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Cloning and sequencing of 5′ flanking sequence from the gene encoding 2S storage protein, from two Brassica species
Gene, 133(1993)301-302 0 1993 Elsevier Science Publishers
GENE
B.V. All rights reserved.
0378-l 119/93/$06.00
301
07390
Brief Notes
Cloning and sequencing of 5’ flanking sequence from the gene encoding 2s storage protein, from two Brassica species* (Promoter; tissue specificity; mustard rape; napin; homology)
Santanu
Dasgupta,
Department of Biochemistry,
Jaydip Dasgupta** Bose Institute, Calcutta-700054,
and Radha K. Mandal India
SUMMARY
Using oligodeoxyribonucleotide primers and the polymerase chain reaction, we have cloned and sequenced about 1.2 kb of upstream sequences from two members of the 2s seed storage protein-encoding gene family from Brassica juncea and B. oleracea. The two sequences bear more than 90% homology and have characteristic seed-specific promoter motifs. The high degree of sequence conservation indicates that this napin-encoding gene family evolved earlier than the divergence of the three primary Brassica species and their amphidiploids, and the sequences have been conserved due to some metabolic constraints in seed development.
The 2S storage protein (napin) is one of the major seed storage proteins in the Brassica species (Lonnerdal and Janson, 1972). In view of the wide interest in rapeseed mustard as a source of quality nutritive proteins, these proteins and their genes have been studied by a number of groups (Crouch and Sussex, 1981; Crouch et al., 1983; Josefsson et al., 1987; Kridl et al., 1991; Dasgupta and Mandal, 1991). With our interest in improving seed-protein quality of Indian cultivated rapeseed mustard, we decided to clone the 5’ upstream sequences of 2S napin genes from B. juncea and B. oleracea for subsequent use for constructing a homologous seed-specific expression system. Using oligo-nucleotide primers corresponding to the two end regions of the published 5’ flanking sequence Correspondence to: Dr. R.K. Mandal, Department Institute, Calcutta-700054, India. Tel. (91-33)
of Biochemistry, Bose 3779219; Fax (91-33)
34-3886. *On request, the authors will supply detailed experimental the conclusions reached in this Brief Note. **Present address: Department of Agronomy, University
evidence for of Kentucky,
KY 40506, USA. Tel. (606) 257-3637. Abbreviations: B., Brassica; bp, base pair(s); GCG, Genetics Computer Group (Madison, WI, USA); kb, kilobase or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide.
of the napin gene (Josefsson et al., 1987), the 5’ flanking sequences from B. juncea var. RLM 198 and B. oleracea var. capitata were amplified from genomic DNA by polymerase chain reaction (PCR) and cloned into pBSKS(+) vector (Stratagene, La Jolla, CA, USA). The sequences of the 1137 bp cloned were determined by the dideoxy method and computer-alligned by GCG program. The sequences have many general and seed-specfic transcription regulatory motifs (Shirsat, 1991). A possible TATA box lies from nt 1066 to 1072, and a putative CAAT box from nt 1048 to 1052. The G-box motif (CACGTG) is present from nt 1033 to 1038 which is