Cloning and analysis of the inulinase gene from Kluyveromyces cicerisporus CBS4857

A highly expressed inulinase gene, KcINU1 was cloned and sequenced from Kluyveromyces cicerisporus CBS4857 a strain which secrets high levels of inulinase into the growth medium. The result of DNA sequencing showed that KcINU1 contained a 1665 bp ORF, coding for a 555 amino acid protein, in which a 23 amino acid signal peptide was included. The sequence has the GenBank Accession no. AF 178979. The analysis of conserved domain in the ORF indicated there was a consensus sequence about 470 amino acids long. The 0.7 Kb promoter and 0.9 Kb terminator were also cloned and sequenced.     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

DNA sequence analysis of a putative primary replicon from a cryptic plasmid pNM214 of a hydrocarbon utilizing marine <Emphasis Type="Italic">Bacillus</Emphasis> strain NM21

Marine Bacillus strain NM21 isolated from hydrocarbon-contaminated site at Naval Harbour, Mumbai grows on high-speed diesel as a source of carbon and energy. This bacterium harbours four plasmids in it. The smallest plasmid, pNM214 was digested with EcoRI enzyme and cloned in pUC19 vector. The clone Om4 containing largest insert of >3.5 kb was sequenced by primer walking. DNA sequence analysis showed this fragment to be homologous to replication initiation protein (rep) gene and dso (double strand origin) of different plasmids from Bacillus subtilis and Bacillus pumilus species. The putative rep gene sequence of pNM214 showed 74.3–91.6% DNA identity to B. subtilis plasmids (pTA1015, pTA1060 and pTA1040) and 86.3% to 88.9% DNA identity to B. pumilus plasmids (pPL7065, pPL10 and pSH1452). The translated amino acid sequence of rep shows that it contains all the three conserved motifs present in the Rep protein of pC194 family of plasmids. DNA sequence comparison of putative dso of pNM214 with other bacillus plasmids belonging to pC194 group shows that it contains highly conserved nick site sequence 5′-TCTTTTCTTATCTTGATA-3′ and surrounding inverted repeats. Thus, it indicates that pNM214 to be a rolling circle replicating plasmid belonging to the pC194 group. The presence of rep and dso like sequences in the sequenced EcoRI fragment indicate that the cloned fragment contain putative primary replicon of pNM214.     

A phosphoglucose isomerase gene is involved in the Rag phenotype of the yeast Kluyveromyces lactis

Summary The rag2 mutant of Kluyveromyces lactis cannot grow on glucose when mitochondrial functions are blocked by various mitochondrial inhibitors, suggesting the presence of a defect in the fermentation pathway. The RAG2 gene has been cloned from a K. lactis genomic library by complementation of the rag2 mutation. The amino acid sequence of the RAG2 protein deduced from the nucleotide sequence of the cloned RAG2 gene shows homology to the sequences of known phosphoglucose isomerases (PGI and PHI). In vivo complementation of the pgi1 mutation in Saccharomyces cerevisiae by the cloned RAG2 gene, together with measurements of specific PGI activities and the detection of PGI proteins, confirm that the RAG2 gene of K. lactis codes for the phosphoglucose isomerase enzyme. Complete loss of PGI activity observed when the coding sequence of RAG2 was disrupted leads us to conclude that RAG2 is the only gene that codes for phosphoglucose isomerase in K. lactis. The RAG2 gene of K. lactis is expressed constitutively, independently of the growth substrates (glycolytic or gluconeogenic). Unlike the pgi1 mutants of S. cerevisiae, the K. lactis rag2 mutants can still grow on glucose, however they do not produce ethanol.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Production of <Emphasis Type="Italic">Chryseobacterium proteolyticum</Emphasis> protein-glutaminase using the twin-arginine translocation pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The protein glutaminase (PG) secreted by the Gram-negative bacterium Chryseobacterium proteolyticum can deamidate glutaminyl residues in several substrate proteins, including insoluble wheat glutens. This enzyme therefore has potential application in the food industry. We assessed the possibility to produce PG containing a pro-domain in Corynebacterium glutamicum which we have successfully used for production of several kinds of proteins at industrial-scale. When it was targeted to the general protein secretion pathway (Sec) via its own signal sequence, the protein glutaminase was not secreted in this strain. In contrast, we showed that pro-PG could be efficiently produced using the recently discovered twin-arginine translocation (Tat) pathway when the typical Sec-dependent signal peptide was replaced by a Tat-dependent signal sequence from various bacteria. The accumulation of pro-PG in C. glutamicum ATCC13869 reached 183 mg/l, and the pro-PG was converted to an active form as the native one by SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus. The successful secretion of PG via this approach confirms that the Tat pathway of C. glutamicum is an efficient alternative for the industrial-scale production of proteins that are not efficiently secreted by other systems.     

