The nucleotide sequence of the gene encoding the K99 subunit of enterotoxigenic Escherichia coli

Abstract The nucleotide sequence of the gene encoding the K99 fimbrial subunit of enterotoxigenic Escherichia coli was determined. It appeared that the subunit is composed of 159 amino acid residues preceded by a N-terminal signal sequence of 22 amino acid residues. The secondary structure of the mature K99 polypeptide and the location of potential antigenic determinants were predicted. A comparison was made between the amino acid sequence of the K99 subunit and the subunits of other fimbrial adhesins.     

Complete amino acid sequence of jack bean urease

The subunit structure of jack bean urease has been unresolved in spite of many investigations. Thus far, the molecular weight for the native urease seem to range from 480,000 to 590,000 and the values for the monomer range from 30,000 to 97,000. The complete amino acid sequence of jack bean urease has been determined primarily by sequencing cyanogen bromide peptides, which were aligned by overlapping peptides obtained by lysylendopeptidase digestion of the protein and tryptic digestion of the citraconylated protein. The protein contains 840 amino acid residues in a single polypeptide chain and the subunit molecular weight calculated from the sequence is 90,790. The value of 544,740 for the hexamer, consistent with the value of 580,000 determined for intact urease by centrifugal analyses, indicated that urease consists of six subunits. Thirteen of 25 histidine residues in the urease subunit are crowded in the region between residues 479 and 607. Urease is a nickel metalloenzyme and the nickel has an essential role in catalysis by this enzyme. It is noteworthy that cysteine-592, which is recognized as essential for enzymatic activity and is related to the nickel ion in the active center, is located on this histidine-rich sequence.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

The amino-terminal sequence of the catalytic subunit of bovine enterokinase

Bovine enterokinase (enteropeptidase) is a serine protease and functions as the physiological activator of trypsinogen. The enzyme has a heavy chain (115 kD) covalently linked to a light or catalytic subunit (35 kD). The amino acid composition showed that the light chain has nine half-cystine residues (four as intramolecular disulfides) and that one half-cystine was in a disulfide link between the light and heavy subunits. The amino-terminal 27 residues of the S-vinylpyridyl derivative of the light chain were determined by gas-phase Edman degradation. The sequence has homologies with other serine proteases containing one or two chains. The homologies suggest that the catalytic subunit has the same three-dimensional structure and, therefore, the same mechanism of enzymatic action as pancreatic chymotrypsin, trypsin, and elastase. The presence of the conserved amino-terminal activation peptide sequence (IVGG) shows that enterokinase must have a zymogen precursor and that the two-chain enzyme arises from limited proteolysis during posttranslational processing.     

类大麦属高分子量谷蛋白基因Kx的序列测定及表达

利用SDS-PAGE检测了2份类大麦属(Crithopsis delileana)材料的高分子量谷蛋白亚基组成,并对其中1份材料的x型亚基进行了克隆和测序。结果表明,2份材料具有完全相同的蛋白电泳图谱。在小麦的高分子量区域仅检测到一条蛋白质带,与小麦y型亚基的迁移率接近,但克隆测序表明其为x型高分子量谷蛋白亚基,其编码基因命名为Kx。Kx基因编码区序列长度为2052bp．编码长度为661个氨基酸残基的蛋白质,其序列具有典型的x型高分子量谷蛋白亚基的特征。Kx基因能在原核表达系统内正确表达,其表达蛋白与来源于种子中的Kx亚基的迁移率完全一致。Kx亚基与小麦属A、B和D,山羊草属C和U以及黑麦属R染色体组编码的高分子量谷蛋白亚基氨基酸序列非常相似,但在N和C保守区的氨基酸组成以及重复区长度上与它们存在明显差异。聚类分析可将Kx与Ax1聚类为平行的分支。由此可见,来源于C．delileana的Kx基因为一新的x型高分子量谷蛋白亚基基因。     

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus

Summary The structural genes (hup) of the H₂ uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization with the structural genes of the H₂ uptake hydrogenase of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup^-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68108 dalton) and by alignment with the NH₂ amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34 256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology, but to a lesser extent, with the hydrogenase of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues, (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe–4S] ferredoxins.     

