We have cloned and characterized two novel DNA binding proteins designated RAV1 and RAV2 from Arabidopsis thaliana. RAV1 and RAV2 contain two distinct amino acid sequence domains found only in higher plant species. The N-terminal regions of RAV1 and RAV2 are homologous to the AP2 DNA-binding domain present in a family of transcription factors represented by the Arabidopsis APETALA2 and tobacco EREBP proteins, while the C-terminal region exhibits homology to the highly conserved C-terminal domain, designated B3, of VP1/ABI3 transcription factors. Binding site selection assays using a recombinant glutathione S-transferase fusion protein have revealed that RAV1 binds specifically to bipartite recognition sequences composed of two unrelated motifs, 5'-CAACA-3' and 5'-CACCTG-3', separated by various spacings in two different relative orientations. Analyses using various deletion derivatives of the RAV1 fusion protein show that the AP2 and B3-like domains of RAV1 bind autonomously to the CAACA and CACCTG motifs, respectively, and together achieve a high affinity and specificity of binding. From these results, we suggest that the AP2 and B3-like domains of RAV1 are connected by a highly flexible structure enabling the two domains to bind to the CAACA and CACCTG motifs in various spacings and orientations.

A cDNA that encoded a bZIP protein, designated OSBZ8, was isolated from a rice embryo cDNA library by use of degenerate oligonucleotide probes that corresponded to the amino acid sequences conserved among the basic regions of plant G-box-binding factor-type bZIP proteins (GBF). OSBZ8 was shown to have structural features typical of the GBF-type bZIP proteins and to bind to G-box and G-box-like sequences that include ABA-responsive elements (ABREs) which have been functionally identified in the promoters of ABA-inducible genes, such as Em, Osem and Rab16. The accumulation of OSBZ8 mRNA was induced by treatment with ABA of imbibed mature rice embryos, of young plant tissues and of suspension-cultured cells. The accumulation of OSBZ8 mRNA in response to ABA preceded that of Osem and Rab16A mRNAs and was not inhibited by an inhibitor of protein synthesis, cycloheximide. By contrast, the induction of Osem and Rab16A was partially inhibited and almost completely inhibited, respectively, by cycloheximide. These results strongly suggest that OSBZ8 might be involved in the regulation of transcription by ABA in rice.

The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open reading frame of 2,493 nucleotides and encodes 831 amino acids with a deduced molecular weight of 90,020. The deduced ChiB is a modular enzyme composed of a family 18 catalytic domain responsible for chitinase activity, two reiterated domains of unknown function, and a chitin-binding domain (CBD). The reiterated domains are similar to the repeating units of cadherin proteins but not to fibronectin type III domains, and therefore they are referred to as cadherin-like domains. ChiB was purified from the periplasm fraction of Escherichia coli harboring the chiB gene. The molecular weight of the purified ChiB (87,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, was in good agreement with the value (86,578) calculated from the deduced amino acid sequence excluding the signal peptide. ChiB was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl ␤-D-N,N-diacetylchitobioside [4-MU-(GlcNAc) 2 ]. The pH and temperature optima of the enzyme were 6.0 and 45°C, respectively. The K m and V max values for 4-MU-(GlcNAc) 2 were estimated to be 6.3 M and 46 mol/min/mg, respectively. SDS-PAGE, zymogram, and Western blot analyses using antiserum raised against purified ChiB suggested that ChiB was one of the major chitinase species in the culture supernatant of C. paraputrificum. Deletion analysis showed clearly that the CBD of ChiB plays an important role in hydrolysis of native chitin but not processed chitin such as colloidal chitin.Chitinase (EC 3.2.1.14) is a glycosyl hydrolase that catalyzes the degradation of chitin, an insoluble linear ␤-1,4-linked polymer of N-acetylglucosamine. Chitinases are present in a wide range of organisms, including bacteria, insects, viruses, plants, and animals, and play important physiological and ecological roles. On the basis of amino acid sequence homology, chitinases are divided into two unrelated families, families 18 and 19 of glycosyl hydrolases (15). Family 18 includes chitinases from bacteria, fungi, viruses, and animals and chitinases classified in class III or V from plants. On the other hand, family 19 includes almost exclusively plant chitinases classified in classes I, II, and IV but also a bacterial chitinase, Streptomyces griseus HUT 6037 chitinase C (32). To date, various chitinases were isolated from aerobic microorganisms such as Bacillus circulans (1,(55)(56)(57), Serratia marcescens (6,13,16,20), an Aeromonas sp. (41,(51)(52)(53), an Alteromonas sp. (48), Streptomyces plicatus (37), Streptomyces olivaceoviridis (3, 36, 38), and Janthiobacterium lividum (12). Several genes encoding chitinases were cloned in Escherichia coli and characterized in detail along with their translated products (9, 12, 20, 36-38, 41, 43, 49, 56, 57). From these studies, chitinases were found to comprise two or more discrete domains, while the function of each domain has not yet been elucidated. Watanabe et al. reported that B. ...

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BdeltaCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.

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