approximately 66 nt upstream from the transcription start point deduced by analogy with that of napin (Josefsson et al., 1987). This G-box is presumed to act as an activator of transcription in other systems (Williams et al., 1992). The interesting feature of this sequence is the close (over 90%) homology between B. juncea (which is an amphidiploid of B. campestris and B. nigra) and B. oleracea and with that of a napin gene promoter pN36 (Josefsson et al., 1987) isolated from B. napus (an amphidiploid of B. oleracea and B. campestris). The upstream sequences from a genotype of B. campestris (B. rapa) isolated by Kridl et al. (1991) bear a close homology with those of another napin promoter sequenced by Scofield
302 . TCTTCATCOGTGATTGATT~CTTTAAAGACtTATGTTTCTTATCTT~T~CTGAG~AA~ :::::::::::l:::::::::I:::I::I:::::::::::I::::: 1::::::::::1:: TCTTCATCGGTGATTGATTCCTTTAAAGACTTTCICAGTTACCACTTATATTCTTGACTTTCTG
. ..T.A.TT..CAGTTACCiCTTATATTC;GGACTTTCTii : : :I ::l:III:II:III:tI:I
s3 :ll::lll: 100
. ACTGCATCC;CATTTTTCC;ACATTTTAA~TTTCACTAr;TTCTTCTTCTTTGAGGA.A;iAMCAGTTCAGATGGCAGA;ATGTATCAA;: :::: I~I:::::::::::::::~:: l:::II:I::::::::::::I:: :::::::::1t::1: : l:llI ::II::II:Ii:I:::I:Il:t:I ACTG.ATCCTCATTTTTCCAACATTTTAAATTTCACTATTGGCTGAATG . ..CTTCTTCTTTGAOOAGA.AAACAATTCAGATGOCAGAAATGTATCAAC . . CAATGCATA;ATACAAATG;ACCTCTTGT~CTTAAAACATCTATCGGAT~GTTCCATTT~CTTTGTCAT~CAATTAGGTGACTACTTTA~ATTATTCAC~ :::::I:::::::::::::II:I:I::::I:: I::l::II::I:::::IIl:II::II ::I:I:III:IIlII:l 1:11111111::11:11111111 CAATGCATATATACAAATGTACCTCTTGTTCTCATCCATCCMTTA.GTGACTACTTTATATTATTCACT CCTCTTTATTACTATTTTC;TGCGA~GT;GCCAtGTAC; ::::::::::I::::::I::::I:: :::::::I:l:::::I:II::: ::tII::ll ::II:::::I:I:I:::!III:III::I:Illl:ll:lll: CCTCTTTATTACTATTTTCATGCGA.GGTTGCCATGTACATTATATTT.GTAAGGATT.GACGCTATTGAGCGTTTTTCTTCAATTTTCTTTATTTAAGA . . . CATGGGTATG.AATGTGTG;TAGAGTGOO;iTT~ATGAGAT.TCCCG.TCAAAGTGAATTGOCATACCGTCCCCGGGTAAGGGTGACC.CCCCATCCTGG llllllll,l I..,II,,II 1:::1::::::::1: ::I::: ::l:llI I :I :I ::ll:l: I:::I”“” II,.., I :: :II::: CATGGGTATGAAATGTGTGTTAGAGTTGGGTTGAATGAGATAT.ACOTTC.AAGTGAAGTGG~ATACCGTTCTCGAGTAAGGATGACCTACCTATTCTTG GGA.AAATGTTACATTTTA~GGTACTCAG:G.GGAATOG~GT.CCCATT;CTCAAATCC~GGGGAAATG;TICCCTTCA~CATAAAATT~AACC~~~ :: ::::::::::::::: ::t :::: : ::: : :: :: :: ::1::1:: I: :: ::I1 :I: ::::I:l::I:I:::l: AGACAAATGTTACATTTTA..GTA.TCAGAGTAAAAT.GTGTACCTATAACTCAAATTCGArlGACArGTATCCATGTAT~ATTCAACATAAAATTAAA.CCAGCCT . . GGAC.CGGAATCCCCAATTCAAG..GTTTTT..AACCGGCCTTTCGGOAGCCCCCAACCGGACCIATTCCG ::::: 1::::::::::: ::::: ::I: : :I: : :: I: : :: ::: ::: : I GCACTC.GCATCCACATTTCAAGATATTTTTCAAACC.G...TTC..G.OCTCCTATCC..ACC.GGG.....T......G.T.AACAAGACQGATTCC.
1. Alignment
of 1.3-kb 5’ upstream
sequences
of 2s protein
genes from Erassica oleracea
292 284
391 . 488 :llll
II
II
II
I 489 583
I:
::: 584 878
:I::::
II
:I:: 859 755
111::
:I1 758 855
::,ll::
,,,,,.,,$ 1*III*11 856 054
::II:II::::I::I:I 954 . 1052 1052 .
v * CTCCCTTATCGCCTATAAATTAACTCATCC~TTCACTCTTTACTCAAACCAAAACTCATCAATACAAACAAGATTAAAAACATA :::: :::I ::::::::::::::::::I:::I ::::::::::::::::::::::::I::::I :::::::::I:::I::I::::I C_TCCTTTATAGCCTATAAATTAACTCATCCGCTTCACTCTTTACTCAAACCAAAACTCATCAATACAAACAAGATTAAAAACATA .... . .... . Fig.
195
392
GAATTGGGA;GG.TTTTGG&CGAATTC&AA.TTAAAT; . . . . . TG..:..A.C . . C .:...C..GTA;AATTCTGGTiAAGCTGCCAiATTATA.TC;: IIIII II. :l .III.,..I. : l IIIIIIII. II. ,;I IIaII ,I. I~**ImI** III :: :: : : ::::::llll IllIll: :III GAATTTGGA.OGATTTTGACTCAAATTCCCAATTTATATTGACCGTGACTAAATCAACTTTAACTTCTATAATTCTGATTAA~TC~AATTTATATTCC GAACGGCAC;ACCTCCAAA;TTTAAAAAG;CTCTAATCCC :ll;::l ::::::::II::::l:::::I:: : :I::::: ::::::: ::::: :i ll::::lll: ::1:1::::: CAACGGCACTACCTCCAAAATTTATATAGACTCTCATCCCCTTTTAAA.CC.AACTTAGTAAACGTTTTTTTTTTTAATTAAGTTTTTAC . CTTGTTTTGGAAGGGAGTCGTTCATGAGATGCCATG~AGAACATTAGCTA~.CGOTTACACATAGCATGCAGCCGCGtAGCATTGtT;tTCTTCGCC~ II*IIIII ;: ;:l;l;!; .I,,.... :: ..,.II*111#~1IIII*III . . . . . . . . . . . . ..‘....I...: : : :::):::I ::::::::::::::I::: CTTGTTTTTAAAAAGAATCGTTCATAAGAT~ATG~AGAACATTA~TA.CAC.GTTACACATAGE?E~E?GCCOCGOAGAATTGTTTTTCTTCGCCA . CTTGTCCCTCCCTTCAAACACCTAAGAGCTTCT.T.TAAAACAGCCCACACAGCCAATCACAT~T~ATGCATTATT~CACGTGATCGCCAT~AAA; ,111.. 111111111111111111~..II,II .I..,. ,III,.IIII~IIIIIIIII*IIIII : : : :: : :::I:: :::::::::::::::::I::::::::::::::::::I::III:II: CTTGTCACTCCCTTCAAACACCTAAGAGCTTCTCTCTCACAC.G.ACACACATACAATCACATGCGTG~~~~~TTATI*(;A_CAAAT
192
1137 1137
(upper lines) and B. juncea (lower lines) (EMBL Data
Library/Gen Bank accession No. X70333 and X67833 respectively). The TATA box (dotted line), putative CAAT-like box (solid line), G-box (dashed line), seed-specific CATGCA (alternate purine-pyrimidine sequence, asterisk under nt) and presumed transcription start point (arrowhead) are indicated.
and Crouch (1987). Our sequence results therefore indicate that at least this member of the napin gene family evolved earlier than the time of divergence of B. oleracea, B. campestris, B. nigra and their amphidiploids. Research was supported by a UNDP/FAO project (IND/87/017) and DST, Govt. of India. Thanks are due to Dr. Arthur Hunt and Dr. I.B. Maiti of the University of Kentucky (Lexington, KY, USA) for technical help, to Dr. M.L. Crouch, Indiana University (Bloomington, IN, USA), and to Dr. L.-G. Josefsson, Swedish Univ. of Agric. Sci. (Uppsala, Sweden) for napin clones, and to DIC, Bose Inst. for sequence analysis.
Crouch,
M.L., Tenbarge,
K.M., Simon, A.E. and Ferl, R.: cDNA clones
for Brassica napus seed storage proteins : evidence from nucleotide sequence analysis that both subunits of napin are cleaved from a precursor Dasgupta,
polypeptide. S. and Mandal,
J. Mol. Appl. Genet. 2 (1983) 273-283. R.K.: Characterization
of 2s seed storage
protein of Brassica campestris and its antigenic homology with seed proteins of other Cruciferae. Biochem. Int. 25 (1991) 409-417. Josefsson, L.-G., Lenman, M., Ericson, M.L. and Rask, L.: Structure of a gene encoding the 1.7s storage protein, napin, from Brassica napus. J. Biol. Chem. 262 (1987) 12196-12201. Kridl, J.C., McCarter, D.W., Rose, R.E., Scherer, D.E., Knutzon, Radke, S.E. and Knauf, V.C.: Isolation and characterization expressed
napin
gene from Bras&a
rapa.
Seed Sci. Res. 1 (1991)
209-219. Lonnerdal, B. and Janson, J.-C.: Studies on Brassica seed proteins. I. The low molecular weight proteins in rapeseed. Isolation and characterization. Biochim. Biophys. Acta 278 (1972) 1755183. Shirsat, A.H.: Control of gene expression in the developing Grierson,
REFERENCES Crouch, M.L. and Sussex, I.M.: Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro. Planta 153 (1981) 64-74.
D.S., of an
D. (Ed.),
Developmental
Regulation
seed. In:
of Plant
Gene
Expression. Blackie, London, 1991, pp. 153-181. Williams, M.E., Foster, R. and Chua N.-H.: Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding. Plant Cell 4 (1992) 485-496.
