Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich protein in transgenic plants

We have constructed a chimeric gene encoding a Brazil nut methionine-rich seed protein which contains 18% methionine. This gene has been transferred to tobacco and expressed in the developing seeds. Tobacco seeds are able to process the methionine-rich protein efficiently from a larger precursor polypeptide of 17 kDa to the 9kDa and 3 kDa subunits of the mature protein, a procedure which involves three proteolytic cleavage steps in the Brazil nut seed. The accumulation of the methionine-rich protein in the seeds of tobacco results in a significant increase (30%) in the levels of the methionine in the seed proteins of the transgenic plants. Our data indicate that the introduction of a chimeric gene encoding a methionine-rich seed protein into crop plants, particularly legumes whose seeds are deficient in the essential sulfur-containing amino acids, represents a feasible method for improving the nutritional quality of seed proteins.     

Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine

We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations.     

Evidence for a precursor molecule of Brazil nut 2 S seed proteins from biosynthesis and cDNA analysis

Summary Seed storage proteins were extracted from Brazil nut (Bertholletia excelsa H.B.K.) seed embryos at various maturation stages. Salt-soluble and water-soluble proteins (globulins and albumins) were separated by gel chromatography and exhaustive dialysis against water. Both fractions were analysed by one- and two-dimensional polyacrylamide gel electrophoresis. Amino acid analysis revealed that both fractions are unusually high in methionine. The albumins consist of a family of low molecular weight polypeptides that are heterogeneous with respect to pI and are identical to the high methionine 2 S proteins described by Youle and Huang (1981). The biosynthesis of this class of proteins in maturing embryos was followed by in vivo labelling combined with immunological studies. Western blotting with monospecific antibodies against purified 2 S albumins and sequencing of a nearly complete cDNA clone revealed that they are synthesized via a precursor polypeptide.     

Cloning and sequencing of a cDNA encoding the major storage proteins of Theobroma cacao

The major storage proteins, polypeptides of 31 and 47 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), have been identified and partially purified by preparative gel electrophoresis. The polypeptides were both N-terminally blocked, but some N-terminal amino-acid sequence was obtained from a cyanogen bromide peptide common to both polypeptides, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy-DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 566-amino-acid polypeptide of M_r 65 612. The existence of a common precursor to the 31- and 47-kDa polypeptides of this size was confirmed by immunoprecipitation from total poly(A)⁺RNA translation products. The precursor has an N-terminal hydrophobic sequence which appears to be a typical signal sequence, with a predicted site of cleavage 20 amino acids after the start. This is followed by a very hydrophilic domain of 110 amino acids, which, by analogy with the cottonseed -globulin, is presumed to be cleaved off to leave a domain of approx. 47 kDa, very close to the observed size of the mature polypeptide. Like the hydrophilic domain of the cottonseed -globulin the cocoa hydrophilic domain is very rich in glutamine and charged residues (especially glutamate), and contains several Cys-X-X-X-Cys motifs. The cyanogen-bromide peptide common to the 47-kDa and 31-kDa polypeptides is very close to the proposed start of the mature domain, indicating that the 31-kDa polypeptide arises via further C-terminal processing. The polypeptide sequence is homologous to sequences of the vicilin class of storage proteins, previously found only in legumes and cotton. Most of these proteins have a mature polypeptide size of approx. 47 kDa, and are synthesised as precursors only slightly larger than this. Some, however, are larger polypeptides (e.g. -conglycinin from soybean is 72 kDa), usually due to an additional N-terminal domain. In cottonseed the situation appears to parallel that in cocoa in that the vicilin is synthesised as an approx. 70-kDa precursor and then processed to a 47-kDa (and in the case of cocoa also a 31-kDa) mature protein. In this context it is interesting that cotton is closer in evolutionary terms to cocoa than are the legumes, both cotton and cocoa being in the order Malvales.Abbreviations A absorbance - cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies.     

Cloning and sequence analysis of the cDNA encoding a snake neurotoxin precursor

A recombinant plasmid has been constructed containing a sequence of 186 nucleotides encoding a potent neurotoxin found in the venom of the sea-snake Laticauda semifasciata and designated as erabutoxin a. This sequence is flanked, in the upstream region, by a sequence of 60 nucleotides encoding a hydrophobic peptide fragment presumably involved in the secretion process of the neurotoxin. The sequence coding for the toxin ends with a termination codon which is followed by a 3'-untranslated sequence of approximately 240 nucleotides (excluding the poly(A) tract).     

