Molecular cloning and nucleotide sequence of cDNAs encoding the precursors of rat long chain acyl-coenzyme A, short chain acyl-coenzyme A, and isovaleryl-coenzyme A dehydrogenases. Sequence homology of four enzymes of the acyl-CoA dehydrogenase family

cDNAs encoding the entire coding regions of the precursors (p) of rat long chain acyl-CoA (LCAD), short chain acyl-CoA (SCAD) and isovaleryl-CoA dehydrogenase (IVD) have been cloned and sequenced. Three cDNAs for rat liver LCAD together cover a 1440-base pair region. These cDNAs encode the entire 430-amino acid sequence of pLCAD, including the 30-amino acid leader peptide and the 400-amino acid mature LCAD. A single 1773 base pair cDNA for rat SCAD covers the entire coding region (414 amino acids), including the 26-amino acid leader peptide and the 388-amino acid mature peptide. Four identified IVD cDNAs, when combined, encompass a 2104 base region, and encode 424 amino acids including a 30-amino acid leader peptide and the 394-amino acid mature peptide. The identities of all cDNA clones have been confirmed by matching the amino acid sequences predicted from the respective cDNAs to the amino-terminal and tryptic peptide sequences derived from the corresponding purified rat enzyme. Comparison of the sequences of four rat acyl-CoA dehydrogenases, including LCAD, MCAD, SCAD, and IVD, and two of their human counterparts (MCAD and SCAD) reveals a high degree of homology (57 invariant and 92 near invariant residues: 30.6-35.4% of identical residues in pairwise comparisons), suggesting that these enzymes belong to a gene family and have evolved from a common ancestral gene.     

Lack of biological activity of preproendothelin [110-130] in several endothelin assays

A 21 amino acid peptide containing the prepropendothelin sequence from amino acids 110 to 130 and two intrachain disulfide bonds was synthesized and tested for biological activity in the following endothelin assays: 1.) a competition binding assay using [125I]ET-1 and dog heart membranes, 2.) three RIA's using 125I-ET-1, -2 and -3 and the respective anti-ET rabbit antisera; and 3.) a contractile activity bioassay using hamster aortic rings. The synthetic peptide which has been referred to as the "endothelin-like" peptide occurs 36 amino acids C-terminal to endothelin in the prepro-protein sequence. It contains only 40% sequence homology to the three endothelin isoforms, but has the same sequence and cyclization pattern of cysteines at positions 1, 3, 11 and 15. Despite the overall similarity in secondary structure to the three isoforms of endothelin and sarafotoxin S6b, preproendothelin [110-130] had no activity in any of the assays when tested at concentrations of 10(-10)M to 10(-5)M.     

Primary structure of bovine endothelin ETB receptor and identification of signal peptidase and metal proteinase cleavage sites.

A cDNA clone corresponding to the entire coding region of the bovine ETB endothelin receptor mRNA was isolated from a lung cDNA library and sequenced. The cDNA encodes 441 amino acids: 26 constituting an NH2-terminal signal peptide and 415 constituting the mature receptor. The signal peptidase cleavage site was determined by direct amino acid sequencing of purified receptor. A comparison of the predicted amino acid sequence with the available bovine ETA and rat ETB endothelin receptor sequences revealed 63 and 85% homology, respectively. Endothelin receptors of various species are known to be very sensitive to a certain metal proteinase(s) and have been shown to be converted to a lower Mr form in the absence of EDTA. The metal proteinase cleavage site was also determined by direct protein sequencing of the proteolysis product. The amino acid sequence (Ala-Gly-X-Pro-Pro-Arg) surrounding the cleavage site (between Ala-79 and Gly-80) is conserved among the ETB endothelin receptors, explaining the above mentioned proteolytic conversion from the higher to lower Mr forms observed in various species.     

Structure and analysis of the bovine atrial natriuretic peptide precursor gene

The isolation and sequence analysis of the gene encoding the bovine atrial natriuretic peptide (ANP) precursor is described. The bovine-ANP coding sequences are located on three exons which are interrupted by two intervening sequences. Comparison of the bovine, human, rat and mouse ANP gene sequences reveals a common organization of introns and exons and a high degree of sequence homology in the 5'-flanking and coding regions. Examination of the pre-proANP amino acid sequence derived from the bovine gene with those from rat, mouse and human, indicates a high degree of sequence homology in both the amino-terminal and biologically-active carboxy-terminal ANP region. The latter region in the bovine sequence resembles its human counterpart except for a carboxy-terminal Arg-Arg dipeptide.     

甘薯贮藏蛋白(sporam in)A基因编码区克隆及序列同源性分析

采用 PCR技术 ,从我国广泛栽培甘薯品种南薯 88基因组中扩增和克隆到甘薯贮藏蛋白 A基因编码区段 ,并测定了其全部核苷酸序列 .该编码区长 65 7bp,编码一个长 2 1 9个氨基酸残基的蛋白质 ,其中信号肽长 37个氨基酸残基 ,成熟蛋白质长 1 82个氨基酸残基 ,其分子量为 2 0 k D.将该片段的核苷酸序列与已登录在 Gen Bank中的另外 6个甘薯贮藏蛋白 A基因编码区序列进行比较 ,发现其同源性高达 90 % ,说明甘薯贮藏蛋白 A基因编码区序列具有高度保守性 .虽然 7个基因编码区的核苷酸总变异为 1 0 % ,但在每两个基因之间的比较则表明其核苷酸的变异范围小于 7% .     

Comparative studies of the amino acid and nucleotide sequences of pilin derived from Pseudomonas aeruginosa PAK and PAO.

The entire amino acid sequence for Pseudomonas aeruginosa PAO pilin was determined through peptide sequencing and from the complete nucleotide sequence encoding the pilin gene. The precursor PAO pilin is 149 amino acids in length which includes a 6-amino-acid positively charged leader sequence. Comparison of the amino acid sequences of pilin produced by P. aeruginosa PAO and PAK reveals a region of high homology corresponding to the leader peptide and residues 1 to 54 of the mature pilin. The amino acid sequence of the peptide encompassing the major antigenic determinant of PAK differs greatly from that of the equivalent region in PAO. The C-terminal regions of these proteins are semiconserved. Few major differences were found when the predicted secondary structures for PAO and PAK pilins were compared. Major nucleotide sequence variation between the equivalent restriction fragments from PAO and PAK occurred within the areas coding for the peptides containing the immunodominant site for PAK pilin and the C termini.     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH2-terminal amino acid sequence analysis of the G6-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102,598. Identity of the NH2-terminal amino acid sequences among each component of the multiform G6-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G6-amylase gene showed no homology with those of other bacterial alpha-amylases although the consensus amino acid sequences of the active center were well conserved.     

Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae)

We previously purified and determined the partial amino acid sequence of a 4 kDa peptide having high homology with scorpion defensin from the hemolymph of adult fed female soft ticks, Ornithodoros moubata. In this study, the full length sequences of two defensin isoforms were obtained. Deduced amino acid sequences reveal a precursor protein of 73 amino acid residues with a mature portion consisting of 37 amino acid residues. This mature peptide contains six cysteine residues conserved in the same location as other invertebrate defensins. Phylogenetic analysis reveals that Ornithodoros defensin is most closely related to scorpion defensin and other more ancient arthropods. Ornithodoros defensin mRNA is constitutively expressed and up-regulated by blood-feeding and bacterial injection. Ornithodoros defensin gene expression occurs mainly in the midgut. This is the first report of the cloning and gene expression of an antibacterial peptide from the Acari.     

特异种质烟草HZNH的Fe-SOD基因的克隆与表达

超氧化物歧化酶(superoxidedismutase,SOD)是一种广泛存在于动物、植物、微生物体内的金属酶,按其结合的金属性离子可分为Fe SOD、Mn SOD和CuZn SOD三种,它们通过催化超氧阴离子自由基O·-2发生歧化反应,达到清除O·-2的效果,具有防御氧毒性、增强机体抗辐射损伤能力、防衰老,治疗某些肿瘤、炎症、自身免疫疾病等功效,在农业、医药、食品、化工等产业中的应用前景广阔,因此广受国内外科研工作者的关注和重视[1].而试图通过转SOD基因技术来培育高抗逆农作物新品种和基因克隆与表达技术来实现SOD的大规模发酵生产,已成为国内外SOD…     

Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component

Complementary and genomic DNA clones corresponding to the human C-reactive protein (CRP) mRNA and structural gene have been analyzed and compared. Nucleotide sequencing of the coding regions of both cDNA and genomic DNA revealed an additional 19 amino acid peptide not described in the published CRP amino acid sequence. The CRP gene contains a single 278 base pair intron within the codon specifying the third residue of mature CRP. The intron contains a repetitive sequence (GT)15G(GT)3 which is similar to structures capable of adopting the Z-DNA form. A comparison of CRP coding and amino acid sequences with those of serum amyloid P component revealed striking overall homology which was not uniform: a region of limited conservation is bounded by two highly conserved regions.     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G₆-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH₂-terminal amino acid sequence analysis of the G₆-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102598. Identity of the NH₂-terminal amino acid sequences among each component of the multiform G₆-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G₆-amylase gene showed no homology with those of other bacterial α-amylases although the consensus amino acid sequences of the active center were well conserved.     

Nucleotide sequence encoding the precursor of the small subunit of ribulose 1,5-bisphosphate carboxylase from Lemna gibba L.G-3

We have sequenced a cDNA clone, pLgSSU, which encodes the small subunit of ribulose 1,5-bisphosphate carboxylase of Lemna gibba L.G-3 a monocot plant. This clone contains a 832 basepair insert which encodes the entire 120 amino acids of the mature small subunit polypeptide (Mr = 14,127). In addition this clone encodes 53 amino acids of the amino terminal transit peptide of the precursor polypeptide and 242 nucleotides of the 3' non-coding region. Comparison of the nucleotide sequence of pLgSSU with Lemna gibba genomic sequences homologous to the 5' end of the cDNA clone suggests that nucleotides encoding four amino-terminal amino acids of the transit peptide are not included in the cDNA clone. The deduced amino acid sequence of the Lemna gibba mature small subunit polypeptide shows 70-75% homology to the reported sequences of other species. The transit peptide amino acid sequence shows less homology to other species. There is 50% homology to the reported soybean sequence and only 25% homology to the transit sequence of another monocot, wheat.     

Studies on the disulfide bridges of sarafotoxins. Chemical synthesis of sarafotoxin S6B and its homologue with different disulfide bridges

Sarafotoxin S6b, a strong vasocontractile peptide with 21 amino acid residues containing two sets of disulfide bridges, was chemically synthesized. The retention time on a reversed-phase HPLC, lethal and vasocontractile activities of natural sarafotoxin S6b agree with those of the synthetic [Cys1-15, Cys3-11]-sarafotoxin S6b. The combination of the disulfide bridges of sarafotoxin S6b is the same as that of endothelin-1, a mammalian vasocontractile peptide, which shows a high degree of sequence homology and shares several common pharmacological properties with sarafotoxins.     

Nucleotide sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene: comparison with the B. amyloliquefaciens gene.

The DNA sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene is reported. Comparison of the inferred amino acid sequence of the B. licheniformis alpha-amylase gene with that of the Bacillus amyloliquefaciens gene shows that whereas the amino acid sequences of the mature proteins have considerable homology, the sequences for the signal peptides are distinct.     

Nucleotide sequence and characterization of the gene for secreted alkaline phosphatase from Lysobacter enzymogenes.

Lysobacter enzymogenes produces an alkaline phosphatase which is secreted into the medium. The gene for the enzyme (phoA) was isolated from a recombinant lambda library. It was identified within a 4.4-kb EcoRI-BamH1 fragment, and its sequence was determined by the chain termination method. The structural gene consists of an open reading frame which encodes a 539-amino-acid protein with a 29-residue signal sequence, followed by a 119-residue propeptide, the 281-residue mature phosphatase, and a 110-residue carboxy-terminal domain. The roles of the propeptide and the carboxy-terminal peptide remain to be determined. A molecular weight of 30,000 was determined for the mature enzyme from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence was compared with sequences available in the current protein data base, and a region of the sequence was found to show considerable homology with sequences in mammalian type 5 iron-containing purple acid phosphatases.     

Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.     

Cloning and Sequence Analysis of a Protease-encoding Gene from the Marine Bacterium Alteromonas sp. Strain O-7

The gene (aprI) encoding alkaline serine protease (AprI; subtilase) from Alteromonas sp. strain O-7 was cloned and sequenced. The nucleotide sequence of aprI has been identified. The deduced amino acid sequence indicated that aprI codes for a precursor of 715 amino acids and the precursor is composed of four regions including a signal peptide, an N-terminal pro-region, a mature protease region and a C-terminal extension region of 215 amino acids as previously described for aprII [H. Tsujibo et al., Gene, 136, 247–251 (1993)]. The amino acid sequence of the mature AprI (AprI-M) showed high sequence homology with those of other class I subtilases. The C-terminal region was characterized by a repeat of 94 amino acids residues, which showed about 50% similarity with those of the C-terminal pro-region of several known proteases from Gram-negative bacteria.