First evidence of methylation in the genome of <Emphasis Type="Italic">Drosophila willistoni</Emphasis>

DNA methylation has been studied abundantly in vertebrates and recent evidence confirms that this phenomenon could be disseminated among some invertebrates groups, including Drosophila species. In this paper, we used the Methylation-Sensitive Restriction Endonuclease (MSRE) technique and Southern blot with specific probes, to detect methylation in the Drosophila willistoni species. We found differential cleavage patterns between males and females that cannot be explained by Mendelian inheritance, pointing to a DNA methylation phenomenon different from the Drosophila melanogaster one. The sequencing of some of these bands showed that these fragments were formed by different DNA elements, among which rDNA. We also characterized the D. willitoni dDnmt2 sequence, through a Mega Blast search against the D. willistoni Trace Archive Database using the D. melanogaster dDnmt2 nucleotide sequence as query. The complete analysis of D. willistoni dDnmt2 sequence showed that its promoter region is larger, its dDnmt2 nucleotide sequence is 33% divergent from the D. melanogaster one, Inverted Terminal Repeats (ITRs) are absent and only the B isoform of the enzyme is produced. In contrast, ORF2 is more conserved. Comparing the D. willistoni and D. melanogaster dDnmt2 protein sequences, we found higher conservation in motifs from the large domain, responsible for the catalysis of methyl transfer, and great variability in the region that carries out the recognition of specific DNA sequences (TRD). Globally, our results reveal that methylation of the D. willistoni genome could be involved in a singular process of species-specific dosage compensation and that the DNA methylation in the Drosophila genus can have diverse functions. This could be related to the evolutionary history of each species and also to the acquisition time of the dDnmt2 gene.     

Binding ofNicotiana nuclear proteins to the subterminal regions of theAc transposable element

Specific binding ofNicotiana nuclear protein(s) to subterminal regions of theAc transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions ofAc, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding. Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif. The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends ofMutator. Moreover, we show that DNA fromDs1 competes for protein binding toAc termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region ofTgm1, Tpn1, En/Spm, Tam3 andDs1-like elements. This suggests that the binding protein(s) might be involved in the transposition process.     

Characterization of Bacillus thuringiensis Cry toxin binding novel GPI anchored aminopeptidase from fat body of the moth Spodoptera litura

Aminopeptidase N (APN) isoforms were identified as candidate receptors for Bacillus thuringiensis Cry toxins from the midgut of several insect species. In this study a partial cDNA encoding aminopeptidase (slfbAPN) was cloned from fat body of the moth Spodoptera litura. In the deduced amino acid sequence the characteristic metallopeptidase sequences, HEXXHX₁₈E and GAMENWG were conserved but the sequence showed only 33–39% identity to other insect APNs, which were also reported to be Cry toxin receptors. The presence of a putative GPI anchor signal sequence at the C-terminus indicated that it is a membrane-anchored protein. The slfbAPN expression was restricted to the fat body as suggested by northern blot analysis of different tissues. Biochemical analyses including immunoblotting, ligand blotting and lectin blotting, demonstrated that slfbAPN is a membrane-anchored glycoprotein in the fat body and it binds to Cry toxins. The nucleotide sequence shown here has been submitted to the GenBank sequence data bank and is available under accession number EF372603.     

