic ribosomal proteins [MRL3 human and rat; Ou, J.-H., Yen, T. S. B., Wang, Y.-F., Kam, W. K. & Rutter, W. J. (1987) Nucleic Acids Res. 15, 8919-8934] are postulated to be true nucleus-encoded mitochondrial ribosomal proteins.
Abstract: The Oenothera mitochondrial genome contains only a gene fragment for ribosomal protein S12 (rps12), while other plants encode a functional gene in the mitochondrion. The complete Oenothera rps12 gene is located in the nucleus. The transit sequence necessary to target this protein to the mitochondrion is encoded by a 5'-extension of the open reading frame. Comparison of the amino acid sequence encoded by the nuclear gene with the polypeptides encoded by edited mitochondrial cDNA and genomic sequences of other plants suggests that gene transfer between mitochondrion and nucleus started from edited mitochondrial RNA molecules. Mechanisms and requirements of gene transfer and activation are discussed.
Abstract: We have determined the N-termini of 26 proteins of the large ribosomal subunit from yeast mitochondria by direct amino acid micro-sequencing. The N-terminal sequences of proteins YmL33 and YmL38 showed a significant similarity to eubacterial ribosomal (r-) proteins L30 and L14, respectively. In addition, several proteins could be assigned to their corresponding yeast nuclear genes. Based on a comparison of the protein sequences deduced from the corresponding DNA regions with the N-termini of the mature proteins, the putative leader peptides responsible for mitochondrial matrix-targeting were compiled. In most leader sequences a relative abundance of aromatic amino acids, preferentially phenylalanine, was found.
Abstract: Using synthetic oligonucleotides deduced from the N-terminal amino acid sequence of purified mitoribosomal protein (mt r-protein) YmL27, the corresponding nuclear gene MRP-L27 of the yeast Saccharomyces cerevisiae has been cloned and sequenced. The MRP-L27 gene codes for 146 amino acids and is located on chromosome X. The mature YmL27 protein consists of 130 amino acids - after cleaving the putative mitochondrial signal peptide - with a net charge of +17 and a calculated relative molecular mass of 14,798 Da. The YmL27 protein as well as the yeast mitoribosomal protein YmL31, which had been characterized and its gene (MRP-L31) cloned previously, is essential for mitochondrial function as shown by the inability of gene disrupted mutants for the MRP-L27 or MRP-L31 genes to grow on non-fermentable carbon sources.
Abstract: The N-terminal amino acid sequence of a large subunit protein, termed YmL33, of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae was determined. The data were obtained to synthesize two kinds of oligonucleotide primers, which were used in the polymerase chain reaction to amplify and clone the nuclear gene for this protein. By nucleotide sequencing, the cloned gene, MRP-L33, was found to encode a basic protein of 11 kDa with 98 amino acid residues. The protein encoded by this gene appears to have no leader sequence at its N terminus. The N-terminal two-thirds of the deduced amino acid sequence showed a significant degree of sequence similarity to ribosomal protein L30 of Escherichia coli and Bacillus stearothermophilus. In addition, the C-terminal one-third showed sequence similarity, though to a lesser extent, to a yeast cytoplasmic ribosomal protein termed L16. By hybridization with the yeast chromosomes and their restriction enzyme fragments, the MRP-L33 gene was concluded to exist on chromosome XIII as a single-copy gene. Disruption of the gene by insertion of a HIS3-containing fragment showed that MRP-L33 was essential for mitochondrial function. The transcriptional level of MRP-L33 in strains with different mitochondrial genetic backgrounds was analyzed in the presence of glucose, galactose, or glycerol.
Abstract: The genes for two large subunit proteins, YmL8 and YmL20, of the mitochondrial ribosome of Saccharomyces cerevisiae were cloned by hybridization with synthetic oligonucleotide mixtures corresponding to their N-terminal amino acid sequences. They were termed MRP-L8 and MRP-L20, respectively, and their nucleotide sequences were determined using a DNA sequencer. The MRP-L8 gene was found to encode a 26.8-kDa protein whose deduced amino acid sequence has a high degree of similarity to ribosomal protein L17 of Escherichia coli. The gene MRP-L20 was found to encode a 22.3-kDa protein with a presequence consisting of 18 amino acid residues. By Southern blot hybridization to the yeast chromosomes separated by field-inversion gel electrophoresis, the MRP-L8 and MRP-L20 genes were located on chromosomes X and XI, respectively. Gene disruption experiments indicate that their products, YmL8 and YmL20 proteins, are essential for the mitochondrial function and the absence of these proteins causes instability of the mitochondrial DNA.
Abstract: According to its cDNA sequence, the product of the DUB1 gene of Dictyostelium discoideum, called ubex52, consists of a ubiquitin monomer with a basic COOH-terminal tail of 52 amino acids that includes a putative zinc finger motif. Antipeptide antibodies raised against the COOH-terminal end of the tail indicated that the ubex52 protein is present in all developmental stages of D. discoideum and that similar proteins with apparent molecular masses of 15 to 17 kDa are found in yeast, wheat germ, Drosophila, and mammals. Subcellular fractionation showed that the D. discoideum and Saccharomyces cerevisiae proteins recognized by the antibodies are associated with the ribosomal fraction. After separation and purification of the 40 and 60 S ribosomal subunits of D. discoideum, the ubex52 protein was exclusively recovered in the small subunit.
Abstract: The nuclear gene for mitochondrial ribosomal protein YmL31 (MRP-L31) of Saccharomyces cerevisiae was cloned using synthetic oligonucleotide mixtures which correspond to the N-terminal amino acid sequence of the mature YmL31. The gene MRP-L31 codes for a basic protein with a calculated molecular mass of 15.5 kDa and resides on chromosome XI. A comparison of the amino acid sequence deduced from the nucleotide sequence of the MRP-L31 gene and the N-terminal sequence of the isolated protein revealed the existence of a leader peptide sequence of 12 amino acid residues. No significant similarity to known ribosomal protein sequences of other organisms was found.
Abstract: Proteins of the small and large subunits of mitochondrial ribosomes from the yeast Saccharomyces cerevisiae were isolated and characterized by two-dimensional gel electrophoresis. Ribosomal proteins of the large subunit were separated by reverse-phase HPLC and up to 37 amino acid residues of the N-terminal sequences of L3, L4, L9 and L31 were determined. No significant homology to ribosomal protein sequences so far determined from other organisms was found.
