Download Nucleotide Sequences of cDNAs Encoding Two

Received for publication November 5, 1991
Accepted December 7, 1991
Plant Physiol. (1992) 98, 1520-1522
0032-0889/92/98/1 520/03/$O1 .00/0
Plant Gene Register
Nucleotide Sequences of cDNAs Encoding Two Members of
the Brazil Nut Methionine-Rich 2S Albumin Gene Family
Susan B. Altenbach*l, Karen W. Pearson, and Samuel S. M. Sun
The Plant Cell Research Institute, Inc., Dublin, California 94568 (S.B.A., K.W.P.); and Department of Plant
Molecular Physiology, University of Hawaii, Honolulu, Hawaii 96822 (S.S.M.S.)
mology to pHS-3 at the DNA level and 88% homology at the
protein level. The protein encoded by pHS-9 contains 16.8%
methionine, and the protein encoded by pHS-6 contains
20.8% methionine.
The 2S albumins ofBrazil nut (Bertholletia excelsa H.B.K.)
are a group of abundant seed proteins containing high concentrations of the sulfur amino acids. Because these proteins
contain about 18% methionine, the albumins have been used
in gene transfer experiments to improve the methionine content of seed proteins from other crop plants (1, 3). The Brazil
nut albumins consist of two subunits, a small subunit of 3 kD
and a large subunit of 9 kD, which are joined by disulfide
linkages. Both subunits are derived from a precursor polypeptide in a series of proteolytic processing events (5, 8). Like
many other seed storage proteins, the Brazil nut albumins are
encoded by a multigene family. Amino acid sequences of five
isoforms of the large subunit (2, 4) and one isoform of the
small subunit (4) have been determined. In addition, the
nucleotide sequences of cDNAs encoding two other isoforms
have been reported (2, 5). Considerable homology is apparent
among proteins encoded by members of the Brazil nut 2S
albumin family. The Brazil nut protein also exhibits strong
structural homology with the 2S albumins of other seeds,
including those from rapeseed, Arabidopsis, sunflower, castor
bean, barley, and lupin (2, 4, 6, 7). Indeed, the positions of
seven to eight cysteine residues have been conserved in albumins from these species. These homologies have been important in defining regions of 2S seed proteins that might tolerate
modifications without adversely affecting processing, transport, or stability of the proteins in seeds. In this paper, the
nucleotide sequences of cDNAs encoding two new isoforms
of the Brazil nut 2S albumin are reported (Fig. 1, Table I).
These sequences are compared with the sequence of cDNA
clone pHS-3, which encodes the entire protein precursor of a
Brazil nut albumin (2). One cDNA, pHS-9, shows 99% homology to pHS-3 at the nucleotide level and 96% homology
at the protein level. The other cDNA, pHS-6, has 95% ho-
LITERATURE CITED
1. Altenbach SB, Kuo CC, Staraci LC, Pearson KW, Wainwright
C, Georgescu A, Townsend J (1992) Accumulation of a Brazil
nut albumin in seeds of transgenic canola results in enhanced
levels of seed protein methionine. Plant Mol Biol 18: 235-245
2. Altenbach SB, Pearson KW, Leung FW, Sun SSM (1987) Cloning and sequence analysis of a cDNA encoding a Brazil nut
protein exceptionally rich in methionine. Plant Mol Biol 8:
239-250
3. Altenbach SB, Pearson KW, Meeker G, Staraci L, Sun SSM
(1989) Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich protein in transgenic plants. Plant Mol Biol 13:
513-522
4. Ampe C, Van Damme J, de Castro L, Sampaio MJ, Van Montagu
M, Vandekerckhove J (1986) The amino acid sequence of the
2S sulfur-rich protein from seeds of Brazil nut (Bertholletia
excelsa H.B.K.). Eur J Biochem 159: 597-604
5. de Castro LAB, Lacerada Z, Aramayo RA, Sampaio MJAM,
Gander ES (1987) Evidence for a precursor molecule of Brazil
nut 2S seed proteins from biosynthesis and cDNA analysis.