The chloroplast genome of mulberry: complete nucleotide sequence, gene organization and comparative analysis

The complete nucleotide sequence of mulberry (Morus indica cv. K2) chloroplast genome (158,484 bp) has been determined using a combination of long PCR and shotgun-based approaches. This is the third angiosperm tree species whose plastome sequence has been completely deciphered. The circular double-stranded molecule comprises of two identical inverted repeats (25,678 bp each) separating a large and a small single-copy region of 87,386 bp and 19,742 bp, respectively. A total of 83 protein-coding genes including five genes duplicated in the inverted repeat regions, eight ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids, were assigned on the basis of homology to predicted genes from other chloroplast genomes. The mulberry plastome lacks the genes infA, sprA, and rpl21 and contains two pseudogenes ycf15 and ycf68. Comparative analysis, based on sequence similarity, both at the gene and genome level, indicates Morus to be closer to Cucumis and Lotus, phylogenetically. However, at genome level, inclusion of non-coding regions brings it closer to Eucalyptus, followed by Cucumis. This may reflect differential selection pressure operating on the genic and intergenic regions of the chloroplast genome.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Communicated by Y. Tsumura     

A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter

Summary The majority of the activation potential of the Saccharomyces cerevisiae TDH3 gene promoter is contained within nucleotides –676 to –381 (relative to the translation initiation codon). An upstream activation sequence (UAS) in this region has been characterized by in vitro and in vivo assays and demonstrated to be composed of two small, adjacent DNA sequence elements. The essential determinant of this upstream UAS is a general regulatory factor 1 (GRF1) binding site at nucleotides –513 to –501. A synthetic DNA element comprising this sequence, or an analogue in which two of the degenerate nucleotides of the GRF1 site consensus sequence were altered, activated 5 deleted TDH3 and CYC1 promoters. The second DNA element of the UAS is a 7 by sequence which is conserved in the promoters of several yeast genes encoding glycolytic enzymes and occurs at positions –486 to –480 of the TDH3 promoter. This DNA sequence represents a novel promoter element: it contains no UAS activity itself, yet potentiates the activity of a GRF1 UAS. The potentiation of the GRFl UAS by this element occurs when placed upstream from the TATA box of either the TDH3 or CYC1 promoters. The characteristics of this element (termed GPE for GRF1 site potentiator element) indicate that it represents a binding site for a different yeast protein which increases the promoter activation mediated by the GRF1 protein. Site-specific deletion and promoter reconstruction experiments suggest that the entire activation potential of the –676 to –381 region of the TDH3 gene promoter may be accounted for by a combination of the GRF1 site and the GPE.     

A novel ras-related rgp1 gene encoding a GTP-binding protein has reduced expression in 5-azacytidine-induced dwarf rice.

Summary Exposure of normal, tall rice (Oryza sativa) seedlings to 5-azacytidine, a powerful inhibitor of DNA methylation in vivo, induced both demethylation of genomic DNA and dwarf plants. Genes that had been affected by treatment were identified by differential screening of a cDNA library, and a ras-related gene, rgp1, was subsequently isolated. The cDNA of rgp1 was found to encode a deduced protein sequence of 226 amino acids with a relative molecular mass of 24850, which was most closely related to the ras-related ypt3 protein of fission yeast, Shizosaccharomyces pombe. The rgp1 protein, expressed in transformed Escherichia coli, clearly showed GTP-binding activity. During seedling growth, rgp1 expression was first observed 14 days after germination, reaching a maximum level between 28 and 42 days, and gradually decreased thereafter until 63 days when it attained the same level of expression as in 14-day-old seedlings. Expression of rgp1 was found to be markedly reduced throughout the growth period of both 5-azacytidine-induced dwarf plants and their progenies, relative to levels in untreated tall control plants. These results suggest that expression of rgp1 may be influenced, either directly or indirectly, by DNA methylation, and that the rgp1 protein may play an important role in plant growth and development.     

