Escherichia coli glutaminyl-tRNA synthetase. II. Characterization of the glnS gene product

Glutaminyl-tRNA synthetase has been purified by a simple, two-column procedure from an Escherichia coli K12 strain carrying the glnS structural gene on plasmid pBR322. The primary sequence of this enzyme as derived from the DNA sequence (see accompanying paper) has been confirmed. Manual Edman degradation was used to identify the NH2-terminal sequence of the protein. Oligopeptides scattered throughout the primary sequence of glutaminyl-tRNA synthetase were sequenced by the gas chromatographic-mass spectrometric method and matched to the theoretical peptides derived from the translated DNA sequence. The expected carboxyl terminus at position 550 was verified by carboxypeptidase B digestion. The primary sequence of glutaminyl-tRNA synthetase contains no extensive sequence repeats. A search was made for sequence homologies between this enzyme and the few other aminoacyl-tRNA synthetases for which primary sequences are available. A single homologous region is shared by at least three of the synthetases examined here.     

Differences between EcoRI nonspecific and "star" sequence complexes revealed by osmotic stress

The binding of the restriction endonuclease EcoRI to DNA is exceptionally specific. Even a single basepair change ("star" sequence) from the recognition sequence, GAATTC, decreases the binding free energy of EcoRI to values nearly indistinguishable from nonspecific binding. The difference in the number of waters sequestered by the protein-DNA complexes of the "star" sequences TAATTC and CAATTC and by the specific sequence complex determined from the dependence of binding free energy on water activity is also practically indistinguishable at low osmotic pressures from the 110 water molecules sequestered by nonspecific sequence complexes. Novel measurements of the dissociation rates of noncognate sequence complexes and competition equilibrium show that sequestered water can be removed from "star" sequence complexes by high osmotic pressure, but not from a nonspecific complex. By 5 Osm, the TAATTC "star" sequence complex has lost almost 90 of the approximately 110 waters initially present. It is more difficult to remove water from the CAATTC "star" sequence complex. The sequence dependence of water loss correlates with the known sequence dependence of "star" cleavage activity.     

The amino acid sequence of porcine intestinal calcium-binding protein.

The complete amino acid sequence of the calcium-binding protein (CaBP) from pig intestinal mucosa has been determined: Ac-Ser-Ala-Gln-Lys-Ser-Pro-Ala-Glu-Leu-Lys-Ser-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Gln-Leu-Ile-Gln-Ala-Glu-Phe-Pro-Ser-Leu-Leu-Lys-Gly-Pro-Arg-Thr-Leu-Asp-Asp-Leu-Phe-Gln-Glu-Leu-Asp-Lys-Asn-Gly-Asn-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. The N-terminal octapeptide sequence was determined by mass spectrometric analysis by Morris and Dell. The first 45 residues of bovine CaBP differ only in six positions from the corresponding sequence of the porcine protein, except that the sequence starts in position two of the porcine sequence. The mammalian intestinal CaBP's belong to the troponin-C superfamily on the basis of an analysis by Barker and Dayhoff.     

Cloned DNA sequences that determine mRNA stability of bacteriophage phi X174 in vivo are functional.

The stability of two species of phi X174 polycistronic mRNA in vivo can be altered by mutating sequences existing immediately upstream of a termination site. The wild type phage contains an mRNA stabilizing sequence ((+) sequence), while the same sequence mutated by insertion ((-) sequence) reduces the stability of the mRNAs. These two sequences were cloned at the 3' ends of gene D or gene B of phi X174 in a pBR322 derivative plasmid. The cloned sequences were functional. The (+) sequence stabilized gene B or gene D mRNA; half-lives of these mRNAs were 7 to 8 min. When the (+) sequence is eliminated ((o) sequence) or replaced with the (-) sequence, the half-lives of the mRNA were reduced to about 1 to 2 min. The stabilization of mRNAs caused an increased production of these proteins.     

The Short-chain Dehydrogenase/Reductase Engineering Database (SDRED): A classification and analysis system for a highly diverse enzyme family

The Short-chain Dehydrogenases/Reductases Engineering Database (SDRED) covers one of the largest known protein families (168 150 proteins). Assignment to the superfamilies of Classical and Extended SDRs was achieved by global sequence similarity and by identification of family-specific sequence motifs. Two standard numbering schemes were established for Classical and Extended SDRs that allow for the determination of conserved amino acid residues, such as cofactor specificity determining positions or superfamily specific sequence motifs. The comprehensive sequence dataset of the SDRED facilitates the refinement of family-specific sequence motifs. The glycine-rich motifs for Classical and Extended SDRs were refined to improve the precision of superfamily classification. In each superfamily, the majority of sequences formed a tightly connected sequence network and belonged to a large homologous family. Despite their different sequence motifs and their different sequence length, the two sequence networks of Classical and Extended SDRs are not separate, but connected by edges at a threshold of 40% sequence similarity, indicating that all SDRs belong to a large, connected network. The SDRED is accessible at https://sdred.biocatnet.de/.     

Some KpnI family members are associated with the Alu family in the human genome

The structures of the termini and their flanking regions of two human KpnI family members were investigated. The two differed in length, but the starting sequence at one terminal (defined as the 5' terminal) was found to be common to both members. The Alu family sequence was found in the 5' flanking regions. The KpnI family sequence started several base-pairs downstream from the 3' end of the Alu family sequence. In both cases, the Alu family sequence was not flanked by the direct repeat sequence common to the Alu family. These two members showed no sequence homology in 3' terminal regions. Interestingly, the Alu family plus the KpnI family unit was found to be flanked by a direct repeat sequence of several base-pair length. Based on these findings, relationship between the Alu family and KpnI family is discussed.     

Amino acid sequence of human von Willebrand factor

The complete amino acid sequence of human von Willebrand factor (vWF) is presented. Most of the sequence was determined by analysis of the S-carboxymethylated protein. Some overlaps not provided by the protein sequence analysis were obtained from the sequence predicted by the nucleotide sequence of a cDNA clone [Sadler, J.E., Shelton-Inloes, B.B., Sorace, J., Harlan, M., Titani, K., & Davie, E.W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6391-6398]. The protein is composed of 2050 amino acid residues containing 12 Asn-linked and 10 Thr/Ser-linked oligosaccharide chains. One of the carbohydrate chains is linked to an Asn residue in the sequence Asn-Ser-Cys rather than the usual Asn-X-Ser/Thr sequence. The sequence of von Willebrand factor includes several regions bearing evidence of internal gene duplication of ancestral sequences. The protein also contains the tetrapeptide sequence Arg-Gly-Asp-Ser (at residues 1744-1747), which may be a cell attachment site, as in fibronectin. The amino- and carboxyl-terminal regions of the molecule contain clusters of half-cystinyl residues. The sequence is unique except for some homology to human complement factor B.     

The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67.

The amino acid sequence of a trimethoprim-resistant dihydrofolate reductase (EC 1.5.1.3) specified by the R-plasmid R67 is described. The sequence was deduced from automatic and manual sequence analysis of the intact protein, the fragments produced by cyanogen bromide cleavage, and peptides derived from the largest cyanogen bromide fragment by digestion with trypsin, Staphylococcus aureus V8 proteus, chymotrypsin, and Lysobacter enzymogenes alpha-lytic protease. The complete sequence comprises 78 residues in a single polypeptide chain of molecular weight 8444. No evidence of heterogeneity was obtained, indicating that all subunits of the native enzyme are identical. Comparison of the sequence with that of all known dihydrofolate reductases shows no significant sequence homology.     

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Sequences of the cyanogen bromide peptides and complete sequences of the enzyme.

The complete amino acid sequence of dihydrofolate reductase from an amethopterin-resistant strain of Lactobacillus casei has been determined by sequence analysis of peptides produced by cleavage with cyanogen bromide, trypsin, staphylococcal protease, and myxobacter protease. Comparison of this sequence with those of reductases from other bacterial sources shows that the enzymes are homologous. The Lactobacillus casei reductase sequences shows a 29% sequence identity with that of the Escherichia coli enzyme and a 34% identity with the sequence of the enzyme from Streptococcus faecium. The NH2-terminal 68 residues of the L. casei reductase show a 54% sequence identity with that of the enzyme from S. faecium.     

Sequence of an expressed human beta-tubulin gene containing ten Alu family members

The complete sequence of a functionally expressed human beta-tubulin gene (5 beta) is presented. The amino acid sequence encoded by this gene constitutes a distinct isotype, differing from a previously described human beta-tubulin sequence at 21 positions throughout the polypeptide chain. The beta-tubulin coding sequence in 5 beta is interrupted by three intervening sequences of 1014, 117 and 4826 nucleotides. The largest of these contains ten members of the Alu family of middle repetitive sequences. Together, these regions account for sixty percent of this intervening sequence. Two of the Alu elements are juxtaposed head to tail, and share the same flanking direct repeat. The ten Alu sequences are substantially divergent, both from each other and from an Alu consensus sequence, and several contain deletions of up to half the entire sequence.     

The amino acid sequence of plastocyanin from Cucurbita pepo L. (vegetable marrow)

The amino acid sequence of plastocyanin from marrow was determined. It consists of a single polypeptide chain of mol.wt. 10284 containing 99 amino acid residues. The sequence was determined by using a Beckman 890C automatic sequencer and by dansyl–phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. The sequence is in good agreement with the amino acid composition, except that fewer residues of glutamic acid were found in the sequence than were suggested by the composition. Evidence for histidine-37 was weaker than for the rest of the sequence. A `tree' of phylogenetic affinities was constructed by using several higher-plant plastocyanin sequences.     

Cloning and sequencing of the gene encoding cytochrome c553 from Desulfovibrio vulgaris Hildenborough.

The gene encoding cytochrome c553 from Desulfovibrio vulgaris Hildenborough was cloned by using two synthetic deoxyoligonucleotide probes. The amino acid sequence derived from the sequence of the gene differs from that reported by Bruschi and LeGall (Biochim. Biophys. Acta 271:48-60, 1972). Renewed protein sequencing confirmed the correctness of the DNA-derived sequence. The gene sequence indicates cytochrome c553 to be synthesized as a precursor protein with an NH2-terminal signal sequence of 24 residues.     

Molecular cloning and sequence analysis of the ribosomal ‘A’ protein gene from the archaebacterium,Halobacterium halobium

The ribosomal ‘A’ protein gene of Halobacterium halobium has been cloned and the nucleotide sequence of the DNA fragment containing the ‘A’ protein gene has been determined. The amino-acid sequence of the protein deduced from the nucleotide sequence was established from manual sequence analysis of the protein and structural data provided by peptides derived from cleavage of the protein with various proteinases. The ‘A’ protein consisted of 114 amino acids with a molecular weight of 11562 and was characterized mainly by a high amounts of alanine and acidic amino acid in the C-terminal half of the molecule. The coding sequence of the gene was preceded by a predicted Shine-Dalgarno sequence and two terminal codons. There was no intron or insertion sequence in the coding sequence. Following the terminal codon of the ‘A’ gene, there was a structure reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 71%. Inspection of the codon usage for the ‘A’ gene revealed 85% preference for G or C at the third codon position.     

Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom.

The complete amino acid sequence of fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom, has been determined. This is the first report of the sequence of a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom. The majority of the sequence was established by automated Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. The amino-terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue, and the sequence of this region of the molecule was determined by mass spectrometry. Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891, as determined by the sequence. Its sequence is homologous to the sequence of the hemorrhagic toxin Ht-d of Crotalus atrox venom and with the sequences of two metalloproteinases from Trimeresurus flavoviridis venom. Microheterogeneity in the sequence was found at both the amino-terminus and at residues 189 and 192. All six cysteine residues in fibrolase are involved in disulfide bonds. A disulfide bond between cysteine-118 and cysteine-198 has been established and bonds between cysteines-158/165 and between cysteines-160/192 are inferred from the homology to Ht-d. Secondary structure prediction reveals a very low percentage of alpha-helix (4%), but much greater beta-structure (39.5%). Analysis of the sequence reveals the absence of asparagine-linked glycosylation sites defined by the consensus sequence: asparagine-X-serine/threonine.     

阴道毛滴虫氢化酶体腺苷酸激酶基因的克隆及序列分析

目的-克隆阴道毛滴虫氢化酶体腺苷酸激酶(AK)基因,并测定其序列,进行序列分析。方法-根据AK基因已知序列设计合成一对引物,应用PCR技术从阴道毛滴虫基因组DNA中扩增出AK基因,并将其克隆入pMD18-T simple载体。阳性克隆的重组质粒经酶切及PCR鉴定后,用双脱氧链末端终止法进行基因序列测定。应用BLAST软件辅助分析所测基因与Genbank中阴道毛滴虫氢化酶体AK序列的同源性。结果-PCR扩增得到特异的阴道毛滴虫氢化酶体腺苷酸激酶基因序列。酶切及PCR鉴定获得了正确的PT-AK重组质粒。测序表明,所克隆的AK基因大小为690bp,编码229个氨基酸。序列分析表明,所测基因与Genbank中阴道毛滴虫氢化酶体AK序列具有高度同源性(99．9％)。结论-克隆了阴道毛滴虫氢化酶体腺苷酸激酶基因,序列测定及同源性分析表明,所测基因与Genbank中阴道毛滴虫氢化酶体AK序列具有高度同源性。     

羊驼性别决定基因sry部分片段的克隆与序列分析

从成年羊驼血液提取基因组DNA,参照哺乳动物sry(sex-determining region on the Y chromosome)基因的同源保守区域设计特异性引物,用PCR技术成功扩增羊驼sry基因的部分片段,且全部实验雄性个体均成功扩增,而雌性个体则无任何特异性片段,说明所扩增基因片段具雄性特异性。对扩增序列所编码蛋白序列分析显示所扩增的片段编码的蛋白序列在哺乳动物sryHMG-box(high mobility group box)蛋白超家族的HMG-box区域,说明扩增的为sry基因片段。用所扩增片段与其他哺乳动物同源序列分析显示,由sry基因构建的系统进化树,与传统的动物分类关系相近,说明由sry基因的HMG-box构建系统树是分析物种亲缘关系的有效工具。     

The primary structure of histone H3 from wheat

Wheat embryo histone H3 has been isolated and purified and the elucidation of the complete amino-acid sequence is described. Peptides were generated by cleavages with CNBr, S. aureus V8 proteinase, endoproteinase Lys-C and trypsin. The peptides were purified by HPLC and the sequence determined by solid-state and gas-phase sequencing methodology. The amino-acid sequence of the protein is identical to pea embryo histone H3 and the sequence deduced from the nucleotide sequence of a wheat embryo histone gene (Tabata T. et al. (1984) Mol. Gen. Genet. 196, 397-400).