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Isolation and characterization of a cDNA encoding a high mobility group protein HMG-1 from Canavalia gladiata D.C.1
Biochimica et Biophysica Acta 1396 Ž1998. 47–50
Short sequence-paper
Isolation and characterization of a cDNA encoding a high mobility group protein HMG-1 from CanaÕalia gladiata D.C. 1 Sumiko Yamamoto
),2
, Takao Minamikawa
Department of Biology, Tokyo Metropolitan UniÕersity, Minami-ohsawa, Hachioji, Tokyo 192-03, Japan Received 8 August 1997; accepted 2 October 1997
Abstract A cDNA clone encoding a HMG-1 protein from maturing seeds of CanaÕalia gladiata was isolated and characterized with respect to its sequence, genomic organization and the expression pattern in seeds. The predicted polypeptide had the characteristic conserved motifs of the HMG-1r2 protein including N-terminal basic region, one HMG-box and polyacidic carboxy terminus. Southern blot analysis suggested that the HMG-1 gene is a single copy gene. Northern blot analysis indicated that the HMG-1 gene was expressed both in maturing and germinated seeds. q 1998 Elsevier Science B.V. Keywords: High mobility group protein; HMG-box; Seed maturation; Seed germination; Single-copy gene; Ž CanaÕalia gladiata.
The HMG-1r2 proteins, one of the three groups of high mobility group ŽHMG. protein w1x, contain Nterminal basic region, HMG-box DNA-binding domain and C-terminal polyacidic region. cDNA clones of the plant HMG-1r2 protein were isolated from maize w2x, soybean w3x, Vicia faba w4x, Pharbitis nil w5x, pea w6x and wheat ŽdeFroidmont, EMBL database. . The plant HMG-1r2 proteins contain only one HMG-box in contrast to animal HMG-1r2 proteins which have two. The HMG-1r2 proteins bind to ArT-rich sequences in vitro and prefer singlestranded to double-stranded DNA w7x. In addition, the
)
Corresponding author. Fax: Ž81. 298 54 6009. The nucleotide sequence reported in this paper has been submitted to the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AB000637. 2 Present address: Plant Molecular Biology Laboratory, Molecular Biology Department, National Institute of Bioscience and Human Technology, Tsukuba, 305 Japan. 1
bindings of the HMG-1r2 protein to distorted DNA, such as four-way junctions or cisplatin-damaged DNA have been reported w8,9x. A number of ArT-rich sequences are found in the 5X-upstream regions of many plant genes including seed storage protein genes from CanaÕalia gladiata D.C. w10x. We previously reported the isolation of cDNAs encoding the HMG-Y protein from maturing seeds of C. gladiata w11x and the binding of the recombinant HMG-Y protein to ArT-rich sequences of the promoter of the seed storage protein genes w12x. Here we report the cloning of a cDNA encoding the HMG-1 protein from C. gladiata that is also expected to interact with ArT-rich sequences. Cloning of C. gladiata HMG-1 cDNA. Poly ŽA.q RNA fraction was prepared from maturing seeds of 40 days after flowering ŽDAF. as described previously w11x, and cDNA was synthesized for the PCR template. The middle portion of C. gladiata HMG-1 cDNA was generated by the polymerase chain reaction ŽPCR. method using 5X primer Ž 5X-AAAC-
0167-4781r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 2 2 6 - 1
48
S. Yamamoto, T. Minamikawar Biochimica et Biophysica Acta 1396 (1998) 47–50
CAAAGAGGCCTCCCAGTGCT-3X . and 3X primer Ž5X-TTCATCCTCATCATTGACTTCAGA-3X . , which were synthesized on the basis of the conserved regions of the HMG-box and acidic domain of Vicia faba HMG cDNA, respectively w4x. The nucleotide sequence of the cDNA insert subcloned into the pCRII vector ŽInvitrogen. was determined, and the primers for 5X-RACE and 3X-RACE were synthesized according to the sequence obtained Ž5R1: 5XATACTCCTCCTTTTTCTTCTCCGCC-3X ; 5R2: 5XGAAGGGAGCCTTCTCTGCATCAGA-3X and 3R1: GATGCAGAGAAGGCTCCCTTCGTTG-3X . . The 5X-end of the C. gladiata HMG-1 cDNA was obtained by 5X-RACE w13x using 5R1 and 5R2 with a commercial kit ŽClontech.. The 3X-end of the C. gladiata HMG-1 cDNA was obtained by 3X-RACE w13x using 3R1, 3R2 Ž 5X-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3X . and 3R3 Ž 5XGACTCGAGTCGACATCG-3X . as PCR primers. The nucleotide sequences of the cDNA subcloned into the pCRII vector ŽInvitrogen. were determined on both strands by using ABI 373 DNA sequencer Ž Applied Biosystems. . The nucleotide sequence of the HMG-1 cDNA from C. gladiata consists of 426 bp of the