Cloning and expression analysis of SKn-type dehydrin gene from bean in response to heavy metals

A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and coppper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H₂O₂). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.     

cDNA Clone, fusion expression and purification of the novel gene related to ascorbate peroxidase from Chinese wild Vitis pseudoreticulata in E. coli

Powdery mildew, caused by Uncinula necator Burr, is one of the most seriously damaging diseases of grapevine all over the world. To gain the novel gene and investigate the resistance mechanism in Chinese Wild Vitis pseudoreticulata clone Baihe-35-1, mRNA differential display was employed to study the differential expression of the resistant gene to the disease of it when inoculated by Uncinula necator under natural field conditions, 5′ RACE and 3′ RACE have been used to clone the whole cDNA sequences of VpAPX, the novel gene related to Ascorbate Peroxidase which involved in resistant to the disease, is composed of specific sequence 1077 bp and has an open reading frame of 750 bp coding for 250 amino acid residues with a molecular weight of 27.566 kDa. The VpAPX gene was obtained by polymerase chain reaction (PCR) with the special primers synthesized according to the sequences of cDNA, and further cloned it into the pGEM-T easy vector. The cloned VpAPX gene was cut out again with two restriction enzymes and was inserted into the prokaryotic expression vector pGEX-4T-1, then transferred into E. coli BL21. As result, GST-VpAPX fusion protein was successfully expressed by induction of IPTG and purified by GST affinity resin. After injecting rabbit, the polyclonal antibodies were produced. Western blot analyses showed that the antibody reacted specifically to GST-VpAPX fusion protein and the titer for this antibody is 10⁵. This research made the foundation to transform the VpAPX gene into grape plants for follow research in processing. Ling Lin, Xiping Wang: These two authors contributed equally to this work.     

The b-32 protein from maize endosperm, an albumin regulated by the O2 locus: Nucleic acid (cDNA) and amino acid sequences

Summary The cDNA coding for the b-32 protein, an albumin expressed in maize endosperm cells under the control of the O2 and O6 loci, has been cloned and the complete amino acid sequence of the protein derived. A lambda gt11 cDNA library from mRNA of immature maize endosperm was screened for the expression of the b-32 protein using antibodies against the purified protein. One of the positive clones obtained was used to isolate a full-length cDNA clone. By Northern analysis, the size of the b-32 mRNA was estimated to be 1.2 kb. Hybrid-selected translation assays show that the message codes for a protein with an apparent molecular weight of 30–35 kDa. The nucleotide sequence shows that several internal repeats are present. The protein has a length of 303 amino acid residues (mol. wt. 32430 dalton) and its sequence shows the following features: no signal peptide is observable; it contains seven tryptophan residues, an amino acid absent in maize storage proteins; polar and hydrophobic residues are spread along the sequence; several pairs of basic residues are present in the N-terminal region; the secondary structure allows the prediction of two structural domains for the b-32 protein that would fold up giving rise to a globular shape. The cloning of this gene may help in understanding the role of the O2 and O6 loci in regulating the deposition of zein, the major storage protein of maize endosperm.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

The secY-rpmJ region of the spc ribosomal protein operon in Escherichia coli: structural alterations affecting secY expression

Summary A unique feature of the spc ribosomal protein operon is that its region distal to the promoter contains a gene (secY) for an integral membrane protein, followed by an open reading frame termed X which has recently been proposed to encode a new ribosomal protein (protein B). We now show that the open reading frame X indeed directs the synthesis of a protein with electrophoretic mobilities similar to the B protein, and this supports the proposal that X may be more appropriately called rpmJ. Insertion of a plasmid sequence into the secY-rpmJ boundary of the chromosome caused a reduced expression of secY probably by destabilizing the secY part of the message. The results of complementation experiments suggested that a normal level of expression of rpmJ is not required for growth or protein secretion.