[PubMed] [Google Scholar]Beckles DM, Smith AM, ap Rees T. encode the same protein. By comparison of the rice EST sequences, two classes could be identified, one of which is homologous to the RGP1 clone described by Dhugga et al. (1997). Each of the isolated wheat cDNA clones is homologous to the other class, designated RGP2. To obtain cDNA clones corresponding to RGP1, a new specific antisense primer was designed. Using this primer, an clones. The complete sequence of the longest clone from each class was determined. To obtain full-length rice cDNAs from both classes, rice cDNA libraries from etiolated shoot and 7-d-old somatic embryo were screened with two EST clones corresponding to both classes. From both classes, one cDNA clone containing the complete coding region was sequenced. The deduced amino acid sequences of both cDNA clones from wheat and rice are shown in Figure ?Figure11 in comparison with the amino acid sequences of homologous proteins from some other plant species. Both RGPs from the first class have a deduced molecular mass of 41 kD and a pI of 6.1. The proteins from the second class have a molecular mass of 39 kD and pI of 6.6 for the wheat protein and 6.4 for the rice protein. The region around the Arg residue that was determined to be the HDAC-IN-7 glucosylation site (Singh et al., 1995) is highly conserved in all proteins. The overall homology between the proteins is given in Table ?TableI.I. These data clearly show that the sequences fall into two classes. The class consisting of the RGP1 proteins is 87% to 93% identical (90%C96% similarity), whereas the RGP2 class is 63% to 88% identical (69%C91% similarity). Proteins from different classes are comparatively less homologous and 47% to 51% identical (57%C61% similarity). Open in a separate window Figure 1 Sequence comparison of RGP protein sequences from wheat, rice, potato, pea, Arabidopsis, maize ((Fig. ?(Fig.2A)2A) and (Fig. ?(Fig.2B).2B). Multiple DNA fragments were hybridizing with resulted in two to four bands in each lane. Considering the hexaploidy of wheat, we assume that is present as a single-copy gene per haploid genome. Open in a separate window Figure 2 Southern-blot analysis of and (A) or (B) cDNA inserts. Positions and sizes in kb of or in Yeast RGPs from several plant species have been shown to react with UDP sugars in an autocatalytic manner (Dhugga et al., 1997; Delgado et al., 1998). To test whether RGP1 and RGP2 from wheat and rice are autocatalytic self-glucosylating proteins, these HDAC-IN-7 proteins were separately produced in using the pET expression system. After induction, crude extracts were analyzed by SDS-PAGE (Fig. ?(Fig.3A)3A) for the presence of the cloned gene products. The soluble extracts from expressing contain proteins of the correct size, but these were present in very small amounts (Fig. ?(Fig.3A,3A, lanes 2 and 4). An appropriate band was visible in gels loaded with the soluble protein extracts containing RGP2 from wheat or rice (Fig. ?(Fig.3A,3A, lanes 3 and 5, respectively). Large amounts of RGP2 were present in the insoluble fractions, whereas RGP1 was not detected in the insoluble fractions (results not shown). Open in a separate window Figure 3 RGP1 and RGP2 produced in expressing RGP1 or RGP2 from wheat or rice and separated by SDS-PAGE. Proteins were stained with Coomassie Brilliant Blue (A) or blotted onto nitrocellulose and analyzed for the presence of recombinant protein with anti-RGP1 antiserum (B) or anti-RGP2 antiserum (C). The following protein preparations were used: control extract containing plasmid pET29b (lane 1), extracts expressing wheat RGP1 (lane 2), wheat RGP2 (lane 3), rice Mouse monoclonal to CMyc Tag.c Myc tag antibody is part of the Tag series of antibodies, the best quality in the research. The immunogen of c Myc tag antibody is a synthetic peptide corresponding to residues 410 419 of the human p62 c myc protein conjugated to KLH. C Myc tag antibody is suitable for detecting the expression level of c Myc or its fusion proteins where the c Myc tag is terminal or internal RGP1 (lane 4), rice RGP2 (lane 5), wheat His-RGP1 (lane 6), wheat His-RGP2 (lane 7), purified wheat His-RGP1 (lanes 8), and purified wheat His-RGP2 (lane 9). Positions and sizes in kD of prestained molecular mass marker proteins (New England Biolabs, Beverly, MA; HDAC-IN-7 lane M) are indicated. In addition, the extracts were analyzed by western blotting using anti-RGP1 antiserum (Fig. ?(Fig.3B)3B) or anti-RGP2 antiserum (Fig. ?(Fig.3C).3C). A few minor HDAC-IN-7 cross-reactive polypeptides were detected in all extracts, including the control extract not expressing RGP1 or RGP2, indicating that the antisera cross-reacted with proteins. However, the overexpressed RGP1 (Fig. ?(Fig.3B)3B) and RGP2 (Fig. ?(Fig.3C)3C) proteins were clearly detected at the correct mass value. The anti-RGP1 antiserum cross-reacted with the thick RGP2 bands (Fig. ?(Fig.3B,3B, lanes 3, 5, and 7). Subsequently, the extracts were tested for glucosylation activity with UDP-[14C]Glc (Fig. ?(Fig.4).4). Control extract contained radiolabeled material, but this was running with the bromphenol blue tracking dye and is smaller than the 16.5-kD polypeptide of the molecular mass marker (Fig. ?(Fig.4,4, lane 1). The extracts containing wheat or rice RGP1 clearly showed a radiolabeled polypeptide.
[PubMed] [Google Scholar]Beckles DM, Smith AM, ap Rees T