Mammalian mitochondrial ribosomal proteins (4). Amino acid sequencing, characterization, and identification of corresponding gene sequences

Mitochondrial ribosomal proteins (MRPs) are required for the translation of all 13 mitochondrial encoded genes in humans. It has been speculated that mutations and polymorphisms in the human MRPs may be a primary cause of some oxidative phosphorylation disorders or modulate the severity and tissue specificity of pathogenic mitochondrial DNA mutations. Although the sequences of most of the yeast MRPs are known, only very few mammalian and nearly no human MRPs have been completely characterized. MRPs differ greatly in sequence, and sometimes biochemical properties, between different species, not allowing easy recognition by sequence homology. Therefore, the Mammalian Mitochondrial Ribosomal Consortium is using a direct approach of purifying individual mammalian (bovine) MRPs, determining their N-terminal and/or internal peptide sequences using different protein sequencing techniques, and using the resulting sequence information for screening expressed sequence tags and genomic data bases to determine human, mouse, and rat homologues of the bovine proteins. Two proteins of the large and three proteins of the small ribosomal subunit have been analyzed in this manner. Three of them represent "new," i.e. formerly unknown mammalian mitochondrial ribosomal protein classes. Only one of these three different MRPs shows significant sequence similarities to known ribosomal proteins. In one case, the corresponding human genomic DNA sequences were found in the data bases, and the exon/intron structure was determined.     

Amino acid sequences of proteins from Leptospira serovar pomona

This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.     

Primary structure of the archaebacterial Methanococcus vannielii ribosomal protein L12. Amino acid sequence determination, oligonucleotide hybridization, and sequencing of the gene

The primary structure of ribosomal protein L12 from Methanococcus vannielii has been determined by direct amino acid sequence analysis with automated liquid phase Edman degradation of the entire protein and manual 4-N,N'-dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate sequencing of fragments obtained by enzymatic digestion and by partial acid hydrolysis. The knowledge of the amino acid sequences of these various fragments allowed the synthesis of two oligonucleotide probes complementary to the 5'- and the 3'-end of the gene, and they were used for hybridization with digested M. vannielii chromosomal DNA. Both oligonucleotide probes gave similar and clear hybridization signals. The plasmid pMvaX1 containing the entire gene of protein L12 was obtained. The nucleotide sequence complemented the partial amino acid sequence, and it is in full agreement with the protein sequence and the amino acid analysis. Comparison of secondary structural elements and hydrophobicity plots of the M. vannielii protein L12 with the known L12 sequences derived from other archaebacterial and eukaryotic sources show strong homologies among these sequences. They contain an exceptional highly conserved hydrophilic sequence area in the C-terminal part of the proteins. In comparison with eubacterial L12 proteins, the conservation is reduced to single amino acid residues. However, the eubacterial L12 proteins have hydrophilic regions similar to those of L12 from M. vannielii. These regions are predicted to be located at the surface of the proteins, as has been proven to be the case in crystallized Escherichia coli L12 protein. It is possible that the strongly conserved hydrophilic sequence regions form part of the factor-binding domain.     

Amino acid sequences of cytotoxin-like basic proteins derived from cobra venoms

Amino acid sequences of cytotoxin-like basic proteins (CLBPs), purified from the venoms of Formosan cobra (Naja naja atra) and Indian cobra (Naja naja), were reinvestigated. The determined sequences differed from those reported previously by Takechi et al. [(1985) Biochem. Int. 11, 795-802; (1987) Biochem. Int. 14, 145-152]. The sequence of CLBPs at residues 25-30 was found to be hydrophilic as compared with those of cytotoxins. The difference in the hydrophobicity of this region might be responsible for the difference in their cytotoxic activities.     

Identification of mammalian mitochondrial ribosomal proteins (MRPs) by N-terminal sequencing of purified bovine MRPs and comparison to data bank sequences: the large subribosomal particle

Bovine mitochondrial ribosomes are presented as a model system for mammalian mitochondrial ribosomes. An alternative system for identifying individual bovine mitochondrial ribosomal proteins (MRPs) by RP-HPLC is described. To identify and to characterize individual MRPs proteins were purified from bovine liver, separated by RP-HPLC, and identified by 2D PAGE techniques and immunoblotting. Molecular masses of individual MRPs were determined. Selected proteins were subjected to N-terminal amino acid sequencing. The peptide sequences obtained were used to screen different databases to identify several corresponding MRP sequences from human, mouse, rat, and yeast. Signal sequences for mitochondrial import were postulated by comparison of the bovine mature N-termini determined by amino acid sequencing with the deduced mammalian MRP sequences. Significant sequence similarities of these new MRPs to known r-proteins from other sources, e.g., E. coli, were detected only for two of the four MRP families presented. This finding suggests that mammalian mitochondrial ribosomes contain several novel proteins. Amino acid sequence information for all of the bovine MRPs will prove invaluable for assigning functions to their genes, which would otherwise remain unknown.     

