Thionin genes specifically expressed in barley leaves

Complementary-DNA (cDNA) clones encoding thionin were identified as one of the most frequent types of clones in a cDNA library constructed from total polyadenylated RNA from young barley leaf cells. One full-length clone codes for a precursor protein that starts with a signal peptide (28 amino acids) followed by the mature thionin (46 amino acids) and terminated by a long acidic extension (63 amino acids). The amino-acid sequence of the leaf thionin is 52% homologous to thionins from barley endosperm and in the C-terminal extension the homology decreases to 41%. In contrast, the leaf thionin is 72% homologous to viscotoxin from mistletoe leaves. Leaf thionin is coded by a multigene family with an estimated nine to eleven genes and analysis of the cDNA clones showed that at least two extremely homologous genes are expressed. Northern hybridization experiments indicate that the leaf thionin genes are not expressed in endosperm and roots. In leaves, the expression of the thionin genes is strongly repressed by light.Abbreviations cDNA complementary DNA - poly(A)RNA polyadenylated RNA     

A plant serpin gene. Structure, organization and expression of the gene encoding barley protein Z4

A 3133-bp nucleotide sequence of the gene Paz1 on chromosome 4 of barley, encoding endosperm protein Z4, has been determined. The sequence includes 1079 bp 5' upstream and 523 bp 3' downstream of the coding region. The 1079-bp 5' upstream region of the gene shows little similarity to 5' regions of other sequences genes expressed in the developing cereal endosperm. The coding sequence is interrupted by one 334-bp-long intron (bases 1497-1830). The deduced amino acid sequence, which was corroborated by peptide sequences, consists of 399 amino acids and has a molecular mass of 43,128 Da. This sequence confirms protein Z4 to be a member of the serpin superfamily of proteins. The similarity with other members of the family expressed as amino acids in identical positions is in the order of 25-30% and pronounced in the carboxy-terminal half of the molecule. Sequence residues assumed to form clusters stabilizing the tertiary structure are highly conserved. Protein Z4 is synthesized in the developing endosperm without a signal peptide and protein Z4 mRNA was evenly distributed among the free and membrane-bound polyribosomes of the endosperm cell. An internal hydrophobic region of 21 amino acids (residues 36-56) may serve as a signal for targeting the polypeptide into the lumen of the endoplasmic reticulum. The gene for protein Z4 could not be detected in the barley variety Maskin and some of its descendants. The 'high-lysine' allees, lys1 (Hiproly barley) and lys3a (Bomi mutant 1508) on chromosome 7, enhance and repress, respectively, the expression of the protein Z4 gene. Also, 1554 bp of another 8-kbp fragment of the barley genome Paz psi, similar to the protein-Z4-coding region, have been determined. Small insertions and deletions and the presence of an internal stop codon identify this fragment as part of a pseudogene related to the protein Z4 gene.     

Primary structure and differential expression of beta-amylase in normal and mutant barleys

The primary structure of barley endosperm beta-amylase, an enzyme which catalyses the liberation of maltose from 1,4-alpha-D-glucans, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 1754 nucleotides long [excluding the poly(A) tail] and codes for a polypeptide of 535 amino acids with a relative molecular mass of 59,663. The deduced amino acid sequence was compared with the sequences of ten peptides obtained from the purified enzyme and unambiguous identification was obtained. The N-terminal region of the deduced sequence was identical to a 12-residue cyanogen-bromide-peptide sequence, indicating that beta-amylase is synthesized as the mature protein. A graphic matrix homology plot shows four glycine-rich repeats, each of 11 residues, preceding the C-terminus. Southern blotting of genomic DNA demonstrates that beta-amylase is encoded by a small gene family, while cDNA sequence analysis indicates the presence of at least two types of mRNA in the endosperm. Dot and northern blot analysis show that Hiproly barley contains greatly increased levels of beta-amylase mRNA compared to the normal cultivar Sundance, whereas Ris? mutant 1508 contains only trace amounts. These results correlate well with the deposition of beta-amylase during endosperm development in these lines. Low but similar amounts of beta-amylase mRNAs sequences were detected in leaves and shoots from normal and mutant barleys, demonstrating that the mutant lys3a (1508) and lysl (Hiproly) genes do not affect the expression of beta-amylase in these tissues.     

