Isolation and characterization of a rice cDNA which encodes the eukaryotic initiation factor 4A.

We isolated a rice cDNA which encodes an open reading frame of 413 amino acids. The deduced amino acid sequence corresponds to eukaryotic initiation factor 4A (eIF-4A) protein. A comparison to the equivalent sequence from tobacco and mouse shows 93.7% and 68.0% homology, respectively.

The primary structure of two proteins from the large ribosomal subunit of rice.

We isolated two rice cDNAs which encode an open reading frame of 389 amino acids. Their deduced amino acid sequence corresponded to the ribosomal protein (r-protein). A comparison of amino acid sequence shows that the deduced amino acid sequence of one cDNA is homologous to Arabidopsis, yeast and the rat r-protein L3. Another encoded products with a high degree of homology to the rat r-protein L7A and yeast r-protein L4.

A novel variant of translation elongation factor-1beta: isolation and characterization of the rice gene encoding EF-1beta2.

A rice gene encoding a novel isoform of translation elongation factor-1beta subunit (termed EF-1beta2) was isolated and characterized. The gene comprises of eight exons, and encodes a 226-amino-acid protein. Expression of EF-1beta2 mRNA is abundant in seeds and cultured cells, but is considerably low in the tissues of the rice seedling. Antiserum raised against an EF-1beta2 synthetic peptide detected a protein with a relative molecular mass of about 32 kDa, indicating the EF-1beta2 gene is actually expressed in rice tissues. EF-1beta2 showed a close similarity to the cognate subunits from plant (beta and beta').

Isolation and characterization of a rice cDNA encoding the gamma-subunit of translation elongation factor 1B (eEF1Bgamma).

We isolated a rice cDNA clone (refg) encoding the gamma-subunit of translation elongation factor 1B (eEF-1B gamma; the old designation was EF-1 gamma). The refg encodes an open reading frame of 419 amino acids which shows a similarity to the equivalent sequences from animals and yeast. Complex formation analysis, which showed the recombinant protein of refg (His-eEF1B gamma) and formed a complex with GST-eEF-1Bbeta, indicated that the refg encodes rice eEF1B gamma of the eEF1B alphabeta gamma complex. Expression analysis showed that refg mRNA is very abundant in suspension-cultured cells during the exponential phase of growth. A DNA blot analysis indicated that refg is located at a single locus in the rice genome.

Molecular structure of ras-related small GTP-binding protein genes of rice plants and GTPase activities of gene products in Escherichia coli.

We isolated two rice cDNA clones (ric1 and ric2) encoding proteins homologous to the ras-related small GTP-binding protein. The amino acid sequences of ric1 and ric2 are conserved in four regions involved in GTP binding and hydrolysis which are characteristic in the ras and ras-related small GTP-binding protein genes. In addition, two consecutive cysteine residues near the carboxyl-terminal end required for membrane anchoring are also present in ric1 and ric2. The ric1 and ric2 proteins synthesized in Escherichia coli possessed GTPase activity (i.e. hydrolysis of GTP to GDP).

Molecular cloning of the wheat CK2alpha gene and detection of its linkage with Vrn-A1 on chromosome 5A.

The casein kinase CK2 is one of the major multifunctional protein kinases in cells that is expressed ubiquitously and is essential for survival. The alpha-subunit of CK2 is thought to be involved in light-regulated gene expression and rhythmic expression of genes by circadian rhythm in plants. The rice chromosome-3 region containing the photoperiod-response Hd6 gene, an orthologue of the CK2alpha genes of Arabidopsis and maize, is in synteny with the wheat chromosome-5A Vrn-A1 region. This evidence proposes two possibilities, first the wheat Vrn-A1 is an orthologue of the rice CK2alpha, and second the wheat CK2alpha which has not yet been identified is located independently but tightly linked to Vrn-A1. To clarify whether the wheat CK2alpha gene is conserved in the Vrn-A1 region and to elucidate the above two possibilities, we attempted to isolate this gene from the wheat cDNA library and to map it on the chromosome-5A region that is syntenous to the rice Hd6 region. The isolated cDNA clone showed an extremely high homology with the Arabidopsis CK2alpha gene. Using this clone as a probe genomic Southern-blot analyses of the aneuploid lines available in Chinese Spring assigned the wheat homologue of CK2alpha to the long arm of chromosome 5A. Furthermore, a linkage analysis using an F(2) population having recombination in the Vrn-A1 region revealed that the wheat CK2alpha, designated as tck2a, is tightly linked to Vrn-A1 by 1.1 cM

Nucleotide sequence of rice (oryza sativa L.) cDNA homologous to cdc2 gene.