GENE
B.V. All rights reserved.
0378-l 119/93/$06.00
301
07390
Brief Notes
Cloning and sequencing of 5’ flanking sequence from the gene encoding 2s storage protein, from two Brassica species* (Promoter; tissue specificity; mustard rape; napin; homology)
Santanu
Dasgupta,
Department of Biochemistry,
Jaydip Dasgupta** Bose Institute, Calcutta-700054,
and Radha K. Mandal India
SUMMARY
Using oligodeoxyribonucleotide primers and the polymerase chain reaction, we have cloned and sequenced about 1.2 kb of upstream sequences from two members of the 2s seed storage protein-encoding gene family from Brassica juncea and B. oleracea. The two sequences bear more than 90% homology and have characteristic seed-specific promoter motifs. The high degree of sequence conservation indicates that this napin-encoding gene family evolved earlier than the divergence of the three primary Brassica species and their amphidiploids, and the sequences have been conserved due to some metabolic constraints in seed development.
The 2S storage protein (napin) is one of the major seed storage proteins in the Brassica species (Lonnerdal and Janson, 1972). In view of the wide interest in rapeseed mustard as a source of quality nutritive proteins, these proteins and their genes have been studied by a number of groups (Crouch and Sussex, 1981; Crouch et al., 1983; Josefsson et al., 1987; Kridl et al., 1991; Dasgupta and Mandal, 1991). With our interest in improving seed-protein quality of Indian cultivated rapeseed mustard, we decided to clone the 5’ upstream sequences of 2S napin genes from B. juncea and B. oleracea for subsequent use for constructing a homologous seed-specific expression system. Using oligo-nucleotide primers corresponding to the two end regions of the published 5’ flanking sequence Correspondence to: Dr. R.K. Mandal, Department Institute, Calcutta-700054, India. Tel. (91-33)
of Biochemistry, Bose 3779219; Fax (91-33)
34-3886. *On request, the authors will supply detailed experimental the conclusions reached in this Brief Note. **Present address: Department of Agronomy, University
evidence for of Kentucky,
KY 40506, USA. Tel. (606) 257-3637. Abbreviations: B., Brassica; bp, base pair(s); GCG, Genetics Computer Group (Madison, WI, USA); kb, kilobase or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide.
of the napin gene (Josefsson et al., 1987), the 5’ flanking sequences from B. juncea var. RLM 198 and B. oleracea var. capitata were amplified from genomic DNA by polymerase chain reaction (PCR) and cloned into pBSKS(+) vector (Stratagene, La Jolla, CA, USA). The sequences of the 1137 bp cloned were determined by the dideoxy method and computer-alligned by GCG program. The sequences have many general and seed-specfic transcription regulatory motifs (Shirsat, 1991). A possible TATA box lies from nt 1066 to 1072, and a putative CAAT box from nt 1048 to 1052. The G-box motif (CACGTG) is present from nt 1033 to 1038 which is approximately 66 nt upstream from the transcription start point deduced by analogy with that of napin (Josefsson et