Mol Gen Genet 206: 338-343
6. Krebbers E, Herdies L, De Clercq A, Seurinck J, Leemans J,
Van Damme J, Segura M, Gheysen G, Van Montagu M,
Vandekerckhove J (1988) Determination of the processing sites
of an Arabidopsis 2S albumin and characterization of the
complete gene family. Plant Physiol 87: 859-866
7. Lilley GG, Caldwell JB, Kortt AA, Higgins TJ, Spencer D (1989)
Isolation and primary structure for a novel methionine-rich
protein from sunflowerseeds (Helianthus annus L.). In TH
Applewhite, ed, Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs., American Oil Chemists' Society, Champaign, IL, pp
497-502
8. Sun SSM, Altenbach SB, Leung FW (1987) Properties, biosynthesis and processing of a sulfur-rich protein in Brazil nut
(Bertholletia excelsa H.B.K.). Eur J Biochem 162: 477-483
'Present address: USDA-ARS, 800 Buchanan Street, Albany, CA
94710.
1520
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BRAZIL NUT METHIONINE-RICH 2S ALBUMIN GENE FAMILY
pHS-9
pHS-6
pHS-9
pHS-6
45
93
141
189
237
285
333
381
429
482
545
TTATACCCAGAATCACC ATG GCG
TCACC ATG GCG
Met Ala
Met Ala
GC_ CTC CTT
GCC CTC CTT
Ala Leu Leu
Ala Leu Leu
GTC CTC ATG
GTC CTC CTG
Val Leu Met
Val Leu Le
GCC CTC GGC
GTC CTC GGC
Ala Leu Gly
Val Leu Gly
AAG ATT TCA GTT GCG
AAI ATT TCA GTT GTG
Lys Ile Ser Val Ala
A= Ile Ser Val Val
CAC GCC ACC
CAC GCC ACC
His Ala Thr
His Ala Thr
ACC GTC ACC ACC ACA GTG GTG GAG GAG GAG AAC
ACC GTC ACC ACC ACA GTG GTG GAG GAG GAG AAC
Thr Val Thr Thr Thr Val Val Glu Glu Glu Asn
Thr Val Thr Thr Thr Val Val Glu Glu Glu Asn
GAG CAG ATG
GAG CAG ATG
Glu Gln Met
Glu Gln Met
CAG AGA CAG
CAG AGA CAG
Gln Arg Gln
Gln Arg Gln
CAG
CAG
Gln
Gln
GCA GCA
GCA GCA
GCC
GCC
Ala
Ala
CAG GAG GAG TGT CGC
CAG GAl --- TG CGCO
Gln Glu Glu Cys Arg
Gln
Cys Pro
TAC
TAC
Tyr
Tyr
ATG
ATG
Met
Met
TAC CAG ACC ATG CCC AGG CGG GGA
TAC CAG ACC ATG CCC AGG CGG GGA
Tyr Gln Thr Met Pro Arg Arg Gly
AGA CAG CAG ATG GAG GAG AGC
AGA CAG AIG ATG MAG GAG AGC
Arg Gln Gln Met Glu Glu Ser
Met Ly. Glu Ser
Arg Gln
CCG
CCG
Pro
Pro
Tyr Gln Thr Met Pro Arg Arg Gly
GAG CCG CAl ATG AGC GAG
GAG CCG CAC ATG AGC GAG
Glu Pro His Met Ser Glu
Glu Pro His Met Ser Glu
TGC
TGC
Cys
Cys
TGC
TGC
Cys
Cys
ATG
ATG
Met
Met
GAG AGC TGC
GAG AGC TGC
Glu Ser Cys
Glu Ser Cys
AGA
AGA
Arg
Arg
CAG AAG GAG ATG
CAG CAG GAG ATG
Gln LVS Glu Met
Gln gln Glu Met
TGC
TGC
Cys
Cys
CAA CCC CGA GGG
CAA CCC CGA GGG
Gln Pro Arg Gly
Gln Pro Arg Gly
CTG GCC GAG AAT ATC
ATG GCC GAG AAT CTC
Leu Ala Glu Asn Ile
Met Ala Glu Asn Leu
CCC
CCC
Pro
Pro
GAA GGC TTA
GAA GGC TTA
Glu Gly Leu
Glu Gly Leu
GAG GGG ATG
GAG GGG ATG
Glu Gly Met
Glu Gly Met
AGG AGS ATG ATG AQG AITG
AGG ATG ATG ATG AQG AIG
Arg ALg Met Met Arg =
Arg Met Met Met Ara Me
ATG
ATG
Met
Met
GAC
GAC
Asp
Asp
25
41
57
73
89
CAA
CAA
Gln
Gln
105
CGA AGG ATG ATG AGG
CGA AIG ATG ATG AGG
Arg Arg Met Met Arg
Arg = Met Met Arg
121
AAC CTC AGT CCC ATG AGA TGC
AAC CTC AGT CCC CAG AGA TGC
Asn Leu Ser Pro Met Arg Cys
Asn Leu Ser Pro §jn Arg Cys
137
GAG CAG ATG
GAG CAG ATG
Glu Gln Met
Glu Gln Met
CCT TCC CGC TGC
CCT TCC CGC TGC
Pro Ser Arg Cys
Pro Ser Arg Cys
ATG GGT GGC TCC ATT GCC
ATG GDG GGC TCC ATG GCC
Met Gly Gly Ser Ile Ala
Met Ala Gly Ser get Ala
GAG CAG CTG
GAG CAG CTG
Glu Gln Leu
Glu Gln Leu
9
Ala Ala
Ala Ala
GCC TTC CGG