Molecular phylogeny of songbirds (Passeriformes) inferred from mitochondrial 16S ribosomal RNA gene sequences

Phylogenetic relationships among the families of passerine birds have been the subject of many debates. These relationships have been investigated by using a number of different character sets, including morphology, proteins, DNA-DNA hybridization, and mitochondrial DNA gene sequences. Our objective was to examine the phylogenetic relationships of a set of passerine songbirds (Oscines) and to test the taxonomic relationships proposed by. We sequenced 1403 aligned bases encompassing the mitochondrial transfer-RNA-Valine and 16S ribosomal RNA genes in 27 species from 14 families (including a Suboscine outgroup). Our results differ in significant ways from the superfamily designations of Sibley and Ahlquist by questioning the monophyly of the Sylvioidea and by placing the Regulidae in the Corvoidea.     

A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.     

The sialidase gene from Clostridium septicum: cloning, sequencing, expression in Escherichia coli and identification of conserved sequences in sialidases and other proteins

Summary An oligonucleotide mixture corresponding to the codons for conserved and repeated amino acid sequences of bacterial sialidases (Roggentin et al. 1989) was used to clone a 4.3 kb PstI restriction fragment of Clostridium septicum DNA in Escherichia coli. The complete nucleotide sequence of the sialidase gene was determined from this fragment. The derived amino acid sequence corresponds to a protein of 110000 Da. The ribosomal binding site and promoter-like consensus sequences were identified upstream from the putative ATG initiation codon. The molecular and immunological properties of the sialidase expressed by E. coli are similar to those of the sialidase as isolated from C. septicum. The newly synthesized protein is assumed to include a leader peptide of 26 amino acids. On sequence alignment, the sialidases from C. septicum, C. sordellii and C. perfringens show significant homologies. As in other bacterial sialidases, conserved amino acid sequences occur at four positions in the protein. Aside from the consensus sequences, only poor homology to other bacterial and viral sialidases was found. The consensus sequence could be identified even in other, non-sialidase proteins, indicating a common function or the evolutionary relatedness of these proteins.     

Evolutionary conservation of ribosomal protein mRNA sequences: application for expansion of corresponding cDNA and gene libraries

Cloned cDNAs, containing ribosomal protein sequences from mouse (five cDNAs) or Xenopus laevis (six cDNAs), were used to estimate the evolutionary conservation, from insects to mammals, of the corresponding mRNA sequences. Northern blot analysis reveals a variable degree of homology between these sequences in different eukaryotes. Thus, among the ribosomal protein cDNA clones utilized, some exhibit complete, others partial, and a few no interphyla cross-hybridization. Melting profile analysis was employed to quantitate this homology. It is proposed that for expansion of eukaryotic ribosomal cDNA and gene libraries, one can exploit the interspecies homology of the corresponding sequences. However, the diverse evolutionary conservation of individual ribosomal protein gene sequences should be taken into account.     

Amino acid sequences of three acyl-binding/lipid-transfer proteins from rape seedlings

The complete amino acid sequence of three acyl-binding/lipid-transfer proteins, AB/LTP I, AB/LTP II and AB/LTP III from germinated rape seeds were determined. AB/LTP I and AB/LTP II consist of 93 residues and the M_r was determined as 9408 by mass spectrometry and calculated as 9406.8 from the sequence. AB/LTP III consists of 92 residues and the M_r was determined as 9424 by mass spectrometry and calculated as 9422.8 from the sequence. The primary structures were determined by automated Edman degradations of the intact proteins and peptides obtained from digestion with trypsin and endoproteinase Asp-N and cyanogen bromide cleavage. Use of ²⁵²Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides.     

Amino acid sequences of transport peptides associated with canine exocrine pancreatic proteins

Using microsequencing techniques and proteins labeled in vitro with tritiated amino acids we have obtained the following NH2-terminal sequences for six canine pancreatic presecretory proteins: pretrypsinogen 1, pretrypsinogen 2+3, prechymotrypsinogen 2, preproelastase1, preporcarboxypeptidase A1, preamylase. Points of cleavage by the transport peptidase, indicated by the vertical arrows, were located from sequences of authentic products synthesized in the presence of membranes of the rough endoplasmic reticulum. All of the identified residues in the pancreatic transport peptides are hydrophobic. Predictions of secondary structure were calculated for each of the transport peptides. The data indicated neither a common primary of secondary structure which could be interpreted as the signal for functional binding of the nascent presecretory protein to the rough endoplasmic reticulum membrane. These findings suggest that the initial interaction with the membrane or membrane receptor may depend in part, on the hydrophobic nature of the transport peptides. Five of the presecretory proteins showed a region with a high probability of forming a beta-turn immediately following the cleavage point. This feature may give the nascent peptide a region of flexibility that would facilitate both its insertion as a loop structure into the membrane and its cleavage by the transport peptidase. The sequences of authentic secretory products derived from a variety of pancreatic tissues suggest that hydrophilic residues are required immediately following the cleavage point in order to allow translocation of the nascent polypeptide chains across the membrane.     