al., 1987). This G-box is presumed to act as an activator of transcription in other systems (Williams et al., 1992). The interesting feature of this sequence is the close (over 90%) homology between B. juncea (which is an amphidiploid of B. campestris and B. nigra) and B. oleracea and with that of a napin gene promoter pN36 (Josefsson et al., 1987) isolated from B. napus (an amphidiploid of B. oleracea and B. campestris). The upstream sequences from a genotype of B. campestris (B. rapa) isolated by Kridl et al. (1991) bear a close homology with those of another napin promoter sequenced by Scofield
302 . TCTTCATCOGTGATTGATT~CTTTAAAGACtTATGTTTCTTATCTT~T~CTGAG~AA~ :::::::::::l:::::::::I:::I::I:::::::::::I::::: 1::::::::::1:: TCTTCATCGGTGATTGATTCCTTTAAAGACTTTCICAGTTACCACTTATATTCTTGACTTTCTG
. ..T.A.TT..CAGTTACCiCTTATATTC;GGACTTTCTii : : :I ::l:III:II:III:tI:I
s3 :ll::lll: 100
. ACTGCATCC;CATTTTTCC;ACATTTTAA~TTTCACTAr;TTCTTCTTCTTTGAGGA.A;iAMCAGTTCAGATGGCAGA;ATGTATCAA;: :::: I~I:::::::::::::::~:: l:::II:I::::::::::::I:: :::::::::1t::1: : l:llI ::II::II:Ii:I:::I:Il:t:I ACTG.ATCCTCATTTTTCCAACATTTTAAATTTCACTATTGGCTGAATG . ..CTTCTTCTTTGAOOAGA.AAACAATTCAGATGOCAGAAATGTATCAAC . . CAATGCATA;ATACAAATG;ACCTCTTGT~CTTAAAACATCTATCGGAT~GTTCCATTT~CTTTGTCAT~CAATTAGGTGACTACTTTA~ATTATTCAC~ :::::I:::::::::::::II:I:I::::I:: I::l::II::I:::::IIl:II::II ::I:I:III:IIlII:l 1:11111111::11:11111111 CAATGCATATATACAAATGTACCTCTTGTTCTCATCCATCCMTTA.GTGACTACTTTATATTATTCACT CCTCTTTATTACTATTTTC;TGCGA~GT;GCCAtGTAC; ::::::::::I::::::I::::I:: :::::::I:l:::::I:II::: ::tII::ll ::II:::::I:I:I:::!III:III::I:Illl:ll:lll: CCTCTTTATTACTATTTTCATGCGA.GGTTGCCATGTACATTATATTT.GTAAGGATT.GACGCTATTGAGCGTTTTTCTTCAATTTTCTTTATTTAAGA . . . CATGGGTATG.AATGTGTG;TAGAGTGOO;iTT~ATGAGAT.TCCCG.TCAAAGTGAATTGOCATACCGTCCCCGGGTAAGGGTGACC.CCCCATCCTGG llllllll,l I..,II,,II 1:::1::::::::1: ::I::: ::l:llI I :I :I ::ll:l: I:::I”“” II,.., I :: :II::: CATGGGTATGAAATGTGTGTTAGAGTTGGGTTGAATGAGATAT.ACOTTC.AAGTGAAGTGG~ATACCGTTCTCGAGTAAGGATGACCTACCTATTCTTG GGA.AAATGTTACATTTTA~GGTACTCAG:G.GGAATOG~GT.CCCATT;CTCAAATCC~GGGGAAATG;TICCCTTCA~CATAAAATT~AACC~~~ :: ::::::::::::::: ::t :::: : ::: : :: :: :: ::1::1:: I: :: ::I1 :I: ::::I:l::I:I:::l: AGACAAATGTTACATTTTA..GTA.TCAGAGTAAAAT.GTGTACCTATAACTCAAATTCGArlGACArGTATCCATGTAT~ATTCAACATAAAATTAAA.CCAGCCT . . GGAC.CGGAATCCCCAATTCAAG..GTTTTT..AACCGGCCTTTCGGOAGCCCCCAACCGGACCIATTCCG ::::: 1::::::::::: ::::: ::I: : :I: : :: I: : :: ::: ::: : I GCACTC.GCATCCACATTTCAAGATATTTTTCAAACC.G...TTC..G.OCTCCTATCC..ACC.GGG.....T......G.T.AACAAGACQGATTCC.
1. Alignment
of 1.3-kb 5’ upstream
sequences
of 2s protein
genes from Erassica oleracea
292 284
391 . 488 :llll
II
II
II
I 489 583
I:
::: 584 878
:I::::
II
:I:: 859 755
111::
:I1 758 855
::,ll::
,,,,,.,,$ 1*III*11 856 054
::II:II::::I::I:I 954 . 1052 1052 .
v * CTCCCTTATCGCCTATAAATTAACTCATCC~TTCACTCTTTACTCAAACCAAAACTCATCAATACAAACAAGATTAAAAACATA :::: :::I ::::::::::::::::::I:::I ::::::::::::::::::::::::I::::I :::::::::I:::I::I::::I C_TCCTTTATAGCCTATAAATTAACTCATCCGCTTCACTCTTTACTCAAACCAAAACTCATCAATACAAACAAGATTAAAAACATA .... . .... . Fig.
