The nucleotide sequence of a cDNA clone encoding the wheat Em protein.

The nucleotide sequence of a cDNA clone isolated from developing wheat embryos and encoding the Em protein is reported. The entire coding region for Em and the 3' non-translated flank are contained within this clone. The amino acid sequence deduced for Em is very rich in glycine (18 mol%) as well as both basic and acidic residues. The molecular weight of the protein is ca. 9,900 daltons. The deduced sequence is supported by direct amino acid sequencing of cyanogen bromide cleavage fragments obtained from purified Em protein. Em is shown by Southern blots to be a product of a gene family of approximately ten members.     

Cloning and Characterization of Full-length Triticin cDNA and Genes from Wheat Varieties K-68 and Chinese Spring

Full-length cDNA and genes for triticin protein were cloned and characterized from wheat varieties K-68 and Chinese Spring differing considerably in grain colour, total protein content, grain hardness, milling behaviour and baking characteristics. Wheat variety K-68 possesses excellent chapatti (unleavened bread) making quality in contrast to Chinese Spring. The nucleotide and deduced amino acid sequences of the full-length triticin cDNA and genes were compared with those of other legumin genes. Although minor variations in the nucleotide sequences were observed when compared with the published sequence of the partial triticin cDNA clone λTri-25, the deduced amino acid sequence of the full-length triticin cDNA clone (Tri-cK68) revealed large variation in the Hyper Variable Region. The deduced amino acid sequence of the full-length triticin cDNA clone Tri-cK68 revealed two lysine-rich regions in the triticin protein. Comparative analysis of the nucleotide sequences of the triticin genes with the cDNA clone λTri-25 revealed the presence of a stretch of 31 nucleotides in the 5′ UTR of λTri-25 having exact complementarity with a stretch of nucleotides of the same length in the 3′ UTR of the full length triticin genes cloned from the wheat varieties K-68 (Tri-gK68) and Chinese Spring (Tri-gCS). Analysis of the nucleotide sequence of triticin promoter (Tri-pK68) revealed the presence of several elements responsible for seed-specific expression and responsiveness to light.     

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.     

Cauliflower mosaic virus promoters direct efficient expression of a bacterial G418 resistance gene in Schizosaccharomyces pombe

We have isolated and sequenced a cDNA clone which contains the entire coding sequence of the precursor to a subunit of wheat phosphoribulokinase (PRKase). (The enzyme is a homodimer). The cDNA contains 1533 bp and has an open reading frame of 1212 nucleotides. This encodes a protein with an amino-terminal transit sequence of 53 amino acids, while the part that forms the mature protein contains 351 amino acids and has a molecular weight of 39,200 daltons. A comparison of the wheat amino acid sequence with that already known for the mature protein of spinach reveals that there are identical residues in 86% of the positions but their transit peptides differ substantially from one another. The mature wheat and spinach proteins are identical in a segment of over 50 amino acids near the amino-terminus which is the region believed to be involved in ATP binding and in regulation by light of the catalytic activity of the enzyme. We further demonstrate that the expression of PRKase mRNA in wheat leaves is regulated in a developmental, tissue-specific and light dependent manner. We also show that the light-induced increase in the steady-state levels of this mRNA is dependent on the developmental stage of the leaf.     

A novel proline-rich protein from wheat

A cDNA (WPRP1) encoding a wheat proline-rich protein has been isolated and sequenced. The amino acid composition shows 45% proline, with high levels of methionine, lysine and glutamic acid. The derived 378 residue amino acid sequence has a highly repetitive structure which is unlike those of other proline-rich proteins. The WPRP1 cDNA clone was used to determine the copy number and chromosomal location of the WPRP1 gene by restriction fragment length polymorphism analysis of wheat inbred lines. Although WPRP1 is encoded by a single-copy gene it is also a representative of a larger family of related sequences. RNA gel blot analysis showed that expression of WPRP1 is highest in rapidly growing tissue which together with its amino acid composition suggests a structural role for the encoded protein.     

The cDNA sequence of a neutral horseradish peroxidase

A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.     

An early meiosis cDNA clone from wheat

Meiosis occupies only a very short period of the life cycle of higher plants but it is a crucial process ensuring the correct passage and maintenance of genetic information from parent to offspring. A clone (designated pAWJL3) has been isolated from a cDNA library generated from RNA prepared from young wheat florets at early meiosis. The clone was identified through cross-hybridisation to a cDNA clone from maize that, in turn, had been isolated by hybridisation to a Lilium meiosis-specific cDNA clone. The genes encoding the sequence represented in the wheat cDNA clone have been assigned to chromosomes in wheat. The clone, pAWJL3, represents a small family of genes with about 20 members located on the short arms of group 3 and 5 chromosomes. The chromosomal regions harbour genes known to control chromosomal pairing in wheat. DNA prepared from a deletion mutation affecting one of the major genes controlling pairing, Ph2 located on the short arm of 3DS, lacks the 3DS-specific members of the pAWJL3 family bands. The genes are shown to be expressed only after leptotene and predominantly at zygotene and pachytene of meiosis I. The deduced amino acid sequence encoded by the cDNA clone shows two domains, one with three leucine-rich, 24-amino acid repeats and the other with four leucine heptad repeats that resemble those found in basic leucine zipper proteins.     

A chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant cells

A cDNA clone, pMA1949, detects two mRNA species in wheat seedling tissue that are late embryogenesis-abundant (LEA) and dehydration stress-inducible. Sequence analysis of the pMA1949 clone shows it to be a 991 bp partial cDNA encoding a polypeptide of 317 amino acids with homology to two group 3 LEA proteins, carrot (DC8) and a soybean protein encoded by pGmPM2 cDNA. Molecular analysis of the deduced protein reveals a 33 kDa acidic and extremely hydrophilic protein with potential amphiphilic -helical regions. In addition, the protein contains eleven similar, contiguous repeats of 11 amino acids, which are separated by 118 amino acids from two additional and unique repeats of 36 residues each at the carboxyl end of the protein. Comparisons of sequences of reported group 3 LEA proteins revealed that there are two types, separable by sequence similarity of the 11 amino acid repeating motifs and by the presence or absence of a certain amino acid stretch at the carboxyl terminus. Based on resuls from these comparisons, we propose a second type of group 3 LEA proteins, called group 3 LEA (II).     

Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli

The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by ubiquitin activating enzyme, E1. Previously, we purified and characterized multiple species of E1 from wheat germ. We now describe the isolation and characterization of a cDNA clone encoding E1 from wheat. This clone (UBA1) was isolated from a cDNA expression library with anti-wheat E1 antibodies. It contained an open reading frame coding for 1051 amino acids and directed the synthesis of a protein that comigrated with a wheat germ E1 of 117 kDa. UBA1 was confirmed as encoding E1 by (i) comparison of the peptide map of the protein product of UBA1 synthesized in Escherichia coli with that of purified E1 from wheat, and (ii) amino acid sequence identity of peptides generated from purified E1 with regions of the derived amino acid sequence of UBA1. The isolation of two additional cDNAs closely related to UBA1 indicated that E1 was encoded by a small gene family in wheat. Nonetheless, a single poly(A+) mRNA size class of 4 kilobases hybridized with UBA1. When expressed in E. coli, the product of UBA1 catalyzed the formation of a thiol ester linkage between ubiquitin and an ubiquitin carrier protein. The ability of E. coli containing UBA1 to synthesize an active protein will allow us to identify domains important for E1 function using in vitro mutagenesis.     

Isolation and sequence analysis of a complementary DNA encoding rat liver L-gulono-gamma-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis

L-Gulono-gamma-lactone oxidase, one of the microsomal flavin enzymes, catalyzes the last step of L-ascorbic acid biosynthesis in many animals; however, it is missing in scurvy-prone animals such as humans, primates, and guinea pigs. A cDNA clone for this enzyme was isolated by screening a rat liver cDNA expression library in lambda gt11 using antibody directed against the enzyme. The cDNA clone contained 2120 nucleotides and an open reading frame of 1320 nucleotides encoding 440 amino acids of the protein with a molecular weight of 50,605. The amino-terminal sequence (residues 1-33) of the enzyme isolated from rat liver completely coincided with the corresponding part of the deduced amino acid sequence. The identity of the cDNA clone was further confirmed by the agreement of the composition of the deduced amino acids with that determined by amino acid analysis of the enzyme. Hydropathy analysis of the deduced amino acid sequence revealed several hydrophobic regions, suggesting that they anchor the protein into the microsomal membrane. The deduced amino acid sequence showed no obvious homology with the flavin-binding regions of other eight flavoenzymes.     

Human ribosomal protein S3a: cloning of the cDNA and primary structure of the protein.

The amino acid (aa) sequence of human ribosomal protein S3a (hRPS3a) was deduced partially from the nucleotide sequence of the corresponding cDNA and confirmed by direct aa sequencing from the N terminus of the purified hRPS3a protein. The cDNA clone was isolated from a cDNA expression library in the pEX vector using antibodies. The hRPS3a protein has 263 aa and its calculated M(r) is 29 813.     

Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus.

We have isolated a cDNA clone encoding a novel calcium-binding protein of the recoverin family from rat brain cDNA library. This clone (PCB11) has 588 nucleotides in the open reading frame including the termination codon, 174 nucleotides of the 5' leader and 800 nucleotides of the 3' noncoding region. The complete amino acid sequence deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22,574 Daltons, and contains three putative calcium-binding domains of the EF-hand structure. The deduced amino acid sequence has a striking sequence homology to those of the retinal recoverin family (recoverin, visinin, P26, 23kD protein, S-modulin) and the brain-derived recoverin family (P23k, 21-kDa CaBP and neurocalcin). Northern blot, in situ hybridization, immunoblot and immunohistochemical analyses revealed that the protein is exclusively expressed in pyramidal layer of the hippocampus. The protein was therefore designated hippocalcin.     

