Biomedical subjects

L Grohmann

Publications and source records attributed to L Grohmann.

32 records · Page 2Linked to original sources

PubMed1994

The 42.5 kDa subunit of the NADH: ubiquinone oxidoreductase (complex I) in higher plants is encoded by the mitochondrial nad7 gene.

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.

Amino Acid Sequence↗

PubMed1994

Purification of the NADH:ubiquinone oxidoreductase (complex I) of the respiratory chain from the inner mitochondrial membrane of Solanum tuberosum.

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.

Amino Acid Sequence↗

PubMed1994

The yeast nuclear gene MRP-L13 codes for a protein of the large subunit of the mitochondrial ribosome.

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.

Amino Acid Sequence↗

PubMed1993

The mitochondrial genome on its way to the nucleus: different stages of gene transfer in higher plants.

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.

Cell Nucleus↗

PubMed1992

The mitochondrial gene encoding ribosomal protein S12 has been translocated to the nuclear genome in Oenothera.

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.

Amino Acid Sequence↗

PubMed1992

YmL9, a nucleus-encoded mitochondrial ribosomal protein of yeast, is homologous to L3 ribosomal proteins from all natural kingdoms and photosynthetic organelles.

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.

Amino Acid Sequence↗

PubMed1991

Extended N-terminal sequencing of proteins of the large ribosomal subunit from yeast mitochondria.

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.

Amino Acid Sequence↗

PubMed1991

The nuclear coded mitoribosomal proteins YmL27 and YmL31 are both essential for mitochondrial function in yeast.

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.

Amino Acid Sequence↗

PubMed1991

Cloning and analysis of the nuclear gene for YmL33, a protein of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae.

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.

Amino Acid Sequence↗

PubMed1990

Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae.

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.

Amino Acid Sequence↗

PubMed1989

Molecular cloning of the nuclear gene for mitochondrial ribosomal protein YmL31 from Saccharomyces cerevisiae.

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.

Amino Acid Sequence↗

PubMed1989

An extended ubiquitin of Dictyostelium is located in the small ribosomal subunit.

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.

Dictyostelium↗

PubMed1988

Mitochondrial ribosomes of yeast: isolation of individual proteins and N-terminal sequencing.

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.

Amino Acid Sequence↗

PubMedDate not supplied

Alternative microbial testing: a novel DNA-based detection system for specified microorganisms in pharmaceutical preparations.

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.

DNA, Bacterial↗