coding region, 19 bp of the 5X untranslated region and 309 bp of 3X untranslated region. The cDNA encodes the protein of 141 amino acids and the theoretical molecular mass is calculated as 15.9 kDa. Comparison of the amino acid sequence of C. gladiata HMG-1 protein with those of other plant HMG-1 proteins. The predicted polypeptide of C. gladiata HMG-1 ŽCgHMG-1. had the characteristic conserved motifs of the HMG-1r2 protein including N-terminal basic region, one HMG-box w14x and polyacidic carboxy terminus. Identities of CgHMG-1 to P. nil HMG, pea HMG, V. faba HMG, soybean SB11, wheat HMG and maize HMG, were 57, 48, 58, 56, 54 and 51%, respectively Ž Fig. 1. . Genomic DNA analysis. Southern blot analysis was performed using the HMG-1 cDNA probe that was generated by the PCR method. One band was detected in the DNA digestion with EcoRI, HindIII and BglII, suggesting a single-copy gene for CgHMG-1 ŽFig. 2. . The result coincides with that reported for the maize HMG-1 protein which was encoded by one or a few genes of high homology w2x, whereas multiple genes encoding HMG-1 protein were present in P. nil w5x.
Fig. 1. Comparison of amino acid sequences of the HMG-1 protein from C. gladiata, CgHMG-1, and other plant HMG-1 proteins. The alignment was done using the program ClustalW w16x. Gaps were introduced for optimal alignment. Residues conserved among the seven sequences are boxed. The values in the parentheses indicate total numbers of the amino acid residues. The sources of the sequences are as follows: P. nil HMG w5x, pea HMG w6x, Vicia faba HMG w4x, soybean SB11 w3x, wheat HMG ŽdeFroidmont, EMBL database. and maize HMG w2x. The DDBJ, EMBL and GenBank accession number of CgHMG-1 is AB000637.
S. Yamamoto, T. Minamikawar Biochimica et Biophysica Acta 1396 (1998) 47–50
49
Expression pattern of the C. gladiata HMG-1 gene. Northern blot analysis was performed to examine the developmental stage-specific expression of mRNA for CgHMG-1 ŽFig. 3. . The size of the mRNA was estimated to be 0.7 kb. In maturing seeds, a high level of mRNA was observed at 30 DAF, but the mRNA was scarcely detected at 50 DAF and 80 DAF. mRNA for the HMG-1 protein was expressed prior to those for seed storage protein which were expressed at about 40 DAF w15x. This supports the possibility that HMG-1 protein is involved in the expression of the seed storage protein genes. In germinated seeds, the expression of mRNA for CgHMG-1 was observed 2 days after imbibition started ŽDAI.. The level then increased at 6 DAI and a much higher level was observed at 10 DAI, at which the primary leaf began to expand. The level of mRNA for HMG-Y, which was high at 6 DAI, decreased at 10 DAI w11x. The
Fig. 3. Northern blot analysis of the HMG-1 mRNA from C. gladiata. PolyŽA.q RNA fractions were isolated from maturing Ž30, 50 and 80 DAF. and germinated Ž2, 6 and 10 DAI. seeds. PolyŽA.q RNA Ž1.5mg per lane. was denatured with glyoxal, separated on 1.4% agarose gel, transferred to a nylon filter and hybridized with the same probe as used in Southern blot analysis. Autoradiography was carried out at y808C for five days with an intensifying screen Župper panel.. The nylon filter was stained with a solution of 0.5 M sodium acetate ŽpH 5.4. and 0.04% methylene blue Žlower panel..
data of a cDNA for the HMG-1 protein will allow the further investigations on the roles of HMG-1 and HMG-Y proteins in the expression of seed storage protein genes in C. gladiata plant. Fig. 2. Southern blot analysis of the gene encoding HMG-1 protein from C. gladiata. Genomic DNA Ž30 mg per lane. was digested with restriction enzymes, separated on 1% agarose gel, denatured, transferred to a nylon filter and hybridized with the 32 P-labeled probe which was generated by the PCR method on the basis of Vicia faba HMG cDNA w4x. The washing was performed twice in 2=SSC Ž0.15 M NaClr0.015 M sodium citrate pH 7.6. containing 0.1% SDS at 658C. DNA size markers are shown on the right.
References w1x K.D. Grasser, Plant J. 7 Ž1995. 185–192. w2x K.D. Grasser, G. Feix, Nucleic Acids Res. 19 Ž1991. 2573– 2577. w3x T. Laux, R.B. Goldberg, Nucleic Acids Res. 19 Ž1991. 4769.
50
S. Yamamoto, T. Minamikawar Biochimica et Biophysica Acta 1396 (1998) 47–50
w4x K.D. Grasser, T. Wohlfarth, H. Baumlein, G. Feix, Plant ¨ Mol. Biol. 23 Ž1993. 619–625. w5x C.C. Zheng, A.Q. Bui, S.D. O’Neill, Plant Mol. Biol. 23 Ž1993. 813–823. w6x C.I. Webster, L.C. Packman, K.-H. Pwee, J.C. Gray, Plant J. 11 Ž1997. 703–715. w7x J.M. Wright, G.H. Dixon, Biochemistry 27 Ž1988. 576–581. w8x M.E. Bianchi, M. Beltrame, G. Paonessa, Science 243 Ž1989. 1056–1059. w9x P.M. Pil, S.J. Lippard, Science 256 Ž1992. 234–237. w10x S. Yamamoto, M. Nishihara, H. Morikawa, D. Yamauchi, T. Minamikawa, Plant Mol. Biol. 27 Ž1995. 729–741.
w11x S. Yamamoto, T. Minamikawa, Plant Mol. Biol. 33 Ž1997. 537–544. w12x S. Yamamoto, T. Minamikawa, Plant Sci. 124 Ž1997. 165– 173. w13x M.A. Frohman, M.K. Dush, G.R. Martin, Proc. Natl. Acad. Sci. U.S.A. 85 Ž1988. 8998–9002. w14x H.-M. Jantzen, A. Admon, S.P. Bell, R. Tjian, Nature 344 Ž1990. 830–836. w15x D. Yamauchi, K. Nakamura, T. Asahi, T. Minamikawa, Eur. J. Biochem. 170 Ž1988. 515–520. w16x J.D. Thompson, D.G. Higgins, T.J. Gibson, Nucleic Acids Res. 22 Ž1994. 4673–4680.
Short sequence-paper
Isolation and characterization of a cDNA encoding a high mobility group protein HMG-1 from CanaÕalia gladiata D.C. 1 Sumiko Yamamoto
),2
, Takao Minamikawa
Department of Biology, Tokyo Metropolitan UniÕersity, Minami-ohsawa, Hachioji, Tokyo 192-03, Japan Received 8 August 1997; accepted 2 October 1997
Abstract A cDNA clone encoding a HMG-1 protein from maturing seeds of CanaÕalia gladiata was isolated and characterized with respect to its sequence, genomic organization and the expression pattern in seeds. The predicted polypeptide had the characteristic conserved motifs of the HMG-1r2 protein including N-terminal basic region, one HMG-box and polyacidic carboxy terminus. Southern blot analysis suggested that the HMG-1 gene is a single copy gene. Northern blot analysis indicated that the HMG-1 gene was expressed both in maturing and germinated seeds. q 1998 Elsevier Science B.V. Keywords: High mobility group protein; HMG-box; Seed maturation; Seed germination; Single-copy gene; Ž CanaÕalia gladiata.
The HMG-1r2 proteins, one of the three groups of high mobility group ŽHMG. protein w1x, contain Nterminal basic region, HMG-box DNA-binding domain and C-terminal polyacidic region. cDNA clones of the plant HMG-1r2 protein were isolated from maize w2x, soybean w3x, Vicia faba w4x, Pharbitis nil w5x, pea w6x and wheat ŽdeFroidmont, EMBL database. . The plant HMG-1r2 proteins contain only one HMG-box in contrast to animal HMG-1r2 proteins which have two. The HMG-1r2 proteins bind to ArT-rich sequences in vitro and prefer singlestranded to double-stranded DNA w7x. In addition, the
)
Corresponding author. Fax: Ž81. 298 54 6009. The nucleotide sequence reported in this paper has been submitted to the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AB000637. 2 Present address: Plant Molecular Biology Laboratory, Molecular Biology Department, National Institute of Bioscience and Human Technology, Tsukuba, 305 Japan. 1
bindings of the HMG-1r2 protein to distorted DNA, such as four-way junctions or cisplatin-damaged DNA have been reported w8,9x. A number of ArT-rich sequences are found in the 5X-upstream regions of many plant genes including seed storage protein genes from CanaÕalia gladiata D.C. w10x. We previously reported the isolation of cDNAs encoding the HMG-Y protein from maturing seeds of C. gladiata w11x and the binding of the recombinant HMG-Y protein to ArT-rich sequences of the promoter of the seed storage protein genes w12x. Here we report the cloning of a cDNA encoding the HMG-1 protein from C. gladiata that is also expected to interact with ArT-rich sequences. Cloning of C. gladiata HMG-1 cDNA. Poly ŽA.q RNA fraction was prepared from maturing seeds of 40 days after flowering ŽDAF. as described previously w11x, and cDNA was synthesized for the PCR template. The middle portion of C. gladiata HMG-1 cDNA was generated by the polymerase chain reaction ŽPCR. method using 5X primer Ž 5X-AAAC-
0167-4781r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 2 2 6 - 1
48
S. Yamamoto, T. Minamikawar Biochimica et Biophysica Acta 1396 (1998) 47–50
CAAAGAGGCCTCCCAGTGCT-3X . and 3X primer Ž5X-TTCATCCTCATCATTGACTTCAGA-3X . , which were synthesized on the basis of the conserved regions of the HMG-box and acidic domain of Vicia faba HMG cDNA, respectively w4x. The nucleotide sequence of the cDNA insert subcloned into the pCRII vector ŽInvitrogen. was determined, and the primers for 5X-RACE and 3X-RACE were synthesized according to the sequence obtained Ž5R1: 5XATACTCCTCCTTTTTCTTCTCCGCC-3X ; 5R2: 5XGAAGGGAGCCTTCTCTGCATCAGA-3X and 3R1: GATGCAGAGAAGGCTCCCTTCGTTG-3X . . The 5X-end of the C. gladiata HMG-1 cDNA was obtained by 5X-RACE w13x using 5R1 and 5R2 with a commercial kit ŽClontech.. The 3X-end of the C. gladiata HMG-1 cDNA was obtained by 3X-RACE w13x using 3R1, 3R2 Ž 5X-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3X . and 3R3 Ž 5XGACTCGAGTCGACATCG-3X . as PCR primers. The nucleotide sequences of the cDNA subcloned into the pCRII vector ŽInvitrogen. were determined on both strands by using ABI 373 DNA sequencer Ž Applied Biosystems. . The nucleotide sequence of the HMG-1 cDNA from C. gladiata consists of 426 bp of the
coding region, 19 bp of the 5X untranslated region and 309 bp of 3X untranslated region. The cDNA encodes the protein of 141 amino acids and the theoretical molecular mass is calculated as 15.9 kDa. Comparison of the amino acid sequence of C. gladiata HMG-1 protein with those of other plant HMG-1 proteins. The predicted polypeptide of C. gladiata HMG-1 ŽCgHMG-1. had the characteristic conserved motifs of the HMG-1r2 protein including N-terminal basic region, one HMG-box w14x and polyacidic carboxy terminus. Identities of CgHMG-1 to P. nil HMG, pea HMG, V. faba HMG, soybean SB11, wheat HMG and maize HMG, were 57, 48, 58, 56, 54 and 51%, respectively Ž Fig. 1. . Genomic DNA analysis. Southern blot analysis was performed using the HMG-1 cDNA probe that was generated by the PCR method. One band was detected in the DNA digestion with EcoRI, HindIII and BglII, suggesting a single-copy gene for CgHMG-1 ŽFig. 2. . The result coincides with that reported for the maize HMG-1 protein which was encoded by one or a few genes of high homology w2x, whereas multiple genes encoding HMG-1 protein were present in P. nil w5x.
Fig. 1. Comparison of amino acid sequences of the HMG-1 protein from C. gladiata, CgHMG-1, and other plant HMG-1 proteins. The alignment was done using the program ClustalW w16x. Gaps were introduced for optimal alignment. Residues conserved among the seven sequences are boxed. The values in the parentheses indicate total numbers of the amino acid residues. The sources of the sequences are as follows: P. nil HMG w5x, pea HMG w6x, Vicia faba HMG w4x, soybean SB11 w3x, wheat HMG ŽdeFroidmont, EMBL database. and maize HMG w2x. The DDBJ, EMBL and GenBank accession number of CgHMG-1 is AB000637.