Barley embryo globulin 1 gene,Beg1: Characterization of cDNA,chromosome mapping and regulation of expression

We report identification of a 2189 by cDNA clone from barley corresponding to a single-copy gene, Beg1 (Barley embryo globulin), on chromosome 4, which encodes a storage globulin. In barley, the major protein reserve in the aleurone layer belongs to the 7S globulin class of proteins found in many seeds. Electrophoretically and antigenically similar proteins are present in the barley embryo. Accumulation of Beg1 mRNA was noted beginning 15–20 days post-anthesis in both the aleurone layer and embryo of the developing barley grain but not in the starchy endosperm. A high level of Beg1 mRNA is also present in the mature imbibed aleurones, which can be repressed by treatment with gibberellic acid. This repressive effect of gibberellin on the levels of Beg1 mRNA is confirmed in the gibberellin response-constitutive mutant, slender, whose aleurone layers do not accumulate Beg1 mRNA even in the absence of applied gibberellic acid. The deduced primary translation product of the Beg1 mRNA is a 637 amino acid (72 kDa) protein with homology to maize embryo globulin 1 (GLB1) and a partial sequence of a wheat 7S globulin. The internal amino acid sequence of BEG1 closely matches the N-terminal sequence of isolated barley aleurone globulin. Seven imperfect tandem repeats of 16 amino acids each are present near the N-terminus of BEG1, which conform to the consensus HGEGEREEEXGRGRGR, and contribute to the observed unusual amino acid composition of this protein. A second, distinct barley globulin gene, Beg2, which is homologous to maize Glb2, was detected by Northern and Southern analysis. Beg-2 and Beg1 are regulated differently which may indicate variation in storage or utilization properties among the barley globulins.     

Barley embryo globulin 1 gene, Beg1: Characterization of cDNA, chromosome mapping and regulation of expression

We report identification of a 2189 by cDNA clone from barley corresponding to a single-copy gene, Beg1 (Barley embryo globulin), on chromosome 4, which encodes a storage globulin. In barley, the major protein reserve in the aleurone layer belongs to the 7S globulin class of proteins found in many seeds. Electrophoretically and antigenically similar proteins are present in the barley embryo. Accumulation of Beg1 mRNA was noted beginning 15–20 days post-anthesis in both the aleurone layer and embryo of the developing barley grain but not in the starchy endosperm. A high level of Beg1 mRNA is also present in the mature imbibed aleurones, which can be repressed by treatment with gibberellic acid. This repressive effect of gibberellin on the levels of Beg1 mRNA is confirmed in the gibberellin response-constitutive mutant, slender, whose aleurone layers do not accumulate Beg1 mRNA even in the absence of applied gibberellic acid. The deduced primary translation product of the Beg1 mRNA is a 637 amino acid (72 kDa) protein with homology to maize embryo globulin 1 (GLB1) and a partial sequence of a wheat 7S globulin. The internal amino acid sequence of BEG1 closely matches the N-terminal sequence of isolated barley aleurone globulin. Seven imperfect tandem repeats of 16 amino acids each are present near the N-terminus of BEG1, which conform to the consensus HGEGEREEEXGRGRGR, and contribute to the observed unusual amino acid composition of this protein. A second, distinct barley globulin gene, Beg2, which is homologous to maize Glb2, was detected by Northern and Southern analysis. Beg-2 and Beg1 are regulated differently which may indicate variation in storage or utilization properties among the barley globulins.     

Isolation and Characterization of a Thionin Proprotein-processing Enzyme from Barley

Thionins are plant-specific antimicrobial peptides that have been isolated from the endosperm and leaves of cereals, from the leaves of mistletoes, and from several other plant species. They are generally basic peptides with three or four disulfide bridges and a molecular mass of ∼5 kDa. Thionins are produced as preproproteins consisting of a signal peptide, the thionin domain, and an acidic domain. Previously, only mature thionin peptides have been isolated from plants, and in addition to removal of the signal peptide, at least one cleavage processing step between the thionin and the acidic domain is necessary to release the mature thionin. In this work, we identified a thionin proprotein-processing enzyme (TPPE) from barley. Purification of the enzyme was guided by an assay that used a quenched fluorogenic peptide comprising the amino acid sequence between the thionin and the acidic domain of barley leaf-specific thionin. The barley TPPE was identified as a serine protease ({"type":"entrez-protein","attrs":{"text":"BAJ93208","term_id":"326526063","term_text":"BAJ93208"}}BAJ93208) and expressed in Escherichia coli as a strep tag-labeled protein. The barley BTH6 thionin proprotein was produced in E. coli using the vector pETtrx1a and used as a substrate. We isolated and sequenced the BTH6 thionin from barley to confirm the N and C terminus of the peptide in planta. Using an in vitro enzymatic assay, the recombinant TPPE was able to process the quenched fluorogenic peptide and to cleave the acidic domain at least at six sites releasing the mature thionin from the proprotein. Moreover, it was found that the intrinsic three-dimensional structure of the BTH6 thionin domain prevents cleavage of the mature BTH6 thionin by the TPPE.     