GCC TTC CGG
Ala Phe Arg
Ala Phe Arg
ATG CTC AGC CAC TGC CGG ATG
ATG CTC AGC CAC TGC CGG ATG
Met Leu Ser His Cys Arg Met
Met Leu Ser His Cys Arg Met
1521
GGG TTC TGA ATCTGCCACTAGCCAGTGCTGTA
GGG TTC TGA ATCAGCCACTAGCCAGTGCTGTA
Gly Phe
Gly Phe
Figure 1. Nucleotide sequences and deduced
amino acid sequences of cDNA clones pHS-9
and pHS-6, which encode two members of the
Brazil nut 2S albumin gene family. Nucleotide
and amino acid residues that differ from a third
family member, pHS-3, reported previously (2),
are underlined. Two regions in the large subunit
of the protein in which methionine residues are
clustered are indicated in boxes. The putative
processing sites involved in the maturation of
the precursor are indicated by vertical lines.
146
AATGTTAATAAGGCTCTCACAAACTAGCTCTTTGTTGGCTTTTGGCCGGAGACTAGGGTGTGG
AATGTTAATAAGGCTCTCACAAACTAGCGCTLTGTLGGCTTTTGGCCGGAGACTAGGGTGTGG
GATTAATAATAATAGCACACTATCGTGTGTTCTCAGCTTCAAGGAC
GATTAATAATAATAGCACAITATCGTGTGTTITC
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1 522
ALTENBACH ET AL.
Plant Physiol. Vol. 98, 1992
Table I. Characteristics of cDNAs Encoding Brazil Nut 2S Methionine-Rich Albumins
Organism:
Brazil nut, Bertholletia excelsa H.B.K.
Location in Genome:
Multigene family of unknown location.
Gene, Function, Pathway:
Water-soluble seed storage protein unusually rich in methionine.
Techniques:
cDNA cloning, restriction fragment subcloning, dideoxynucleotide sequencing of both DNA strands.
Method of Isolation, Subsequent Identification:
Isolated from cDNA library of 9-month-old developing seeds by hybridization with 18-base oligonucleotide probe based on amino acid sequence of protein, nucleotide sequence compared to amino acid
sequence of purified protein.
Expression Characteristics:
Developmentally regulated. cDNAs hybridize to a mRNA of approximately 700 bases in 8- to 9month-old developing Brazil nut seeds.
Features of cDNA Structure:
Translation start site at nucleotide 18 and stop site at nucleotide 456 in pHS-9. Start site at nucleotide
6 and stop site at nucleotide 441 in pHS-6.
Codon Usage:
60% (G + C) content in coding region of both cDNAs.
Structural Features of the Protein:
The mature protein consists of two subunits joined together by disulfide linkages. In pHS-9, the small
subunit is encoded by nucleotides 127 to 209 and the large subunit is encoded by nucleotides 225
to 443. The protein is synthesized as a 146-amino acid precursor that is processed extensively. The
first 22 amino acids of the precursor represent a signal peptide that is processed cotranslationally.
Portions of the precursor are removed from the NH2 terminus (amino acids 23-36), the COOH
terminus (amino acids 143-146) and the region separating the two subunits (amino acids 65-69).
The protein encoded by pHS-6 contains one less amino acid in the small subunit. Methionine residues
are clustered in two regions of the large subunit, between amino acids 98 and 104 and between 116
and 121 (pHS-9).
Antibodies:
Both monoclonal and polyclonal antibodies specific for the Brazil nut methionine-rich albumin have
been prepared by this laboratory.
GenBank Accession Nos.:
M80400 (pHS-9) and M80399 (pHS-6)
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