Cloning and sequence analysis of a rat liver cDNA encoding acyl-peptide hydrolase

Acyl-peptide hydrolase catalyzes the removal of an N alpha-acetylated amino acid residue from an N alpha-acetylated peptide. Two overlapping degenerate oligonucleotide probes based on the sequence of a CNBr tryptic peptide, derived from purified rat acyl-peptide hydrolase, were synthesized and used to screen a rat liver lambda gt11 cDNA library. A 2.5-kilobase cDNA was cloned and sequenced. This clone contained 2364 base pairs of rat acyl-peptide hydrolase sequence but lacked a translational initiation codon. Using a 220-base pair probe derived from near the 5'-end of this almost full-length cDNA to rescreen the library, full-length clones were isolated, which contained an in-frame ATG codon at nucleotides 6-8 and encoded the NH2-terminal sequence, Met-Glu-Arg-Gln.... The DNA sequence encoded a protein of 732 amino acid residues, 40% of which were confirmed by protein sequence data from 19 CNBr or CNBr tryptic peptides. The isolated enzyme is NH2-terminally blocked (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), and based on the NH2-terminal protein sequence deduced from the DNA sequence and the sequence of the most NH2-terminal CNBr peptide, it is likely that the NH2-terminal residue is an acetylated methionine residue, since such residues are frequently juxtaposed to glutamyl residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). The RNA blot analysis revealed a single message of 2.7 kilobases in various rat tissues examined. Although this enzyme is known to be inhibited by diisopropyl fluorophosphate and acetylalanine chloromethyl ketone (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), no strong similarity in protein sequence has been found with other serine proteases. This result suggests that acyl-peptide hydrolase may be a unique serine protease.     

Cloning, sequence analysis, and expression of a cDNA encoding a plastid-localized heat shock protein in maize

We have cloned and characterized a cDNA encoding a maize (Zea mays L.) heat shock protein (HSP), HSP26. The mRNA of HSP26 is present as a single mRNA species of 1.1 kilobase pairs in size and is detectable when maize seedlings are treated at 40°C but not at 28°C. Accumulation of HSP26 mRNA was detected after 10 minutes of incubation at 40°C, reaching the maximum level after 1 hour. Comparison of the deduced amino acid sequence of maize HSP26 to other HSPs indicated a strong homology to the sequences of two nuclear encoded HSPs that are transported into the chloroplasts during heat shock: pea HSP21 and soybean HSP22. Maize HSP26 was also found to cross-react with anti-pea chloroplast HSP21 antibodies. Because of the sequence homology between maize HSP26, soybean HSP22, and pea HSP21, in vitro chloroplast protein import experiments were conducted. The in vitro synthesized maize HSP26 is specifically imported to the soluble fraction of the chloroplast and processed to a smaller polypeptide. The sequence homology and antibody cross-reactivity between maize HSP26 and pea HSP21 have allowed us to conclude that maize HSP26 is a nuclear-encoded, plastid-localized protein in maize.     

伴花生球蛋白cDNA克隆及其在花生发育种子中的表达

利用伴花生球蛋白多克隆抗体,免疫筛选花生品种汕油523成熟子叶中期cDNA文库得到6个阳性克隆.经过DNA序列测定和同源性分析确定为2组(Ahyα和Ahyβ) ,2组序列之间的同源性为97%.Ahyβ与花生过敏原Ara h1 p17以及Ahyα与花生过敏原Ara h1p41b的核苷酸相同性达到99%以上.以Ahy-βcDNA为探针的Northern blot分析结果表明,伴花生球蛋白基因在发育的花生种子中大量表达,而在幼苗的叶片中不表达.对成熟中期花生子叶表达序列标签(EST)分析,获得了包括5种花生球蛋白、2种伴花生球蛋白、6种conglutin蛋白的EST共70条,占总转录本的17%.     

Protein sorting and expression of a unique soybean cotyledon protein,GmSBP, destined for the protein storage vacuole

The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a re-characterization of GmSBP via sequence homology, cell biology, immunolocalization, and semi-quantitative analysis. The GmSBP proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and -conglycinin established several storage protein-like characteristics for GmSBP. All three proteins were present in a prevacuolar compartment and protein storage vacuole. All three proteins increased in expression during seed development and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, -conglycinin (/ subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitative differences between GmSBP and glycinin may be attributed to the unconserved order and spacing of cis-acting regulatory elements present within the promoter regions. Ultimately, GmSBP is transported to the mature protein storage vacuole. The biological function of GmSBP within the protein storage vacuole remains uncertain, but its localization is a remnant of its evolutionary link to a globulin-like or vicilin-like ancestor that gave rise to the 7S family of storage proteins.     

Cloning and sequence analysis of cDNA encoding Rhizopus niveus lipase.

Complementary DNA encoding Rhizopus niveus lipase (RNL) was isolated from the R. niveus IF04759 cDNA library using a synthetic oligonucleotide corresponding to the amino acid sequence of the enzyme. A clone, which had an insert of 1.0 kilobase pairs, was found to contain the coding region of the enzyme. The lipase gene was expressed in Escherichia coli as a lacZ fusion protein. The mature RNL consisted of 297 amino acid residues with a molecular mass of 32 kDa. The RNL sequence showed significant overall homology to Rhizomucor miehei lipase and the putative active site residues were strictly conserved.     

Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells

A cDNA clone, pHGR21 encoding poly-ubiquitin, was isolated from a human ovarian granulosa cDNA library. This clone contained three complete, and part of a fourth, ubiquitin coding sequence joined head to tail with no spacer sequences. Northern analysis employing a restriction fragment comprising a complete ubiquitin coding unit indicated the existence of two mRNA species of 1.1kb and 2.8kb. Sequence comparison of pHGR21 with the known two human ubiquitin genes revealed differences to the human ubiquitin-3 repeat gene but significant homology to the human ubiquitin-9 repeat gene. The untranslated 3'-region and the adjacent ubiquitin coding repeat were found to be identical to that of the human ubiquitin-9 repeat gene. The other 3 ubiquitin coding repeats were of close homology to the fourth ubiquitin coding repeat of the human ubiquitin-9 repeat gene. These findings suggest the existence of yet another human poly-ubiquitin gene.     

Cloning and sequence analysis of cDNA encoding a precursor for rat brain natriuretic peptide

Brain natriuretic peptide (BNP) is a new type of natriuretic peptide, which has so far been identified only in porcine brain and atrium. Immunological observations suggest that rat and porcine BNP may have structural difference according to species. To identify rat BNP, we constructed a rat atrial cDNA library, and screened for clones encoding rat BNP-precursor by using part of porcine BNP cDNA as a probe. By sequencing a cloned cDNA, the amino acid sequence of rat BNP-precursor comprising 121 residues was deduced as carrying a 26-residue putative signal peptide at the N-terminus and a region homologous to porcine BNP-32 at the C-terminus. In addition, remarkably high homology between rat and porcine BNP-precursors was observed in the 3'-untranslated AT-rich region. Comparing sequences of precursors of ANP and BNP thus far identified, structural and processing features characteristic of the BNP family were discussed.     

Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide

Brain natriuretic peptide (BNP) is a novel diuretic-natriuretic and vasorelaxant peptide originally isolated from porcine brain. In contrast to mammalian atrial natriuretic peptide (ANP), immunological characterization suggests that mammalian BNPs show structural species differences. In order to determine the amino acid sequence of human BNP, we constructed a human cardiac atrium cDNA library and screened for clones hybridizing with porcine BNP cDNA. By sequence analysis of cDNA encoding a putative human BNP precursor, an amino acid sequence of human prepro-BNP of 134 residues has been deduced, in which a minimum bioactive unit highly homologous to porcine BNP-32 is present at the carboxy-terminus.     

Cloning of a complete cDNA encoding human aromatase: immunochemical identification and sequence analysis

A complete cDNA clone encoding a human aromatase was isolated from a human placental cDNA library in lambda gt11. An antibody to the polypeptide specified by the isolated clone was prepared, and Western blot analysis and antibody inhibition experiments of human placental aromatase activity confirmed the identification of the clone as aromatase cDNA. The isolated aromatase cDNA clone of 3030 bp with two unique EcoRI sites contained a 3'-noncoding region of 1397 bp, an open reading frame of 1509 bp encoding 503 amino acid residues, and a 5'-noncoding region of 124 bp. Analysis of the amino acid sequence of aromatase and comparison of aromatase with other forms of cytochrome P-450 indicated that this enzyme is a unique form of the cytochrome P-450 superfamily.     

Cloning and sequence determination of a cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase.

A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.