S. Yamamoto, T. Minamikawar Biochimica et Biophysica Acta 1396 (1998) 47–50
49
Expression pattern of the C. gladiata HMG-1 gene. Northern blot analysis was performed to examine the developmental stage-specific expression of mRNA for CgHMG-1 ŽFig. 3. . The size of the mRNA was estimated to be 0.7 kb. In maturing seeds, a high level of mRNA was observed at 30 DAF, but the mRNA was scarcely detected at 50 DAF and 80 DAF. mRNA for the HMG-1 protein was expressed prior to those for seed storage protein which were expressed at about 40 DAF w15x. This supports the possibility that HMG-1 protein is involved in the expression of the seed storage protein genes. In germinated seeds, the expression of mRNA for CgHMG-1 was observed 2 days after imbibition started ŽDAI.. The level then increased at 6 DAI and a much higher level was observed at 10 DAI, at which the primary leaf began to expand. The level of mRNA for HMG-Y, which was high at 6 DAI, decreased at 10 DAI w11x. The
Fig. 3. Northern blot analysis of the HMG-1 mRNA from C. gladiata. PolyŽA.q RNA fractions were isolated from maturing Ž30, 50 and 80 DAF. and germinated Ž2, 6 and 10 DAI. seeds. PolyŽA.q RNA Ž1.5mg per lane. was denatured with glyoxal, separated on 1.4% agarose gel, transferred to a nylon filter and hybridized with the same probe as used in Southern blot analysis. Autoradiography was carried out at y808C for five days with an intensifying screen Župper panel.. The nylon filter was stained with a solution of 0.5 M sodium acetate ŽpH 5.4. and 0.04% methylene blue Žlower panel..
data of a cDNA for the HMG-1 protein will allow the further investigations on the roles of HMG-1 and HMG-Y proteins in the expression of seed storage protein genes in C. gladiata plant. Fig. 2. Southern blot analysis of the gene encoding HMG-1 protein from C. gladiata. Genomic DNA Ž30 mg per lane. was digested with restriction enzymes, separated on 1% agarose gel, denatured, transferred to a nylon filter and hybridized with the 32 P-labeled probe which was generated by the PCR method on the basis of Vicia faba HMG cDNA w4x. The washing was performed twice in 2=SSC Ž0.15 M NaClr0.015 M sodium citrate pH 7.6. containing 0.1% SDS at 658C. DNA size markers are shown on the right.
References w1x K.D. Grasser, Plant J. 7 Ž1995. 185–192. w2x K.D. Grasser, G. Feix, Nucleic Acids Res. 19 Ž1991. 2573– 2577. w3x T. Laux, R.B. Goldberg, Nucleic Acids Res. 19 Ž1991. 4769.
50
S. Yamamoto, T. Minamikawar Biochimica et Biophysica Acta 1396 (1998) 47–50
w4x K.D. Grasser, T. Wohlfarth, H. Baumlein, G. Feix, Plant ¨ Mol. Biol. 23 Ž1993. 619–625. w5x C.C. Zheng, A.Q. Bui, S.D. O’Neill, Plant Mol. Biol. 23 Ž1993. 813–823. w6x C.I. Webster, L.C. Packman, K.-H. Pwee, J.C. Gray, Plant J. 11 Ž1997. 703–715. w7x J.M. Wright, G.H. Dixon, Biochemistry 27 Ž1988. 576–581. w8x M.E. Bianchi, M. Beltrame, G. Paonessa, Science 243 Ž1989. 1056–1059. w9x P.M. Pil, S.J. Lippard, Science 256 Ž1992. 234–237. w10x S. Yamamoto, M. Nishihara, H. Morikawa, D. Yamauchi, T. Minamikawa, Plant Mol. Biol. 27 Ž1995. 729–741.
w11x S. Yamamoto, T. Minamikawa, Plant Mol. Biol. 33 Ž1997. 537–544. w12x S. Yamamoto, T. Minamikawa, Plant Sci. 124 Ž1997. 165– 173. w13x M.A. Frohman, M.K. Dush, G.R. Martin, Proc. Natl. Acad. Sci. U.S.A. 85 Ž1988. 8998–9002. w14x H.-M. Jantzen, A. Admon, S.P. Bell, R. Tjian, Nature 344 Ž1990. 830–836. w15x D. Yamauchi, K. Nakamura, T. Asahi, T. Minamikawa, Eur. J. Biochem. 170 Ž1988. 515–520. w16x J.D. Thompson, D.G. Higgins, T.J. Gibson, Nucleic Acids Res. 22 Ž1994. 4673–4680.