195
392
GAATTGGGA;GG.TTTTGG&CGAATTC&AA.TTAAAT; . . . . . TG..:..A.C . . C .:...C..GTA;AATTCTGGTiAAGCTGCCAiATTATA.TC;: IIIII II. :l .III.,..I. : l IIIIIIII. II. ,;I IIaII ,I. I~**ImI** III :: :: : : ::::::llll IllIll: :III GAATTTGGA.OGATTTTGACTCAAATTCCCAATTTATATTGACCGTGACTAAATCAACTTTAACTTCTATAATTCTGATTAA~TC~AATTTATATTCC GAACGGCAC;ACCTCCAAA;TTTAAAAAG;CTCTAATCCC :ll;::l ::::::::II::::l:::::I:: : :I::::: ::::::: ::::: :i ll::::lll: ::1:1::::: CAACGGCACTACCTCCAAAATTTATATAGACTCTCATCCCCTTTTAAA.CC.AACTTAGTAAACGTTTTTTTTTTTAATTAAGTTTTTAC . CTTGTTTTGGAAGGGAGTCGTTCATGAGATGCCATG~AGAACATTAGCTA~.CGOTTACACATAGCATGCAGCCGCGtAGCATTGtT;tTCTTCGCC~ II*IIIII ;: ;:l;l;!; .I,,.... :: ..,.II*111#~1IIII*III . . . . . . . . . . . . ..‘....I...: : : :::):::I ::::::::::::::I::: CTTGTTTTTAAAAAGAATCGTTCATAAGAT~ATG~AGAACATTA~TA.CAC.GTTACACATAGE?E~E?GCCOCGOAGAATTGTTTTTCTTCGCCA . CTTGTCCCTCCCTTCAAACACCTAAGAGCTTCT.T.TAAAACAGCCCACACAGCCAATCACAT~T~ATGCATTATT~CACGTGATCGCCAT~AAA; ,111.. 111111111111111111~..II,II .I..,. ,III,.IIII~IIIIIIIII*IIIII : : : :: : :::I:: :::::::::::::::::I::::::::::::::::::I::III:II: CTTGTCACTCCCTTCAAACACCTAAGAGCTTCTCTCTCACAC.G.ACACACATACAATCACATGCGTG~~~~~TTATI*(;A_CAAAT
192
1137 1137
(upper lines) and B. juncea (lower lines) (EMBL Data
Library/Gen Bank accession No. X70333 and X67833 respectively). The TATA box (dotted line), putative CAAT-like box (solid line), G-box (dashed line), seed-specific CATGCA (alternate purine-pyrimidine sequence, asterisk under nt) and presumed transcription start point (arrowhead) are indicated.
and Crouch (1987). Our sequence results therefore indicate that at least this member of the napin gene family evolved earlier than the time of divergence of B. oleracea, B. campestris, B. nigra and their amphidiploids. Research was supported by a UNDP/FAO project (IND/87/017) and DST, Govt. of India. Thanks are due to Dr. Arthur Hunt and Dr. I.B. Maiti of the University of Kentucky (Lexington, KY, USA) for technical help, to Dr. M.L. Crouch, Indiana University (Bloomington, IN, USA), and to Dr. L.-G. Josefsson, Swedish Univ. of Agric. Sci. (Uppsala, Sweden) for napin clones, and to DIC, Bose Inst. for sequence analysis.
Crouch,
M.L., Tenbarge,
K.M., Simon, A.E. and Ferl, R.: cDNA clones
for Brassica napus seed storage proteins : evidence from nucleotide sequence analysis that both subunits of napin are cleaved from a precursor Dasgupta,
polypeptide. S. and Mandal,
J. Mol. Appl. Genet. 2 (1983) 273-283. R.K.: Characterization
of 2s seed storage
protein of Brassica campestris and its antigenic homology with seed proteins of other Cruciferae. Biochem. Int. 25 (1991) 409-417. Josefsson, L.-G., Lenman, M., Ericson, M.L. and Rask, L.: Structure of a gene encoding the 1.7s storage protein, napin, from Brassica napus. J. Biol. Chem. 262 (1987) 12196-12201. Kridl, J.C., McCarter, D.W., Rose, R.E., Scherer, D.E., Knutzon, Radke, S.E. and Knauf, V.C.: Isolation and characterization expressed
napin
gene from Bras&a
rapa.
Seed Sci. Res. 1 (1991)
209-219. Lonnerdal, B. and Janson, J.-C.: Studies on Brassica seed proteins. I. The low molecular weight proteins in rapeseed. Isolation and characterization. Biochim. Biophys. Acta 278 (1972) 1755183. Shirsat, A.H.: Control of gene expression in the developing Grierson,
REFERENCES Crouch, M.L. and Sussex, I.M.: Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro. Planta 153 (1981) 64-74.
D.S., of an
D. (Ed.),
Developmental
Regulation
seed. In:
of Plant
Gene
Expression. Blackie, London, 1991, pp. 153-181. Williams, M.E., Foster, R. and Chua N.-H.: Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding. Plant Cell 4 (1992) 485-496.