Amino acid sequences of two nonspecific lipid-transfer proteins from germinated castor bean

The amino acid sequence of two nonspecific lipid-transfer proteins (nsLTP) B and C from germinated castor bean seeds have been determined. Both the proteins consist of 92 residues, as for nsLTP previously reported, and their calculated Mr values are 9847 and 9593 for nsLTP-B and nsLTP-C, respectively. The sequences of nsLTP-B and nsLTP-C, compared to the known sequence of nsLTP-A from the same source, are 68% and 35% similar, respectively. No variation was found at the positions of the cysteine residues, indicating that they might be involved in disulfide bridges.     

Amino acid sequences of several Bacillus subtilis proteins modified by apparent guanylylation

Bacillus subtilis cell extracts, prepared at different times during growth, contained several proteins that were apparently guanylylated in vitro with [alpha-32P]-GTP. Four of the proteins were partially purified and the N-terminal amino acid sequences (13 to 20 residues) were determined. One sequence had 84% identity to Bacillus stearothermophilus triosephosphate isomerase, two were 100% identical to the predicted sequences of the B. subtilis ptsI and ptsH genes while no identity was found for the fourth sequence. This apparent guanylylation occurred with proteins involved in glucose metabolism, although the significance is unknown.     

Amino acid sequences and phosphorylation sites of emu and rhea eggshell C-type lectin-like proteins

Avian calcified eggshell layers contain in their organic matrix one or two C-type lectin-like proteins. Previously characterized eggshell proteins of this family are chicken ovocleidin-17 (OC-17), goose ansocalcin and ostrich struthiocalcins 1 and 2 (SCA-1, SCA-2). In this report we present the amino acid sequences of two emu (Dromaius novaehollandiae) (dromaiocalcin-1 and -2; DCA-1, DCA-2) and of two rhea (Rhea americana) (rheacalcin-1 and -2; RCA-1, RCA-2) C-type lectin-like eggshell proteins, thus doubling the data set for comparison of these major specific eggshell proteins. The ratite proteins can be divided into two groups. Group 1, comprising SCA-1, DCA-1 and RCA-1, shows by 70--77% identity of sequences, the lack of phosphorylation, and a variable number (7--9) of cysteines. Group 2, consisting of SCA-2, DCA-2 and RCA-2, shows 78--85% identical sequences, 2--3 phosphorylated serines located at almost identical sites, and contains only the common set of six conserved cysteins characteristic for this family of proteins. While goose ansocalcin fits perfectly into group 1 with a sequence identity of 63--70% to the other members, no phosphorylation, and seven cysteines, chicken OC-17 was assigned to group 2 in spite of only 42--47% sequence identity (and 37--39% to group 1) because of its two phosphorylated serines and its regular set of six cysteines. At present it remains unknown why ratites, but not goose or chicken, require two different types of C-type lectin-like proteins to construct their eggshells.     

小鲵科线粒体16S rRNA基因序列分析及其系统发育

To study the phylogeny of Hynobiidae, we amplified DNA fragments of 470 bp 16S ribosomal RNA (16S rRNA) gene on mitochondrial DNA from Ranodon sibiricus and Ranodon tsinpaensis. PCR products were cloned into PMD18 T vector after purification. These sequences were determined and deposited in the GenBank (accession numbers: AY373459 for Ranodon sibiricus, AY372534 for Ranodon tsinpaensis). By comparing the nucleotide differences of 16S ribosomal RNA sequences among Liua shihi, Pseudohynobius flavomaculatus and Batrachuperus genus from GenBank database, we analyzed the divergences and base substitution among these sequences with the MEGA software. The molecular results support that B. tibetanus, B. pinchonii and B. karlschmidti are classified into three valid species. Liua shihi has closer phylogenetic relationships to Ranodon tsinpaensis than to other species. More our results reveal that Pseudohynobius flavomaculatus is not a synonym of Ranodon tsinpaensis. [Acta Zoologica Sinica 50 (3) : 464 - 469,2004].