We isolated and determined a nucleotide sequence of a rice DNA clone (SS224) denoted to rcdc2. This clone encodes an open reading frame of 302 amino acids and typical three conserved domains that exist in all cdc2 homologues. The evolutionary tree showed that rcdc2 was far from cdc2 and its homologous genes identified in various plants.

Molecular cloning and mapping of casein kinase 2 alpha and beta subunit genes in barley.

Casein kinase 2 (CK2) is a ubiquitous, highly pleiotropic, constitutively active, and messenger-independent Ser/Thr protein kinase. It is found in two different forms: the heterotetrameric CK2, composed of two alpha catalytic subunits and two beta regulatory subunits, and the monomeric CK2 alpha, consisting of the alpha catalytic subunit. In the present study, we isolated barley cDNA clones of the CK2 alpha and beta subunit genes, designated HvCK2A and HvCK2B, respectively. Chromosome assignment, using a set of wheat-barley disomic chromosome addition lines, and RFLP mapping, using two doubled haploid populations, showed that HvCK2A was duplicated on the short arm of chromosome 2H and the long arm of chromosome 5H (designated HvCK2a-2H and HvCK2a-5H, respectively), and a single copy of HvCK2B was located on the long arm of chromosome 1H (designated HvCK2b). A PCR-Southern hybridization experiment demonstrated that the HvCK2A sequence originated from the HvCK2a-5H locus, showing that at least HvCK2a-5H was expressed. The present cDNA sequences and genomic organization of the two subunits will facilitate further functional analysis of CK2 in barley.

Isolation, characterization and mRNA expression of four cDNAs encoding translation elongation factor 1A from rice (Oryza sativa L.).

Four different cDNA clones encoding protein synthesis elongation factor 1A, eEF1A, were isolated from rice (Oryza sativa L.). The genes encoded by these cDNAs were designated rice elongation factor 1A genes refa1, refa2, refa3 and refa4. The genes encoded identical eEF-1A polypeptides and shared high amino acid identity with eEF1A of other eukaryotes. Southern blot analysis suggested that some of these refa genes may be organized in a cluster on the same chromosome within a short distance. PCR analysis of rice genomic DNA showed that refa1 and refa4, and refa3 and refa2 are in neighboring locations on the rice genome. The mRNAs of the four refa genes accumulated to nearly equal levels in a variety of tissues and at different stages of growth. Suspension-cultured cells were the most abundant in refa mRNAs. Dormant seeds contained a small amount of the four refa mRNAs. Transcript accumulation was highly induced after seed germination, and the same expression levels were maintained even in old leaf blades of mature plants.

Detection and characterization of glutathione S-transferase activity in rice EF-1betabeta'gamma and EF-1gamma expressed in Escherichia coli.

Plant elongation factor EF-1 consists of four subunits (EF-1alphabetabeta'gamma). EF-1alpha. GTP catalyses the binding of aminoacyl-tRNA to the ribosome. EF-1beta and EF-1beta' catalyze the GDP/GTP exchange on EF-1alpha. GDP. However, the function of EF-1gamma, a subunit detected in eukaryotes, but not in prokaryotes remained unknown. This report demonstrates that rice EF-1betabeta'gamma and recombinant EF-1gamma possess glutathione S-transferase (GST) activity. The EF-1betabeta'gamma- or EF-1gamma-dependent GST activity is about one-fiftieth of the rice GST activity. The Km values of EF-1betabeta'gamma, EF-1gamma, and rice GST for glutathione and 1-chloro-2,4-dinitrobenzene are of about the same order. Although recombinant EF-1gamma is heat labile, active EF-1gamma was obtained by purifying it in the presence of 20% glycerol.

Molecular characterization of cDNA encoding for adenylate kinase of rice (Oryza sativa L.).

Two types of genes (Adk-a, and Adk-b) encoding for adenylate kinase (AK, EC 2.7.4.3.) were isolated from the cDNA library constructed from poly(A)+ RNA of rice (Oryza sativa L.). Two cDNAs were heterogeneous at 5' and 3' ends of non-coding sequences and had possible polyadenylation signals. One of the genes, Adk-a, had 1154 bp sequences encoding 241 amino acid residues, while the other type, Adk-b, contained 1085 bp sequences encoding for 243 amino acid residues. Homology between Adk-a and Adk-b was 73.7% in nucleotide sequences, and 90.8% in amino acid level. Two genes showed about 53% homology to bovine mitochondrial adenylate kinase (AK2) at nucleotide and amino acid levels. Concerning the codon usage of rice AK genes, T was abundant at the third position of a codon in the reading frames. In order to examine the enzyme activity of the protein encoded by the rice cDNA, Adk-a was cloned into an expression vector, pUC119, which was introduced into Escherichia coli strain CV2, a temperature-sensitive mutant of adenylate kinase. We found that the transformant carrying the rice Adk-a gene in the sense orientation recovered cell growth at non-permissive high temperature (42 degrees C) and expressed enzyme activities higher than the untransformed CV2 and the transformant possessing Adk-a cDNA in the antisense orientation. These observations suggest that rice Adk-a codes a biologically active enzyme. Furthermore, sucrose was found to regulate the transcription of AK genes in rice cell cultures. Organ related accumulation of mRNA in whole plants was also found.

Isolation and characterization of a rice cDNA similar to the bovine brain-specific 14-3-3 protein gene.

We isolated a rice cDNA clone which is similar to the bovine brain-specific 14-3-3 protein (an activator protein of tyrosine and tryptophan hydroxylase involved in the synthetic pathway of monoamine) gene. The deduced amino acid sequence of the cDNA indicated a surprising similarity to a potent inhibitor of Ca(2+)-phospholipid-dependent protein kinase C. DNA blot analysis indicated that this gene is located at more than a single locus in rice genome DNA. Expression of this gene is regulated by external stresses.

Isolation and characterization of a rice cDNA similar to the S-phase-specific cyc07 gene.

We isolated a rice cDNA clone (T151) which encodes an open reading frame of 262 amino acids. This clone is similar to the S-phase-specific cyc07 gene of Catharanthus roseus. Expression of this gene is much higher in callus than in seedlings and regulated by external stresses such as high osmotic pressure, salinity, low temperature and submergence.

Isolation and characterization of a rice cDNA which encodes a ubiquitin protein and a 52 amino acid extension protein.

We isolated a rice cDNA clone encoding the ubiquitin protein fused to a ribosomal protein. This clone encodes a single ubiquitin polypeptide and extension protein of 53 amino acids. This extension protein shows a high degree of homology with those of the yeast ubil or ubi2 gene, both of which encode the same protein. Northern blot analysis suggested that the expression pattern of this gene is more similar to other ribosomal protein genes not linked to ubiquitin protein than to the polyubiquitin gene.

Expression of elongation factor 1 beta' in Escherichia coli and its interaction with elongation factor 1 alpha from silk gland.

Silk gland elongation factor 1 (EF-1) consists of four subunits: alpha, beta, beta', and gamma. EF-1 beta beta' gamma catalyzes the exchange of GDP for GTP on EF-1 alpha and stimulates the binding of EF-1 alpha-dependent aminoacyl-tRNA to ribosomes. The carboxy-terminal regions of the EF-1 beta subunits from various species are highly conserved. We examined the region of EF-1 beta' that binds to EF-1 alpha by in vitro binding assays, and examined the GDP/GTP exchange activity using deletion mutants of a GST-EF1 beta' fusion protein. We thereby suggested a pivotal amino acid region, residues 189-222, of EF-1 beta' for binding to EF-1 alpha.

Genomic organization, chromosomal localization, and promoter analysis of the mouse Mail gene.

The Mail (molecule possessing ankyrin repeats induced by lipopolysaccharide) protein is a member of the IkappaB family. It has six ankyrin repeats that are conserved in other IkappaB proteins, such as IkappaB-alpha and Bcl-3. Mail mRNA expression is induced rapidly following lipopolysaccharide (LPS) injection, most notably in the spleen, lung, and lymph nodes of mice, where immune cells, such as lymphocytes and macrophages, are abundant. In this study, we cloned and characterized the Mail gene. The isolated genomic clones span approximately 30 kb and encompass the entire gene. Comparisons with Mail cDNA revealed that the Mail gene consists of 14 exons. Several splice junctions encoding ankyrin repeats are conserved among Mail and other IkappaB family genes. Southern hybridization showed that Mail is a single-copy gene. Using fluorescence in situ hybridization analysis, mouse and rat Mail genes were mapped to Chromosome (Chr) 16C1.2-C1.3 and Chr 11q21.1, respectively. Primer extension determined the transcription start site of Mail. Sequence analysis of the proximal promoter region revealed the presence of a TATA box and putative transcription factor-binding sites, such as those for NF-kappaB and NF-IL6. This region is sufficient to drive high-level reporter gene expression in LPS-stimulated transfected cells.