Os07g0214100

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Annotated Information

Function

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Seed allergenic protein

RAG1 shows complete agreement with the cDNA clone, RA17, suggesting that cDNA clone RA17 is derived from RAG1 gene transcripts. On the other hand, RAG2 displays strong homology with another eDNA clone, RA 14 (Table 1). Two RA genes are located divergently at a distance of 4 kb. The 5' region of RAG1 gene has relatively long direct repeat units (about 170 bp), three 22 bp direct repeat units, and two 12 bp direct repeat units (Fig. 3). In addition, six and two 8 bp direct repeat units (ATGCAAAA) overlined in Fig. 3 exist in RAG 1 and RAG2 promoters, respectively (Table 2). DNA blotting analysis of completely digested rice genomic DNA was performed as described in Materials and methods using the Nco I-Eco RI eDNA (RA17) probe encoding a complete mature RA. Three hybridizing bands were observed in lanes 2 and 3 (Fig. 4). About 6 kb fragment in The synthetic oligonucleotide primer is complementary to 17 nucleotide sequence (numbered 62 to 78 in Fig. 3) that corresponds to the potential signal sequence of the clone, RAG1. Total RNA prepared from rice seed at 20 DAF was analyzed by using the synthetic oligonucleotide primer. The result of primer extension analysis is shown in Fig. 5. Primer-extended products detected here would be derived mainly from RAG1 gene tran-scripts because the population of its cDNA, RA17, is dominant in the cDNA library. The 5' end of RA gene transcripts was expected to be at the position of A (numbered -26 in Fig. 3), though other minor bands were also detected, suggesting that the transcriptional initiation site of RAG1 is 26 bp upstream of the translational initiation codon, ATG. The distance between the transcriptional initiation site and the translational initiation site of RAG 1 gene complies with that of most plant genes . In the 5' region, the putative TATA box and CAAT box exists about 45 bp and 147 bp upstream of the transcriptional initiation site, respectively. The transcriptional initiation site of RAG2 gene might be the same as that of RAG1 gene since the nucleotide sequence around comparable region resembles each other. The size of RA gene family is relatively small compared with that of the prolamine genes, which encode the predominant protein fraction of the major cereals though glutelin is predominant in rice seed. Rice glutelin genes were reported to be encoded by about 15 to 24 copies based on DNA blotting analysis . However, the size difference of their multigene families is not consistent with the relative abundances of their gene products. This difference might occur depending on the control of their transcription or translation level. Comparison of two genes, RAG1 and RAG2, showed that though RAG1 was truncated, coding regions displayed 80 identity, and the 5'- flanking regions (from -1000 to + 1 in Fig. 3) revealed 57~o identity. The 5' region of RAG1 (numbered -1000 to -935) showed significant divergence from the comparable sequence of RAG2. However, two regions were highly conserved between the two genes (nucleotide number from -933 through -804 and from -297 through -3 of RAG1 and from -927 through -795 and from -297 through -3 of RAG2, respectively). The one conserved region arranged downstream was found to be located around TATA box, hence this region might play an important role for transcriptional initiation. Moreover, one 169 bp direct- repeat unit (underlined in Fig. 3), of the clone, RAG1, was involved in the conserved region. RAG1, RAG2 (recombination activating gene): These are closely linked (11p13) and encode the proteins of lymphocyte-specific recombination of the V(D)J sequences of immunoglobulin genes. The functional part of RAG1 is the core sequence whereas other tracts can be deleted without affecting recombination. Mutation in RAG results in the inability to form functional antigen receptors on B and T cells and antibodies. The recombinational cleavage takes place between the coding sequence of the immunoglobulin genes and the so-called recombinational signal sequence (RSS) nucleotides. Recombination usually occurs between the original coding sequences or it may take place by the rejoining of one coding end, the signal sequence, which originally belonged to the other coding end (hybrid joint) or the same codingand signal sequence end can be reunited (open-andshut joint). Besides RAGs, the joining reaction requires the double-strand repair protein XRCC4, a DNA-dependent protein kinase and the Ku protein(- s). For recombination, an accessory protein HMG1 or HMG2 is also required. Nucleotides may be added or deleted in each type of joining. RAG1 and RAG2 are actually transposons but after the joining of the V(D)J ends, normally following the formation of the antibody genes, RAGs are inactivated. RAG proteins seem homologous to the Tc1 transposon of Caenorhabditis. The V(D)J recombinase shows a sequence similarity to retroviral integrase superfamily (Zhu L et al 2004 Nature [Lond] 432:995). RAG1 and RAG2 genes are coordinately regulated by cell-type specific elements upstream of RAG2. The 5′ promoter upstream sequences in RAG2 regulate B and T cells differently. For T cells there are four T cell receptors (TCR) and for the B cell to recombine there are threeimmunoglobulin loci. ▶antibody, ▶immunoglobulins, ▶junctional diversification, ▶NHEJ, ▶combinatorial diversification, ▶RSS, ▶V(J)D recombinase, ▶XRCC4, ▶Ku, ▶DNA-PK, ▶TCR, ▶hybrid dysgenesis, ▶reticulosis familial histiocytic, ▶integrase;