Isolation and characterization of a cDNA clone encoding human aromatic L-amino acid decarboxylase

The nucleotide sequence of a cDNA clone that includes the entire coding region of human aromatic L-amino acid decarboxylase gene is presented. A human pheochromocytoma cDNA library was screened using an oligonucleotide probe which corresponded to a partial amino acid sequence of the enzyme purified from the human pheochromocytoma. The isolated cDNA clone encoded a protein of 480 amino acids with a calculated molecular mass of 53.9 kDa. The amino acid sequence Asn-Phe-Asn-Pro-His-Lys-Trp around a possible cofactor (pyridoxal phosphate) binding site is identical in human, Drosophila, and pig enzymes.     

Cloning and expression of a cDNA coding for a rat liver plasma membrane ecto-ATPase. The primary structure of the ecto-ATPase is similar to that of the human biliary glycoprotein I

The amino acid sequence of the ecto-ATPase from rat liver was deduced from analysis of cDNA clones and a genomic clone. Immunoblots with antibodies raised against a peptide sequence deduced from the cDNA sequence indicated that the determined amino acid sequence is that of the ecto-ATPase. The deduced sequence predicts a 519-amino acid protein with a calculated molecular mass of 57,388 daltons. There are 16 potential asparagine-linked glycosylation sites in the protein. Hydropathy analysis of the deduced amino acid sequence indicates that the protein has two hydrophobic stretches. One is located at the N-terminal and the other is near the C-terminal end. A full-length clone encoding the ecto-ATPase was expressed transiently in mouse L cells and human HeLa cells. The cell lysate from the transfected cells contained immunoreactive ecto-ATPase and Ca2+-stimulated ATPase activities. The expressed protein is glycosylated and has an apparent molecular weight (100,000) similar to that of the rat liver plasma membrane ecto-ATPase.     

The nucleotide sequence of a complete chicken delta-crystallin cDNA

The nucleotide sequence of a full length cDNA of delta-crystallin mRNA from chicken lens has been determined using a delta-crystallin cDNA clone (pB delta 11), which represents the mRNA sequence of 1530 nucleotides from the poly(A) junction but does not contain the 5'-terminal sequence of 44 nucleotides of the mRNA. The 5'-terminal sequence of the mRNA, absent in the cDNA clone, has been determined with a stretch of cDNA sequence by the primer extension procedure. The amino acid sequence deduced from the nucleotide sequence is consistent with the amino acid sequences of several tryptic peptides, the total amino acid composition, and the mol. wt. of delta-crystallin estimated by SDS-polyacrylamide gel electrophoresis. The computer-assisted analysis predicts high alpha-helical content throughout the polypeptide. Sequence analyses have revealed that gene 1 encodes the mRNA from which the cDNA clone was derived.     

Nucleotide sequence encoding the precursor of the small subunit of ribulose 1,5-bisphosphate carboxylase from Lemna gibba L.G-3

We have sequenced a cDNA clone, pLgSSU, which encodes the small subunit of ribulose 1,5-bisphosphate carboxylase of Lemna gibba L.G-3 a monocot plant. This clone contains a 832 basepair insert which encodes the entire 120 amino acids of the mature small subunit polypeptide (Mr = 14,127). In addition this clone encodes 53 amino acids of the amino terminal transit peptide of the precursor polypeptide and 242 nucleotides of the 3' non-coding region. Comparison of the nucleotide sequence of pLgSSU with Lemna gibba genomic sequences homologous to the 5' end of the cDNA clone suggests that nucleotides encoding four amino-terminal amino acids of the transit peptide are not included in the cDNA clone. The deduced amino acid sequence of the Lemna gibba mature small subunit polypeptide shows 70-75% homology to the reported sequences of other species. The transit peptide amino acid sequence shows less homology to other species. There is 50% homology to the reported soybean sequence and only 25% homology to the transit sequence of another monocot, wheat.     

Characterization of a cDNA clone encoding a Brassica napus 12 S protein (cruciferin) subunit. Relationship between precursors and mature chains

Cruciferin (12 S globulin) is a large, neutral, oligometric protein synthesized in rapeseed (Brassica napus) during seed development. It is the major seed protein and is composed of six subunit pairs. Each of these pairs is synthesized as a precursor containing one heavy alpha-chain and one light beta-chain. Electrophoretic analysis of cruciferin showed that four different alpha- and four different beta-chains exist. A cruciferin clone was selected from an embryo cDNA library. This clone, pCRU1, contains a 1518-base pair open reading frame corresponding to a truncated NH2-terminal signal sequence followed by an alpha-chain of 296 and a beta-chain of 190 amino acid residues. Individual cruciferin chains as well as peptides thereof were subjected to NH2-terminal amino acid sequence analysis. The sequences obtained from a specific alpha- and beta-chain pair (alpha 1 and beta 1) showed total identity with the deduced amino acid sequence from pCRU1. Further comparisons revealed that a previously characterized cruciferin cDNA clone encodes one of the precursors for the closely related alpha 2/ alpha 3-beta 2/beta 3 subunits. The deduced amino acid sequences of the two cDNA clones display 64% similarity.