Cloning and sequence analysis of the Erythrina corallodendron lectin cDNA.

Examination of the hemagglutinating activity of extracts from seeds of Erythrina corallodendron at various maturation stages revealed that the level of lectin increases markedly past mid-maturation. Seeds at this stage of maturation served as a source of mRNA for the construction of an expression cDNA library in the vector lambda Zap, which generates fusion proteins with an N-terminal portion of beta-galactosidase. The library was screened with rabbit polyclonal anti-ECorL antiserum. Four immunopositive clones were isolated. Western blot analysis of cell extracts from one of the clones (pIEcL-B) showed a 36 kDa protein that reacted with the antiserum, as well as with a mouse monoclonal antibody raised against the lectin. DNA sequence analysis by the chain termination method revealed that clone pIEcl-C has an insert of 1017 bp with the entire coding sequence of ECorL, beginning with an initiation codon ATG at position 26 and ending with stop codon TAA at position 868. This fragment encodes a polypeptide of 281 amino acids consisting of a signal leader sequence of 25 amino acids and a mature protein of 256 amino acids. The deduced amino acid sequence from this fragment is identical to the sequence of the first 244 amino acids of ECorL, as determined at the protein level, except at 7 positions.     

Nucleotide sequence of cDNA containing the complete coding sequence for human lysosomal glucocerebrosidase

Complementary DNA clones for human glucocerebrosidase were isolated from a human hepatoma library in lambda gt11. The complete nucleotide sequence of the 1805-base pair cDNA insert has been determined. In addition to 5' and 3' untranslated regions (51 and 206 base pairs, respectively), the cDNA insert contains 1548 base pairs that completely encode human glucocerebrosidase. All possible N-linked glycosylation sites are identified. Examination of the 19 amino acids of the leader polypeptide beginning with the ATG at position 52 revealed a hydrophobic core and a carboxyl-terminal glycine at the peptidase cleavage site, features consistent with the leader sequences described for other human translocated proteins. The Mr of 57,000 calculated from the 516 amino acids deduced from cDNA sequence is in good agreement with that identified by immunoprecipitation following in vitro translation of human placental mRNA.     

Extreme divergence of a novel wheat thionin generated by a mutational burst specifically affecting the mature protein domain of the precursor.

A new type of neutral thionin (type V), specifically expressed in developing wheat endosperm, has been found to be encoded by a set of single-copy genes located in the long arms of chromosomes 1A, 1B and 1D, within less than 10,000 base-pairs of those corresponding to the highly basic type-I thionins. Divergence between types I and V has occurred through a process of accelerated evolution that has affected the amino acid sequence of the mature thionin but not the precursor domains corresponding to the N-terminal signal peptide and the long C-terminal acidic peptide. This process involved a deletion and a non-synonymous nucleotide substitution rate equal to the synonymous rate in the thionin sequence.     

Cloning and partial characteristics of the barley D-hordein sequence]

A number of clones containing major endosperm-specifically transcribed gene copies were selected from a cDNA library developed on the basis of barley endosperm mRNA. Approx. 30% of the recombinant clones carried sequences homologous to mRNA of various cereal storage proteins. Some of them appeared to be related to cDNA clones of wheat and barley storage proteins. The typical insert length ranged from 0.3 to 1.7 kB. A couple of clones among them were selected which revealed positive hybridization with all probes used. The positive signals disappeared after stringent washing of the filters. The nucleotide sequences of two representatives of the group were determined and corresponding amino acid sequence deduced after subsequent computer analysis. The comparison with known cereal storage protein genes revealed relatively high homology level with the central part of wheat high molecular weight (HMW) glutenine subunit genes. The fact suggests the cloned gene to belong to barley D-hordein family.     