Expression

Please input expression information here. Stage- and tissue-specific expression of RA gene To investigate the expression of RA genes, total RNA and protein were prepared from rice seeds at different stages of maturation (5, 10, 15, 20, 25, and 30 DAF), and from other tissues. RNA and protein preparations were used for RNA blotting and immunoblotting analyses, respectively. The mRNA of RA was detected only in ripening seed (lane 4 in Fig. 6A) but not in leaves, stalks, and roots (lanes 1, 2, and 3, respectively, in Fig. 6A). RA mRNA accumulated from 10 DAF and reached its maximum level at 15 to 20 DAF (lanes 3 and 4 in Fig. 6B). Immunoblotting analysis showed that the protein synthesis was consistent with the accumulation of RA mRNAs (data not shown). In conclusion, RA gene expression is controlled in transcription level.

Evolution

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You can also add sub-section(s) at will. Recently many promoters of the plant genes have been characterized, and many transcription factors and cis-elements have already been identified.Several motifs already identified in the plant genes also exist in the 5'-flanking region of RA genes. RAG 1 and RAG2 have eight and two octamer direct repeats (Fig. 3 and Table 2), respectively.This consensus sequence, ATGCAAAA, reminisces the heptamer sequence, TGCAAAA, identified in rice glutelin genes and the -300 bp element in cereal genes . Especially in glutelin promoter, CTTTCGTGTA has been identified as the recognition site of DNAbinding protein . This is similar to the sequences, CTTTGGTCTT and CTTTAGTCTT (nucleotide numbers -91 to -82 and -103 to -94 in Fig. 3) in RAG1 and RAG2 promoter regions, respectively. The accumulation pattern of RA mRNA is also similar to that of glutelin mRNA (Gtl) and thus it is conceived that the regulations of two genes presumably resemble each other. In addition, the 5' region of RAG1 gene has a motif (CATC, nucleotide number from -198 to -195 in Fig. 3) specific for cereal prolamine genes . It is supposed that some common feature of the transcriptional machinery might be shared among prolamine, glutelin and RA genes. Several putative cis elements exist in RA promoter as described above, but it is still unknown whether they are functional or not. Further study on identification of cis elements and their roles is in progress.

