Jiro Kataoka

Morphological changes and hypomethylation of DNA in transgenic tobacco expressing antisense RNA of the S-adenosyl-l-homocysteine hydrolase gene

S-adenosyl-l-homocysteine hydrolase (SAHH) is a key enzyme in the regulation of intracellular methylation reactions. To investigate the role of SAHH in methylation reactions and morphogenesis in planta, we have made transgenic plants expressing antisense RNA of tobacco SAHH. The transgenic plants displayed distinct morphological changes including a floral homeotic change. We hypothesized that the changes were caused by increased levels of cytokinin. In those transgenic plants, we observed that a repetitive DNA sequence appeared less methylated than controls. We speculated that altered gene expressions by the hypomethylation of DNA might be involved in the changes.

Isolation and analysis of a genomic clone encoding a pokeweed antiviral protein

Partial cDNAs encoding a pokeweed antiviral protein were obtained by polymerase chain reaction from the poly(A)+ RNA of seeds, leaves, and roots using two specific primers based on the amino acid sequence of a pokeweed antiviral protein from the seeds (PAP-S). Using the cDNAs as a radioactive probe, 17 and 39 positive plaques were isolated from libraries containing the genomic DNA of Phytolacca americana digested with Bam HI partially and completely, respectively. The plaques were grouped into nine types by Southern hybridization. The type α genomic clone encodes a protein of 294 amino acids. Its amino acid sequence is similar but not identical to that of PAP-S. A comparison of the two amino acid sequences suggested that the deduced protein contains extrapeptides of 24 and 9 amino acids at the NH2 and the COOH terminals, respectively. The putative protein was expressed in Escherichia coli and shown to depurinate the specific adenine of wheat 25S rRNA, indicating that the protein encoded by a type α genomic clone is a functional protein exhibiting RNA N-glycosidase activity.

Inducible expression by plant hormones of S-adenosyl-l-homocysteine hydrolase gene from Nicotiana tabacum during early flower bud formation in vitro

Abstract cDNA clones corresponding to genes induced during the early stage of flower bud formation in thin cell layer (TCL) cultures of tobacco were isolated by differential screening. Sequence analysis showed that one of the clones encoded S -adenosyl- l -homocysteine hydrolase (SAHH), which is known to be an enzyme regulating intracellular transmethylation reactions. Recently, it was also found to be a cytokinin-binding protein. An SAHH gene was isolated by screening a tobacco genomic library with the cDNA as a probe. Northern blot analysis showed that expression of the SAHH gene was greatest in pistils and roots, and could be induced by kinetin and IAA. Kinetin and IAA are required for in vitro flower differentiation in TCL culture. The promoter sequence of the gene was fused to the β-glucuronidase (GUS) reporter gene and introduced in suspension-cultured cells, which rendered expression of GUS inducible by kinetin. Possible involvement of SAHH in regulation of gene expression, cytokinin signal transduction and flower differentiation is discussed.

Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP)

Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A4324 in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes.Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.

Escherichia coli ribosome is inactivated by Mirabilis antiviral protein which cleaves the N-glycosidic bond at A2660 of 23 S ribosomal RNA.

Ribosome-inactivating proteins (RIPs) are known to inactivate eukaryotic ribosomes, which results in the inhibition of protein synthesis, but there has been no evidence that they inactivate the ribosomes of Escherichia coli. Recently, Mirabilis antiviral protein (MAP), a RIP, has been shown to inhibit the protein synthesis of E. coli as well as eukaryotes. To elucidate its mechanism, E. coli ribosomes treated with MAP were analyzed by polyacrylamide/agarose composite gel electrophoresis and RNA sequencing using reverse transcriptase with DNA primer. The 23 S rRNAs, with an A260 value for ribosomes of 15, were completely cleaved in vitro by a 30 minute treatment with MAP at a concentration of 100 nM at 37 degrees C and a subsequent treatment with aniline. However, they were not affected by ricin A-chain under the same conditions. The primer extension of DNA polymerization stopped before A2660 of 23 S rRNA in RNA sequencing. Furthermore, both 16 S and 23 S rRNAs were cleaved by the MAP and aniline treatments when naked E. coli rRNAs were used as substrates, and the primer extension stopped before bases A2660 and A1014, respectively, in RNA sequencing. As the A2660 region has been shown to interact with the elongation factors EF-Tu and EF-G these results indicate that MAP cleaves the N-glycosidic bond at A2660 in E. coli 23 S RNA resulting in the inactivation of the ribosome.

Nucleotide sequence of a genomic gene encoding tritin, a ribosome-inactivating protein from Triticum aestivum.

A genomic gene of tritin, a ribosome-inactivating protein (RIP) from Triticum aestivum, was cloned using a barley RIP gene as a probe. The 5′-non-coding region has potential TATA boxes and three sequences homologous to the binding sequence of the transcriptional activator protein Opaque-2 which activates maize RIP gene expression. The cloned DNA encoded tritin consits of 275 amino acids with no secretion signal sequence. The coding region of tritin was expressed in Escherichia coli using lac promoter and yielded a protein similar to the native one, as determined by SDS-polyacrulamide gel electrophoresis and immunological analysis.

Expression of a pokeweed antiviral protein in Escherichia coli and its characterization

Two expression vectors were constructed to produce a putative mature α‐pokeweed antiviral protein (α‐PAP) in Escherichia coli with its NH2‐ and COOH‐terminal extrapeptides excised. One was for its intracellular expression with a methionine at its NH2‐terminal. The other was for its secretion using an ompA signal peptide. The former product was purified from the total soluble proteins of the transformant with a yield of 1.74 mg/liter and the latter had a yield of 5.55 mg/liter. Both products exhibited RNA N‐glycosidase activity on wheat ribosomes and inhibitory activity to protein synthesis in a rabbit reticulocyte system.

Comparative study of screening with subtracted probe and differential screening on isolation of flower-specific cDNA clones from Nicotiana sylvestris

Abstract A simple and efficient screening procedure for isolation of tissue-specific cDNA clones is described. A plasmid cDNA library was constructed from mRNAs extracted from Nicotiana sylvestris flower buds. Flower-specific cDNA clones were identified after screening of the cDNA library with subtracted probe enriched for flower-specific sequences. Comparison of this procedure with the conventional differential screening indicated that it has the advantages of direct isolation of highly specific cDNA clones, as well as identification of less abundant specifically expressed clones. The isolated cDNA clones were classified into 14 independent cross-hybridization groups. Northern blot analysis with the cDNA clones as probes, distinguished three types of mRNA expression patterns: (1) specific expression in stamen during a restricted period of flower development; (2) expression in the reproductive organs; (3) expression in both the structural and reproductive flower organs. Sequencing revealed that 6 of the groups correspond to known genes, but the remaining 8 were not homologous to the sequences in the data bank.

Improved Crystals of the Toxic Protein MAP by Protein Engineering Towards the Host Specificity

Mirabilis anti-viral protein (MAP) is a ribosome-inactivating protein from Mirabilis jalapa L. Since MAP is effective over a broad spectrum of species, the protein is difficult to express in heterologous hosts such as Escherichia coli. Recently, we obtained a MAP mutant, Y72F which exhibits a lower (1/100) activity against E. coli ribosomes while retaining almost full activity against mammalian cells [Habuka, Miyano, Kataoka, Tsuge & Noma (1992). J. Biol. Chem. 267, 7758-7760]. For the crystallographic studies, the Y72F MAP expression vector with an OmpA leading sequence was constructed and expressed in E. coli. The Y72F MAP mutant was then isolated and purified from the cell culture medium. Crystals were grown using the crystallization conditions for the native MAP crystals [Miyano et al. (1992). J. Mol. Biol. 226, 281-283]: 50% ammonium sulfate containing 50 mM ammonium citrate and 2 mM adenine sulfate, pH 5.4. The crystals belong to space group P3(1)21 (or P3(2)21) with a = b = 104.1 and c = 134.3 A. The crystals are isomorphous with the wild-type crystals but diffract to higher resolution. Imaging-plate photographs of the Y72F mutant showed sharp intense spots without the streaking observed in the native crystals.