Subcellular localization of type I thionins in the endosperms of wheat and barley

Summary Thionins are cysteine-rich polypeptides of about 5,000 Da. Localization at the subcellular level of type I endosperm thionins has been carried out by immunogold labeling, using an antibody that recognizes type I thionin variants. In developing wheat and barley caryopses, sectioned at different times between 13 and 24 days after flowering, this type of thionins was only detected around protein bodies from cells of the starchy endosperm, using light microscopy. Electron microscopy revealed that these proteins were located in electron-dense spheroids in the periphery of protein bodies, at the earlier stages, whereas later the label appeared also as a thin layer around these organelles.Abbreviations DAF days after flowering - RER rough endoplasmic reticulum     

Isolation and characterization of a complementary DNA clone coding for the E1 beta subunit of the bovine branched-chain alpha-ketoacid dehydrogenase complex: complete amino acid sequence of the precursor protein and its proteolytic processing

A 1.7-kb cDNA clone encoding the entire precursor of the E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1 beta subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequence analysis revealed that the isolated cDNA clone contained the 5'-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3'-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1 beta subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1 beta with that of the human pyruvate dehydrogenase (PDH) complex E1 beta subunit revealed a high degree of sequence homology throughout the two enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Propeptide of human protein C is necessary for gamma-carboxylation

Protein C is one of a family of vitamin K dependent proteins, including blood coagulation factors and bone proteins, that contains gamma-carboxyglutamic acid. Sequence analysis of the cDNAs for these proteins has revealed the presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from the growing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. In the present study, deletion mutants have been constructed in the propeptide region of the cDNA for human protein C, and the cDNAs were then expressed in mammalian cell culture. These deletions included the removal of 4, 9, 12, 15, 16, or 17 amino acids comprising the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These experiments have shown that protein C is readily synthesized in mammalian cell cultures, processed, and secreted as a two-chain molecule with biological activity. Furthermore, the pre portion or signal sequence in human protein C is 18 amino acids in length, and the pro portion of the leader sequence is 24 amino acids in length. Also, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) is important for gamma-carboxylation of protein C, while the present data and those of others indicate that the carboxyl-terminal portion of the propeptide (residues -1 through -4) is important for the removal of the pro leader sequence by proteolytic processing.     

The gene for trypsin inhibitor CMe is regulated in trans by the lys 3a locus in the endosperm of barley (Hordeum vulgare L.)

Summary A cDNA encoding trypsin inhibitor CMe from barley endosperm has been cloned and characterized. The longest open reading frame of the cloned cDNA codes for a typical signal peptide of 24 residues followed by a sequence which is identical to the known amino acid sequence of the inhibitor, except for an Ile/Leu substitution at position 59. Southern blot analysis of wheat-barley addition lines has shown that chromosome 3H of barley carries the gene for CMe. This protein is present at less than 2%–3% of the wild-type amount in the mature endosperm of the mutant Risø 1508 with respect to Bomi barley, from which it has been derived, and the corresponding steady state levels of the CMe mRNA are about I%. One or two copies of the CMe gene (synonym Itc1) per haploid genome have been estimated both in the wild type and in the mutant, and DNA restriction patterns are identical in both stocks, so neither a change in copy number nor a major rearrangement of the structural gene account for the markedly decreased expression. The mutation at the lys 3a locus in Risø 1508 has been previously mapped in chromosome 7 (synonym 5H). A single dose of the wild-type allele at this locus (Lys 3a) restores the expression of gene CMe (allele CMe-1) in chromosome 3H to normal levels.     

Synthesis and processing of thionin precursors in developing endosperm from barley (Hordeum vulgare L.)

Thionin is a lysine-rich polypeptide (mol. wt. 5000) which is synthesized in developing barley endosperm from ˜8 days to ˜30 days after anthesis. Two thionin precursors (THP1 and THP2) have been identified using monospecific antibodies (A-TH) prepared against the mature protein. THP1, which is the only polypeptide recognized in vitro by A-TH, is encoded by a 7.5S mRNA obtained from membrane-bound polysomes, and its alkylated derivative has an apparent mol. wt. of 17 800. THP2, which is selected together with mature thionin by A-TH among labelled proteins in vivo, differs from THP1 in apparent mol. wt. (17 400 alkylated) and in electrophoretic mobility at pH 3.2. Both THP1 and THP2 are competed out of the antigen-antibody complex by purified thionin. The conversion of THP2 into thionin, which has been demonstrated in a pulse-chase experiment in vivo, is a post-translational process. As it has not been possible to detect THP1 in vivo it is assumed that it is converted co-translationally into THP2. Final deposition of thionin as an extrinsic membrane protein, possibly associated with the endoplasmic reticulum, has been tentatively established on the basis of subcellular fractionation experiments.     

Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments

Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high-performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half-cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII.