Expression of a wheat alpha-gliadin gene in Saccharomyces cerevisiae.

A vector was constructed that directs the expression of foreign genes in the yeast Saccharomyces cerevisiae. This vector contains an expression site that was constructed by in vitro modification of the iso-1-cytochrome c (CYC1) gene of S. cerevisiae. The expression of heterologous sequences can be experimentally controlled by catabolite control sequences, promoter and transcription initiation sequences and termination sequence derived from the CYC1 gene. A portion of a genomic wheat alpha-gliadin gene consisting of the entire 861 bp of protein-coding sequence, 18 bp of 5' leader sequence and 54 bp of 3'-noncoding sequence was inserted into the expression site. A CYC1::alpha-gliadin transcript of approx. 1050 nucleotides was synthesized in transformed yeast under the control of the CYC1 regulatory region. The transcripts terminated within the alpha-gliadin 3'-noncoding region, near a nucleotide sequence similar to the yeast transcription termination consensus sequence. The alpha-gliadin was immunochemically detected in total protein extracts from transformed cells and accounted for approx. 0.1% of the total cellular protein. The size of alpha-gliadin synthesized in yeast is the same as that of mature wheat alpha-gliadin. This is consistent with recognition and cleavage of the signal peptide by yeast. Due to the amino acid composition of alpha-gliadin, the availability of glutamine tRNA is a potential translational limitation to high-level synthesis in yeast.

Sequence analysis of a functional member of the Em gene family from wheat.

We report the complete sequence of one functional member of the Em gene family whose expression in wheat embryos is regulated by a complex set of environmental and developmental controls, including the phytohormone abscisic acid (ABA). The Em coding region contains one short intron, and there is an inverted repeat in the transcribed 3'-flanking region. A 646 bp fragment from the 5' promoter, which was previously shown to direct ABA-regulated expression in transformed tobacco tissue and rice cells, is characterized by: (1) three stretches of between 33 and 73 nucleotides of A/T rich (greater than 86%) boxes, (2) one copy of an eight bp palindrome (CATGCATG) which is identical to the RY repeat found in the 5' promoters of many legume genes expressed during embryo development, (3) 15 copies of a six bp repeat (PuCACGPy), found primarily in the 5' region, and (4) two sequences in the ABA-response region, CGAGCAG and a CACGT motif, both of which are conserved in 5' non-coding regions of other plant genes that are expressed in response to ABA and/or in embryos. These sequence comparisons are discussed in relation to the regulation of Em gene expression and other ABA-regulated genes.

Differential expression of alpha-amylase genes in germinating rice and barley seeds.

Steady-state levels of mRNA from individual alpha-amylase genes were measured in the embryo and aleurone tissues of rice (Oryza sativa) and two varieties of barley (Hordeum vulgare L. cv. Himalaya and cv. Klages) during germination. Each member of the alpha-amylase multigene families of rice and barley was differentially expressed in each tissue. In rice, alpha-amylase genes displayed tissue-specific expression in which genes RAmy3B, RAmy3C, and RAmy3E were preferentially expressed in the aleurone layer, genes RAmy1A, RAmy1B and RAmy3D were expressed in both the embryo and aleurone, and genes RAmy3A and RAmy2A were not expressed in either tissue. Whenever two or more genes were expressed in any tissue, the rate of mRNA accumulation from each gene was unique. In contrast to rice, barley alpha-amylase gene expression was not tissue-specific. Messenger RNAs encoding low- and high-pI alpha-amylase isozymes were detectable in both the embryo and aleurone and accumulated at different rates in each tissue. In particular, peak levels of mRNA encoding high-pI alpha-amylases always preceded those encoding low-pI alpha-amylases. Two distinct differences in alpha-amylase gene expression were observed between the two barley varieties. Levels of high-pI alpha-amylase mRNA peaked two days earlier in Klages embryos than in Himalaya embryos. Throughout six days of germination, Klages produced three times as much high-pI alpha-amylase mRNA and nearly four times as much low-pI alpha-amylase mRNA than the slower-germinating Himalaya variety.

Structure-function studies on phytochrome. Preliminary characterization of highly purified phytochrome from Avena sativa enriched in the 124-kilodalton species.

A new protocol for the purification of phytochrome from etiolated Avena sativa L. seedlings in 15-25% yield is described. This preparation exhibits molecular properties dissimilar to those previously reported from oats. Phytochrome prepared by this method contains a dominant species having a Mr = 124,000 (70% of total) on sodium dodecyl sulfate-polyacrylamide gels, in addition to a chromoprotein species with Mr = 118,000 (30%). A blocked NH2 terminus is indicated by amino acid sequencing of the purified chromoprotein preparation. By ultraviolet-visible spectroscopy, this preparation displays a 730-nm absorption maximum for Pfr, an enhanced APfr730/APfr668 ratio under saturating red light illumination, and a reduced amount of dark reversion. Circular dichroism reveals a new absorption band for Pfr in the blue spectral region. These spectral properties and electrophoretic behavior are consistent with available information on phytochrome in vivo or in rapidly prepared extracts from oat seedlings. Thus, the molecular properties described here may more accurately reflect those of phytochrome in vivo in etiolated A. sativa shoots than those of phytochrome species isolated by previous methodologies.

Chromosomal localization and genomic organization of α-amylase genes in rice (Oryza sativa L.).

Genes for α-amylase, alcohol dehydrogenase, andEm, an ABA-regulated gene expressed late in embryogenesis, were localized on rice chromosomes by the analysis of primary trisomies. The validity of the mapping approach was confirmed usingAdh-1 as a control. TheAdh-1 gene has previously been assigned to chromosome 11 using conventional techniques. In this study we confirm this assignment and report an additional locus for alcohol dehydrogenase (Adh-2) on chromosome 9. The α-amylase genes were located on chromosomes 1, 2, 6, 8, and 9 while theEm gene was mapped to chromosome 5. To facilitate trisomic analysis and correlation of cloned genes with bands observed on Southern blots, a nomenclature for the rice α-amylase genes has been proposed. In addition to mapping nine cloned α-amylase genes, we have identified two previously uncloned α-amylase genes as part of this study. Polymorphism for α-amylase genes belonging to each of the three subfamilies was observed between M202 and IR36. The maximum degree of polymorphism was found among genes belonging to the RAmy3 subfamily, which also has the most diverse group of genes.

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.

Structure of a rice beta-glucanase gene regulated by ethylene, cytokinin, wounding, salicylic acid and fungal elicitors.

A rice beta-glucanase gene was sequenced and its expression analyzed at the level of mRNA accumulation. This gene (Gns1) is expressed at relatively low levels in germinating seeds, shoots, leaves, panicles and callus, but it is expressed at higher levels in roots. Expression in the roots appears to be constitutive. Shoots express Gns1 at much higher levels when treated with ethylene, cytokinin, salicylic acid, and fungal elicitors derived from the pathogen Sclerotium oryzae or from the non-pathogen Saccharomyces cereviseae. Shoots also express Gns1 at higher levels in response to wounding. Expression in the shoots is not significantly affected by auxin, gibberellic acid or abscisic acid. The beta-glucanase shows 82% amino acid similarity to the barley 1,3;1,4-beta-D-glucanases, and from hybridization studies it is the beta-glucanase gene in the rice genome closest to the barley 1,3;1,4-beta-glucanase EI gene. The mature peptide has a calculated molecular mass of 32 kDa. The gene has a large 3145 bp intron in the codon for the 25th amino acid of the signal peptide. The gene exhibits a very strong codon bias of 99% G + C in the third position of the codon in the mature peptide coding region, but only 61% G + C in the signal peptide region.

Nucleic acid sequence and chromosome assignment of a wheat storage protein gene.

A cloned gliadin gene was isolated from a wheat genomic library, and 2.4 kb of its primary sequence determined. The gene, alpha-1Y, was found by Southern analysis to be located on chromosome 6A, and its derived amino acid sequence identifies it as a member of the A-gliadin subgroup of alpha-gliadins located on the short arm of that chromosome. alpha-1Y is apparently functional, and contains consensus TATA and CAAT boxes, and polyadenylation signals. This gliadin gene has no introns, and its noncoding flanking regions contain several short repeats and inverted sequences. The gene is contained in a 6.2 kb EcoRI genomic fragment whose apparent copy number varies in different wheat cultivars.

Characterization of an alpha-amylase multigene cluster in rice.

Rice genomic clones containing eight different alpha-amylase genes have been previously classified into five groups based on DNA hybridization studies and restriction site mapping. This report describes the clustering of three Group 3 genes (RAmy3A, RAmy3B and RAmy3C) within 28 kb of genomic DNA. The genes are separated from each other by about 5 kb and transcribed in the same direction. At the protein level, RAmy3B and RAmy3C are 95% homologous while each is 78% homologous to RAmy3A. All three genes have relatively small introns in the first and third positions. RAmy3A; however, has an additional 409 bp intron in the second intron insertion site. Nucleotide sequence comparisons of the coding and 3' flanking regions suggest that clustering of the RAmy3 genes occurred by gene duplication resulting from unequal crossing-over at repetitive sequences. A comparison of the 5' flanking regions revealed several sequences that may be involved in transcription. Expression of RAmy3B/C first appears in the germinating seed after two days and at a higher level after four days. Quantitative primer extension analysis indicates that RAmy3B and RAmy3C contribute 25% and 75%, respectively, of the transcripts from this cluster at four days of germination. No primer extension band specific to RAmy3A transcripts could be detected at this time point. However, RAmy3A PCR products could be amplified from RNA isolated from embryo-derived callus tissue.

The isolation and characterization of a barley 1,3-1,4-beta-glucanase gene.

The barley gene encoding isozyme I of 1,3-1,4-beta-glucanase was isolated and sequenced. The 6260-bp region sequenced included 1885 bp of the 5'-flanking region, the entire coding region, an intron of 2490 bp, and 792 bp of the 3'-flanking region. The 1,3-1,4-beta-glucanase mRNA was found to be regulated at the level of RNA accumulation by both gibberellins (positively) and abscisic acid (negatively) in barley aleurones. The mRNA for isozyme II preferentially accumulated (70%) relative to the mRNA for isozyme I (30%) in poly(A)-rich RNA isolated from material including both the aleurone and the scutellum tissues. The gene family encoding 1,3-1,4-beta-glucanase enzymes in barley was found to be comprised of two closely related genes, isozymes I and II, as well as several related sequences that could be identified by Southern blot analysis. The nucleotide sequence for the 5' untranslated leader and the coding region for the signal peptide of the isozyme II transcript were determined from a cDNA produced by the polymerase chain reaction. The structure of the protein encoded by the isozyme I gene is also discussed.

Classification and characterization of the rice alpha-amylase multigene family.

To establish the size and organization of the rice alpha-amylase multigene family, we have isolated 30 alpha-amylase clones from three independent genomic libraries. Partial characterization of these clones indicates that they fall into 5 hybridization groups containing a total of 10 genes. Two clones belonging to the Group 3 hybridization class have more than one gene per cloned fragment. The nucleotide sequence of one clone from Group 1, lambda OSg2, was determined and compared to other known cereal alpha-amylase sequences revealing that lambda OSg2 is the genomic analog of the rice cDNA clone, pOS103. The rice alpha-amylase genes in Group 1 are analogous to the alpha-Amy1 genes in barley and wheat. lambda OSg2 contains sequence motifs common to most actively transcribed genes in plants. Two consensus sequences, TAACAAGA and TATCCAT, were found in the 5' flanking regions of alpha-amylase genes of rice, barley and wheat. The former sequence may be specific to alpha-amylase gene while the latter sequence may be related to a 'CATC' box found in many plant genes. Another sequence called the pyrimidine box (TCCTTTTTC) was found in the alpha-amylase genes as well as other genes regulated by gibberellic acid (GA). Comparisons based on amino acid sequence alignment revealed that the multigene families in rice, barley and wheat shared a common ancestor which contained three introns. Some of the descendants of the progenitor alpha-amylase gene appear to have lost the middle intron while others maintain all three introns.