Labs working on this gene

Please input related labs here. Department of Food Science and Technology, School of Agriculture, Nagoya University, Chikusa, Nagoya 464-01, Japan; 2Department of Biochemistry, College of Agricultural Chemistry, Kyoto Prefectural University, Shimogamo, Kyoto 606, Japan

References

Please input cited references here. References 1. Adachi T, Yamagata H, Tsukagoshi N, Udaka S: Multipleand tandemly arranged promoters of the cell wall protein gene operon in Bacillus brevis 47. J Bact 171: 10!0-1016 (1989). 2. Barber D, Sanchez-Monge R, Gomez L, Carpizo J, Armentia A, Lopez-Otin C, Juan F, Salcedo G: A barley flour of insect a-amylase is a major allergen associated with baker's asthma disease. FEBS Lett 248:119-122 (1989). 3. Cagampang GB, Cruz LJ, Espiritu SG, Santiago RG, Juliano BO: Studies on the extraction and composition of rice proteins. Cereal Chem 43:145-155 (1966). 4. Color V, Robert LS, Kavanagh TA, Bevan MW, Thompson RD: Localization of sequences in wheat endosperm protein genes which confer tissue-specific expression in tobacco. EMBO J 6:3559-3564 (1987). 5. Forde BG, Heyworth A, Pywell J, Kreis M: Nucleotide sequence of a B 1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize. Nucl Acids Res 13:7327-7339 (1985). 6. Forde BG, Kreis M, Williamson MS, Fry RP, Pywell J, Shewry PR, Bunce N, Miflin B J: Short tandem repeats shared by B- and C-hordein cDNAs suggest a common evolutionary origin for two groups of cereal storage protein genes. EMBO J 4:9-15 (1985). 7. Garcia-Olmedo F, Salcedo G, Sanchez-Monge R, Gomez L, Royo J, Carbonero P: Plant, proteinaceous inhibitors of proteinases and c~-amylases. Oxford Surv Plant Mol Cell Biol 4:275-334 (1987). 8. Gomez L, Martin E, Hernandez D, Sanchez-Monge R, Barber D, Pozo V, Andres B, Armentia A, Lahoz C, Salcedo G, Palomino P: Members of the a-amylase inhibitors family from wheat endosperm are major allergens associated with baker's asthma. FEBS Lett 261:85-88 (1990). 9. 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Masumura T, Sibata D, Hibino T, Kato T, Kawabe K, Takeba G, Tanaka K, Fujil S: cDNA cloning of an mRNA encoding a sulfur-rich 10 kDA prolamin polypeptide in rice seeds. Plant Mol Biol 12:123-130 (1989). 19. Matsuda T, Nomura R, Sugiyama M, Nakamura R: Im 248 munochemical studies on rice allergenic proteins. Agric Biol Chem 55:509-513 (1991). 20. Matsuda T, Suglyama M, Nakamura R, Torii S: Purification and properties of an allergenic protein in rice grain. Agric Biol Chem 52:1465-1470 (1988). 21. Messing J: New M13 vectors for cloning. Meth Enzymol 101:20-78 (1983). 22. Okita TW, Cheesbrough V, Reeves CD: Evolution and heterogeneity of the ~-/fl-type and y-type gliadin DNA sequences. J Biol Chem 260:8203-8213 (1985). 23. Okita TW, Hwang YS, Hnilo J, Kim WT, Aryan AP, Larson R, Krishnan HB: Structure and expression of the rice glutelin multigene family. J Biol Chem 264: 12573- 12581 (1989). 24. Pedersen K, Argos P, Naravana SVL, Larkins BA: Sequence analysis and characterization of a maize gene encoding a high-sulfur zein protein of Mr 15000. J Biol Chem 261:6279-6284 (1986). 25. Prat S, Cortadas J, Puigdomenech P, Palau J: Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2. Nucl Acids Res 13: 1493- 1504 (1985). 26. Reeves CD, Okita TW: Analysis of ~t / //-type gliadin genes from diploid and hexaploid wheats. Gene 52: 257- 266 (1987). 27. Rogers SO, Bendich AJ: Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissue. Plant Mol Biol 5:69-76 (1985). 28. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467 (1977). 29. Shibasaki M, Suzuki S, Nemoto H, Kuroume T: Allergenicity and lymphocyte-stimulating property of rice protein. J Allergy Clin Immunol 64:259-265 (1979). 30. 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