A xylanase,AtXyn1, is predominantly expressed in vascular bundles,and four putative xylanase genes were identified in the Arabidopsis thaliana genome

The cDNA clone RXF12, which encodes a xylanase (EC 3.2.1.8), was isolated from Arabidopsis thaliana. The C-terminal half of the amino acid sequence of the deduced protein, named AtXyn1, showed similarity with the catalytic domain of barley xylanase X-1. The N-terminal half of AtXyn1 also contained three regions with sequences similar to cellulose-binding domains (CBDs). A xylanase assay revealed that transgenic A. thaliana plants expressing exogenous AtXyn1 fused with enhanced green fluorescent protein (EGFP) possessed approximately twice as much xylanase activity as wild-type plants. Observation by fluorescence microscopy of transgenic A. thaliana plants expressing a fusion protein of AtXyn1 and EGFP suggested that AtXyn1 is a cell wall protein. Analysis of the localization of beta-glucuronidase (GUS) activity in transgenic A. thaliana plants containing a chimeric gene with the upstream sequence of the AtXyn1 gene and the GUS gene demonstrated that the AtXyn1 gene is predominantly expressed in vascular bundles, but not in vessel cells. These data suggest that AtXyn1 is involved in the secondary cell wall metabolism of vascular bundle cells. A database search revealed that four putative xylanase genes exist in the A. thaliana genome, besides the AtXyn1 gene. Of these, two also contain several regions with sequences similar to CBDs in their N-terminal regions. Comparison of the amino acid sequences of the five xylanases suggests a possible process for their molecular evolution.     

Exploring evolutionary trends within the Pennellidae (Copepoda: Siphonostomatoida) using molecular data

Systematic Parasitology - The family Pennellidae comprises ecto- and mesoparasitic copepods on marine fishes. Although a preliminary scheme of phylogenetic relationships of pennellids based on...     

Gene cloning, nucleotide sequencing, and purification and characterization of the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3.

The gene encoding the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 was cloned and sequenced. Analysis of 7.3 kb of genomic DNA revealed the presence of six ORFs, one of which (daaA) encodes the D-amino-acid amidase. This enzyme, DaaA, is composed of 363 amino-acid residues (molecular mass 40 082 Da), and the deduced amino-acid sequence exhibits homology to alkaline D-peptidase from Bacillus cereus DF4-B (32% identity), DD-peptidase from Streptomyces R61 (29% identity), and other penicillin-recognizing proteins. The DaaA protein contains the typical SXXK, YXN, and H(K)XG active-site motifs identified in the penicillin-binding proteins and beta-lactamases. The daaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant DaaA enzyme in cell-free extracts of E. coli was 33.6 U. mg-1 with D-phenylalaninamide as substrate, which is about 350-fold higher than in extracts of O. anthropi SV3. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and three column chromatography steps. On gel-filtration chromatography, DaaA appeared to be a monomer with a molecular mass of 40 kDa. It had maximal activity at 45 degrees C and pH 9.0, and was completely inactivated in the presence of phenylmethanesulfonyl fluoride or Zn2+. DaaA had hydrolyzing activity toward D-amino-acid amides with aromatic or hydrophobic side chains, but did not act on the substrates for the DD-peptidase and beta-lactamase, despite their sequence similarity to DaaA. The characteristics of the recombinant DaaA are similar to those found for the native enzyme partially purified from O. anthropi SV3.     

Establishment and characterization of the Komeda diabetes-prone rat as a segregating inbred strain.

The Komeda diabetes-prone (KDP) rat is a spontaneous animal model of human autoimmune type 1 diabetes. By positional cloning of the non-MHC major susceptibility locus lddm/kdp1, we recently identified a nonsense mutation in Cblb and also found that lymphocytes of KDP rats infiltrate into various tissues, indicating autoimmunity. The maintenance and production of KDP rats has been a critical problem owing to the poor reproductive ability of diabetic animals. To solve the problem, we here established the KDP rat as a segregating inbred strain. We first identified animals that were heterozygous at the lddm/kdp1 region in a breeding colony of KDP rats. The heterozygous region spans at least from D11Yok1 to Cblb on rat chromosome 11. By mating between the heterozygous rats, we obtained homozygotes, heterozygotes and wild-types with the expected ratio of 1:2:1 and found that only the homozygotes developed diabetes, suggesting that these genotypes represent those of lddm/kdp1. We then tried to maintain KDP rats by mating between the heterozygotes, which resulted in a segregating inbred strain. Within 210 d of age, about 80% of lddm/kdp1 homozygotes developed diabetes with severe insulitis, while neither heterozygotes nor wild-types developed diabetes. The phenotypic characteristics of the homozygotes are the same as those of progeny of diabetic parents in the original KDP rats. The segregating inbred KDP rat strain described here would serve as a useful animal model for autoimmune diseases, including type 1 diabetes.     

Synthesis of flagellin and hook subunit protein in flagellar mutants of Escherichia coli K12

Summary We have examined Escherichia coli K12 flagellar mutants affected in each of 29 different loci for the synthesis of flagellin and hook subunit protein. Immune precipitation experiments were employed by treating cell extracts with antiserum against each protein. Flagellin was synthesized in mutants defective in genes flaS, flaT, flaU and flbC. The flaE and flaZ mutants produced small amounts of flagellin, while all the other mutants failed to produce any detectable amount of flagellin.Hook subunit protein was found in most mutants including those defective in genes flaA, flaB, flaC, flaD, flaE, flaG, flaH, flaL, flaM, flaN, flaO, flaP, flaQ, flaR, flaS, flaT, flaU, flaV, flaW, flaX, flaY, flaZ, flbA, flbC, flbD, and hag but not in mutants of flaK, flaI, and flbB. The results conform to the predictions made by our previous indirect gene fusion study (Komeda 1982).     

Purification and characterization of a novel (R)-hydroxynitrile lyase from Eriobotrya japonica (Loquat)

A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30-80% (NH(4))(2)SO(4) fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which suggested the presence of a carbohydrate side chain. The purified enzyme was a monomer with a molecular mass of 72 kDa as determined by gel filtration, and 62.3 kDa as determined by SDS-gel electrophoresis. The N-terminal sequence is reported. The enzyme was a flavoprotein containing FAD as a prosthetic group, and it exhibited a K(m) of 161 microM and a k(cat)/K(m) of 348 s(-1) mM(-1) for mandelonitrile. The optimum pH and temperature were pH 5.5 and 40 degrees C respectively. The enzyme showed excellent stability with regard to pH and temperature. Metal ions were not required for its activity, while activity was significantly inhibited by CuSO(4), HgCl(2), AgNO(3), FeCl(3), beta-mercaptoethanol, iodoacetic acid, phenylmethylsulfonylfluoride, and diethylpyrocarbonate. The specificity constant (k(cat)/K(m)) of the enzyme was investigated for the first time using various aldehydes as substrates. The enzyme was active toward aromatic and aliphatic aldehydes, and showed a preference for smaller substrates over bulky one.     

Characterization of the spermidine synthase-related gene family in Arabidopsis thaliana

The Arabidopsis genome contains four genes that encode proteins similar to both spermidine synthase and spermine synthase of other organisms. Our previous study revealed that one of these genes, designated ACAULIS5 (ACL5), encodes spermine synthase and that its null mutation results in a severe defect in the elongation of stem internodes. Here we report the characterization of the other three genes, designated SPDS1, SPDS2 and SPDS3. Our results showed that SPDS1 and SPDS2 possess spermidine synthase activity in yeast spermidine synthase-deficient mutants, but the enzyme activity of SPDS3 remained to be determined. RNA gel blot analysis revealed that all of these genes are expressed in all plant organs but show different responses to exogenous plant hormones, suggesting that they are involved in different aspects of growth by modulating the contents of polyamines in plant cells.     

Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii

We isolated the promoter regions of five methanol-inducible genes (P(AOD1), alcohol oxidase; P(DAS1), dihydroxyacetone synthase; P(FDH1), formate dehydrogenase; P(PMP20), Pmp20; and P(PMP47), Pmp47) from the Candida boidinii genome, and evaluated their strength and studied their regulation using the acid phosphatase gene of Saccharomyces cerevisiae (ScPHO5) as the reporter. Of the five promoters, P(DAS1) was the strongest methanol-inducible promoter whose strength was approximately 1.5 times higher than that of the commonly used P(AOD1) in methanol-induced cells. Although the expression of P(AOD1) and P(DAS1) was completely repressed by the presence of glucose, formate-induced expression of P(FDH1) was not repressed by glucose. Expression under P(PMP47), another methanol-inducible promoter, was highly induced by oleate. The induction kinetics of P(PMP47) and P(DAS1) revealed that methanol induces the expression of peroxisome membrane protein Pmp47, earlier than the expression of matrix enzyme dihydroxyacetone synthase (Das1p), and that this information is contained in the promoter region of the respective gene. This is the first report which evaluates several methanol-inducible promoters in parallel in the methylotrophic yeast.