Characterization of two HMW glutenin subunit genes from Taenitherum Nevski

The compositions of high molecular weight (HMW) glutenin subunits from three species of Taenitherum Nevski (TaTa, 2n = 2x = 14), Ta. caput-medusae, Ta. crinitum and Ta. asperum, were investigated by SDS-PAGE analysis. The electrophoresis mobility of the x-type HMW glutenin subunits were slower or equal to that of wheat HMW glutenin subunit Dx2, and the electrophoresis mobility of the y-type subunits were faster than that of wheat HMW glutenin subunit Dy12. Two HMW glutenin genes, designated as Tax and Tay, were isolated from Ta. crinitum, and their complete nucleotide coding sequences were determined. Sequencing and multiple sequences alignment suggested that the HMW glutenin subunits derived from Ta. crinitum had the similar structures to the HMW glutenin subunits from wheat and related species with a signal peptide, and N- and C-conservative domains flanking by a repetitive domain consisted of the repeated short peptide motifs. However, the encoding sequences of Tax and Tay had some novel modification compared with the HMW glutenin genes reported so far: (1) A short peptide with the consensus sequences of KGGSFYP, which was observed in the N-terminal of all known HMW glutenin genes, was absent in Tax; (2) There is a specified short peptide tandem of tripeptide, hexapeptide and nonapeptide and three tandem of tripeptide in the repetitive domain of Tax; (3) The amino acid residues number is 105 (an extra Q presented) but not 104 in the N-terminal of Tay, which was similar to most of y-type HMW glutenin genes from Elytrigia elongata and Crithopsis delileana. Phylogenetic analysis indicated that Tax subunit was mostly related to Ax1, Cx, Ux and Dx5, and Tay was more related to Ay, Cy and Ry.     

Primary structure of the reaction center from Rhodopseudomonas sphaeroides

The reaction center is a pigment-protein complex that mediates the initial photochemical steps of photosynthesis. The amino-terminal sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single-stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that the H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly biased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch of hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was constructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and the D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.     

The primary structure of a cDNA for PsaN,encoding an extrinsic lumenal polypeptide of barley photosystem I

A cDNA clone encoding a 15.501 Da photosystem I (PSI) subunit of barley was isolated using an oligonucleotide based on the NH₂-terminal amino acid sequence of the isolated protein. The polypeptide, which migrates with an apparent molecular mass of 9.5 kDa on denaturing SDS-PAGE, has been designated PSI-N, and the corresponding gene is PsaN. Analysis of the deduced protein sequence indicates a mature protein of 85 amino acid residues and a molecular mass of 9818 Da. PSI-N is a hydrophilic, extrinsic protein with no predicted membrane-spanning regions. The transit peptide of 60 residues (5683 Da) contains a predicted hydrophobic -helix, suggesting that the protein is routed into the thylakoid lumen. Thus, PSI-N is the second known lumenal protein component associated with PSI, together with PSI-F.     

Comparative analysis of the primary structure of the reaction center-bound cytochrome subunit in purple bacteria

The amino acid sequences of the reaction center-bound cytochrome subunit of six species of purple bacteria were compared. Amino acid residues thought to be important in controlling the redox midpoint potentials of four hemes in Blastochloris (Rhodopseudomonas) viridis were found to be well conserved. As opposed to all other species studied, the amino acid sequence of the cytochrome subunit of B. viridis had several insertions of more than 10 residues at specific regions close to the LM core, suggesting that interaction of the cytochrome subunit with the LM core in most species is different from that in B. viridis. Distribution of charged amino acid residues on the surface of the cytochrome subunit was compared among six species and discussed from the viewpoint of interaction with soluble electron donors.     

The amino acid sequence of the rabbit glycoprotein hormone alpha subunit

The amino acid sequence of the alpha subunit of rabbit (lagomorph) lutropin (lLH) has been determined. Overlapping peptides from trypsin and chymotrypsin digestions were isolated by reverse-phase high-pressure liquid chromatography (HPLC). Sequencing was by the dansyl-Edman procedure. Amide placements were established by HPLC analysis of the PTH amino acid derivatives. The proposed sequence of lLH alpha subunit is (asterisks denote carbohydrate attachment sites): This proposed sequence is highly homologous with the porcine, murine, ovine, and bovine glycoprotein hormone alpha subunit sequences. Two unusual proteolytic cleavages were observed: (1) a cleavage by trypsin between Asn-77 and Ala-78, and (2) a cleavage by chymotrypsin between Ala-45 and Arg-46. Similar enzymatic cleavages were previously reported for equine chorionic gonadotropin alpha subunit by Wardet al. and for these sites in the ovine LH alpha subunit by Liuet al. Chymotrypsin cleaved on the carboxyl side of methionine sulfone residues at positions 51 and 75.     

Energy-transducing nicotinamide nucleotide transhydrogenase: Nucleotide sequences of the genes and predicted amino acid sequences of the subunits of the enzyme fromRhodospirillum rubrum

Based on the amino acid sequence of the N-terminus of the soluble subunit of theRhodospirillum rubrum nicotinamide nucleotide transhydrogenase, two oligonucleotide primers were synthesized and used to amplify the corresponding DNA segment (110 base pairs) by the polymerase chain reaction. Using this PCR product as a probe, one clone with the insert of 6.4kbp was isolated from a genomic library ofR. rubrum and sequenced. This sequence contained three open reading frames, constituting the genesnntA1, nntA2, andnntB of theR. rubrum transhydrogenase operon. The polypeptides encoded by these genes were designated 1, 2, and , respectively, and are considered to be the subunits of theR. rubrum transhydrogenase. The predicted amino acid sequence of the 1 subunit (384 residues; molecular weight 40276) has considerable sequence similarity to the subunit of theEscherichia coli and the N-terminal 43-kDa segment of the bovine transhydrogenases. Like the latter, it has a fold in the corresponding region, and the purified, soluble 1 subunit cross-reacts with antibody to the bovine N-terminal 43-kDa fragment. The predicted amino acid sequence of the subunit of theR. rubrum transhydrogenase (464 residues; molecular weight 47808) has extensive sequence identity with the subunit of theE. coli and the corresponding C-terminal sequence of the bovine transhydrogenases. The chromatophores ofR. rubrum contain a 48-kDa polypeptide, which cross-reacts with antibody to the C-terminal 20-kDa fragment of the bovine transhydrogenase. The predicted amino acid sequence of the 2 subunit of theR. rubrum enzyme (139 residues; molecular weight 14888) has considerable sequence identity in its C-terminal half to the corresponding segments of the bovine and the subunit of theE. coli transhydrogenases.     

A structural model for human dihydrolipoamide dehydrogenase

The hypothesis that dihydrolipoamide dehydrogenases (E₃s) have tertiary structures very similar to that of human glutathione reductase (GR) was tested in detail by three separate criteria: (1) by analyzing each putative secondary structural element for conservation of appropriate polar/nonpolar regions, (2) by detailed comparison of putative active site residues in E₃s with their authentic counterparts in human GR, and (3) by comparison of residues at the putative dimeric interface of the E₃s with the authentic residues in GR. All three criteria are satisfied in a convincing way for the 7 E₃s that were considered, supporting the conclusion that the structural scaffolding and the overall tertiary structure (which determines the location of functional sites and residues) are remarkably similar for the E₃s and for GR. These analyses together with the crystal structures of human erythrocyte GR formed the basis for construction of a molecular model for human E₃. The cofactor FAD and the substrakes NAD and lipoic acid were also included in the model. Unexpectedly, the surface residues in the cleft that holds the lipoamide were found to be highly charged and predominantly acidic, allowing us to predict that the region around the lipoamide in the sub-unit should be basic in nature. The molecular model can be tested by site-directed mutagenesis of residues predicted to be in the dihydrolipoamide acetyltransferase subunit binding cleft. © 1992 Wiley-Liss, Inc.     

Thiolase mRNA translated in vitro yields a peptide with a putative N-terminal presequence

Thiolase is part of the fatty acid oxidation machinery which in plants is located within glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48 539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase.     

Single chain human chorionic gonadotropin,hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the α and β subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGαβ) was used to investigate roles of critical residues of the α subunit in hormone receptor interaction and biological activity. The α subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type β subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCGα P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCGα T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the α subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormone-receptor interactions.     

Sequencing and characterization of a cDNA encoding a ferritin subunit of Colorado potato beetle, Leptinotarsa decemlineata

A differentially expressed cDNA fragment (P311) from Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), was identified by restriction fragment differential display-polymerase chain reaction (RFDD-PCR) technique, and showed a strong similarity to ferritin heavy chain subunits of other organisms. Based on P311, we constructed specific primers and obtained a 840-bp cDNA fragment spanning the open reading frame of CPB ferritin subunit using the rapid amplification of cDNA ends (RACE) technique. The sequence encodes 213 amino acid residues, including a 19 amino acid signal peptide. The sequence has a conserved cysteine in the N-terminus and has the seven conserved residues that comprise the ferroxidase center, which is the feature of heavy chain ferritins of vertebrates. The CPB ferritin subunit has high amino acid sequence identity with the Apriona germari (69.3%), Galleria mellonela (54.5%), Manduca sexta (54.0%), Drosophila melanogaster (53.2%), Calpodes ethlius (51.4%), and Nilaparvata lugens (47.6%) but lower identity with the Anopheles gambiae (38.7%) and Aedes aegypti (37.8%). Using Northern blot analysis, the subunit mRNA was identified from fat body and midgut of 4th instars with much higher mRNA levels found in midgut than that in fat body (2.5-fold). Nevertheless, only the levels of mRNA in fat body was induced by dexamethasone (1.5-fold).     

Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase

Summary The Bacillus subtilis cdd gene encoding cytidine/2-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order rpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis 43 and 37 RNA polymerase holoenzymes during growth and stationary phase.Abbreviations SDS sodium dodecyl sulphate - Ap ampicillin resistance - Tet^r tetracycline resistance - Km^r kanamycin resistance     

Primary structure of a novel subunit in ba3-cytochrome oxidase from Thermus thermophilus

The bax-type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix "subunit IIa" spanning the membrane. The hydrophobic N-terminally blocked protein was isolated in high yield using high-performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C-terminal sequencing of the native protein. The molecular mass of 3,794 Da as determined by MALDI-MS and by ESI requires the N-terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5-Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3-cytochrome oxidase from Natronobacterium pharaonis. The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB).