Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis.

Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A. parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein. A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated. The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced. This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000. Direct sequencing of the purified mature enzyme from A. parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein. The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment).(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA interference of an antimicrobial peptide, gloverin, of the beet armyworm, Spodoptera exigua, enhances susceptibility to Bacillus thuringiensis

Gloverin is known to be an inducible antimicrobial peptide. This study reports a gloverin gene (Seglv) identified from the beet armyworm, Spodoptera exigua. Seglv encodes 175 amino acids with a signal peptide. Its amino acid sequence is highly homologous (>95%) to other known gloverins. Seglv was expressed from egg to adult stages even without immune challenge. Especially, in larval stage, it was expressed in all tested tissues, such as hemocyte, fat body, gut, and epidermis. However, the constitutive expression level was significantly elevated in response to bacterial challenge. Expression of a Toll gene was required for expression of Seglv. A recombinant Seglv protein was synthesized using a bacterial expression system and purified with an affinity chromatography. The recombinant protein showed a specific antibacterial activity against a Gram-positive bacterium, but no activity against a Gram-negative Escherichia coli. Injection of specific double stranded RNA (dsRNA) against Seglv could suppress its expression. Knockdown of Seglv expression induced a significant developmental retardation and resulted in hypotrophy pupae. The larvae treated with dsRNA were much more susceptible to Bacillus thuringiensis than the control larvae. These results suggest that Seglv acts as an antimicrobial peptide especially against Gram-positive bacteria including B. thuringiensis.     

Characterization of an isoform of rice starch branching enzyme, RBE4, in developing seeds

cDNA clones encoding an isoform of starch branching enzyme, RBE4, have been identified from a developing rice seed cDNA library, using a synthetic oligonucleotide probe corresponding to the N-terminal amino acid sequence of RBE4. The cDNA-derived amino acid sequence indicated that RBE4 is initially produced as a precursor protein of 841 amino acids, including a 53-residue transit peptide at the N-terminus. The mature form of RBE4 shared a high degree of sequence identity (80%) with mature RBE3, and possessed an N-terminal extra sequence, as found in RBE3. Northern blot analysis demonstrated that the RBE4 gene is expressed in both leaves and developing seeds. The RBE4 gene was distinguished from the RBE1 and RBE3 genes by expression at the earlier stages of seed development. To examine enzymatic functions of RBE4, recombinant proteins were produced in Escherichia coli cells, and purified by two chromatographic separations. The branched alpha-glucans produced by the recombinant enzymes from potato amylose revealed the different patterns of oligosaccharide chain transfer. The peak of major branches of the products by RBE3 or RBE4 was 6 glucose units, whereas the peaks of major branches of the products by RBE1 were 6 and 11 glucose units. The similar property between RBE3 and RBE4 is supported by high similarity of the amino acid sequences between them.     

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis

The 5' regulatory region and the portion of the structural gene coding for the amino-terminal sequence of alkaline phosphatase I (APase I) were isolated from Bacillus licheniformis MC14 using a synthetic oligodeoxynucleotide deduced from the amino acid sequence of the enzyme. The DNA sequence analysis of this region revealed an open reading frame of 129 amino acids containing the amino-terminal sequence of the mature APase protein. The protein sequence was preceded by a putative signal sequence of 32 amino acid residues. The predicted amino acid sequence of the partial APase clone as well as the experimentally determined amino acid sequence of the enzyme indicated that B. licheniformis APase retains the important features conserved among other APases of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and various human tissues. Heterologous expression studies of the promoter using a fusion with the lacZ gene indicated that it functions as a very strong inducible promoter in B. subtilis that is tightly regulated by phosphate concentration.     

Molecular cloning of the gene encoding an outer-membrane-associated beta-N-acetylglucosaminidase involved in chitin degradation system of Alteromonas sp. strain O-7

The gene encoding beta-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97kDa. A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane. The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial beta-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases.     

Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).     

Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae.

We have cloned and expressed in Escherichia coli a gene encoding a 15,000-apparent-molecular-weight peptidoglycan-associated outer membrane lipoprotein (PAL) of Haemophilus influenzae. The nucleotide sequence of this gene encodes an open reading frame of 153 codons with a predicted mature protein of 134 amino acids. The amino acid composition and sequence of the predicted mature protein agree with the chemically determined composition and partial amino acid sequence of PAL purified from H. influenzae outer membranes. We have also identified a second gene from H. influenzae that encodes a second 15,000-apparent-molecular-weight protein which is recognized by antiserum against PAL. This protein has been shown to be a lipoprotein. The nucleotide sequence of this gene encodes an open reading frame of 154 codons with a predicted mature protein of 136 amino acids and has limited sequence homology with that of the gene encoding PAL. Southern hybridization analysis indicates that both genes exist as single copies in H. influenzae chromosomal DNA. Both genes encode polypeptides which have amino-terminal sequences similar to those of reported membrane signal peptides and are associated primarily with the outer membrane when expressed in E. coli.     

Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase.

The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences. The pattern of identity of amino acid sequences was quite evident in the regions that include structurally and functionally important residues of Bacillus subtilis thermolysin.     

Primary structure of a hormonally regulated beta-glucanase of Nicotiana plumbaginifolia

A cDNA clone for a hormonally regulated beta-glucanase from Nicotiana plumbaginifolia has been isolated by using an oligodeoxynucleotide probe, synthesized to match the previously determined N-terminal amino acid sequence. The cDNA has the complete sequence of the mature protein and contains at least part of a hydrophobic signal peptide. At the amino acid level, the beta-glucanase of N. plumbaginifolia is 73% homologous to a beta(1,3)-glucanase from tobacco and 52% homologous to a beta(1,3;1,4)-glucanase from barley. Southern-blot analysis clearly demonstrated that N. plumbaginifolia contains at least two related genes encoding beta-glucanase. The extent of the complete signal peptide of the cloned beta-glucanase was determined by sequencing part of the corresponding gene. Northern analysis showed that the expression of the beta-glucanase gene is influenced by auxins and cytokinins.     

A novel streptomycete lipase: cloning,sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene

An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.     

Molecular cloning, sequencing, and expression in Escherichia coli of the gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A

The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited by p-chloromercuribenzoic acid, EDTA, o-phenanthroline, HgCl(2), and CuSO(4). The EDTA-inactivated enzyme was completely restored by the addition of Zn(2+) or Co(2+). In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme.     

Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco.     

Isolation of a cDNA encoding a novel GTP-binding protein of Arabidopsis thaliana

A cDNA encoding for a 68 kDa GTP-binding protein was isolated from Arabidopsis thaliana (aG68). This clone is a member of a gene family that codes for a class of large GTP-binding proteins. This includes the mammalian dynamin, yeast Vps1p and the vertebrate Mx proteins. The predicted amino acid sequence was found to have high sequence conservation in the N-terminal GTP-binding domain sharing 54% identity to yeast Vps1p, 56% amino acid identity to rat dynamin and 38% identity to the murine Mx1 protein. The northern analysis shows expression in root, leaf, stem and flower tissues, but in mature leaves at lower levels. Southern analysis indicates that it may be a member of a small gene family or the gene may contain an intron.     

cDNA cloning,characterization and expression of an endosperm-specific barley peroxidase

A barley peroxidase (BP 1) of pI ca. 8.5 and M _r 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from a cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed maximal expression 15 days after flowering, and high levels were found only in the endosperm. BP 1 was not expressed in the leaves.