First complete nucleotide sequence and heterologous gene organization of the two rRNA operons in the phytoplasma genome

Phytoplasmas are cell-wallless Gram-positive low G + C bacteria belonging to the Mollicutes that inhabit the cytoplasm of plants and insects. Although phytoplasmas possess two ribosomal RNA (rrn) operons, only one has been fully sequenced. Here, we determined the complete nucleotide sequence of both rrn operons (designated rrnA and rrnB) of onion yellows (OY) phytoplasma. Both operons have rRNA genes organized as 5'-16S-23S-5S-3' with very highly conserved sequences; the 16S, 23S, and 5S rRNA genes are 99.9, 99.8, and 99.1% identical between the two operons. However, the organization of tRNA genes in the upstream region from 16S rRNA gene and in the downstream region from 5S rRNA gene differs markedly. Several promoter candidates were detected upstream from both operons, which suggests that both operons are functional. Interestingly, both have a tRNA(Ile) gene in the 16S-23S spacer region, while the reported rrnB operon of loofah witches' broom phytoplasma does not, indicating heterogenous gene organization of rrnB within phytoplasmas. The phytoplasma tRNA gene organization is similar to that of acholeplasmas, a closely related mollicute, and different from that of mycoplasmas, another mollicute. Moreover, the organization suggests that the rrn operons were derived from that of a related nonmollicute bacterium, Bacillus subtilis. This data should shed light on the evolutionary relationships and phylogeny of the mollicutes.     

Crystal structure of human ISG20, an interferon-induced antiviral ribonuclease

ISG20 is an interferon-induced antiviral exoribonuclease that acts on single-stranded RNA and also has minor activity towards single-stranded DNA. It belongs to the DEDDh group of RNases of the DEDD exonuclease superfamily. We have solved the crystal structure of human ISG20 complexed with two Mn2+ ions and uridine 5'-monophosphate (UMP) at 1.9 A resolution. Its structure, including that of the active site, is very similar to those of the corresponding domains of two DEDDh-group DNases, the epsilon subunit of Escherichia coli DNA polymerase III and E. coli exonuclease I, strongly suggesting that its catalytic mechanism is identical to that of the two DNases. However, ISG20 also has distinctive residues, Met14 and Arg53, to accommodate hydrogen bonds with the 2'-OH group of the UMP ribose, and these residues may be responsible for the preference of ISG20 for RNA substrates.     

Characteristics of NADPH oxidase genes (Nox2, p22, p47, and p67) and Nox4 gene expressed in blood cells of juvenile Ciona intestinalis

To illuminate the origins of NADPH oxidase (Nox), we identified cDNA clones encoding Nox2, Nox4, p22 phagocyte oxidase (phox), p47phox, and p67phox in a chordate phylogenetically distant to the vertebrates, the sea squirt Ciona intestinalis. We also examined the spatiotemporal expression of these genes in embryos and juveniles. The sequences of the Nox2, Nox4, p22phox, p47phox, and p67phox cDNAs contained open reading frames encoding 581, 811, 175, 461, and 515 amino acids, respectively. The level of identities between the deduced Nox2, Nox4, p22phox, p47phox, and p67phox amino acid sequences and their corresponding human components were 54.0, 31.0, 44.4, 36.0, and 26.2%, respectively. Despite these low identities, the functional domains of the C. intestinalis and human NADPH oxidase and Nox4 are highly conserved. The genomic organizations of the components of the NADPH oxidase gene except for p67phox (a single exon gene) and the Nox4 gene in C. intestinalis are highly similar to those of the corresponding human NADPH oxidase genes. Further, the analyzed part of the C. intestinalis genome and EST database do not seem to present p40phox and Nox5. The Nox2, p22phox, p47phox, and p67phox genes were specifically expressed in the blood cells of juveniles. The Nox4 gene was expressed in blood cells and endostyle of juveniles. These results suggest that C. intestinalis NADPH oxidase components possess potential functional activities similar to those of human, but the manner in which cytosolic phox proteins in C. intestinalis interact is different from that in human.     

Amino acid sequence and characterization of a nuclease (nuclease Le3) from Lentinus edodes

The fruit body of shiitake (Lentinus edodes) produces two acid nucleases, nuclease Le1 and nuclease Le3, both of which are thought to be candidates for the enzyme that produces a flavorful substance, 5'-GMP, and the primary structure of one of the nucleases, nuclease Le1, has been analyzed by both protein chemistry and gene cloning [Biosci. Biotechnol. Biochem. 64, 948-957 (2000)]. In this study the amino acid sequence of nuclease Le3 was analyzed by protein chemistry and gene cloning. Nuclease Le3 is a glycoprotein that contains 280 amino acid residues, and the molecular mass of the protein moiety of nuclease Le3 is 31,045. The nucleotide sequence of the cDNA and genomic DNA encoding nuclease Le3 revealed the presence of an 18-residue putative signal peptide. Nuclease Le3 contains 170, 108, and 98 amino acid residues that are identical to residues of nuclease Le1, nuclease P1, and nuclease S, respectively. The amino acid residues involved in coordination with Zn2+ atoms in nuclease P1 are all conserved in nuclease Le3. Nuclease Le3 contains 9 half-cystine residues, and 7 of them are located in the same positions as in nuclease Le1.     

A new method for fish leucocyte counting and partial differentiation by flow cytometry

A simple and rapid method for analysis of fish blood cells is presented. Carp (Cyprinus carpio) blood was diluted 200 times with Hanks' solution containing 1 microg/ml of DiOC6(3) which is a fluorescent, lipophilic dye. After staining for 10 min, the blood cells were measured by a flow cytometer (FACS). Several blood cell populations were identified by different FL-1 (green fluorescence), FSC (forward scatter), and SSC (side scatter) properties. FL-1 v. SSC or FSC v. SSC dot-plot of stained blood cells displayed five separate cell populations: erythrocytes: a mixture of thrombocytes plus lymphocytes; monocytes; neutrophils; and basophils. The number of each type of blood cell counted by the FACS was in good agreement with those counted microscopically.     

Electrophoretic Studies on Plant Protoplasts II. Relative Amounts of Various Charged Groups on the Surface of Barley Mesophyll Protoplasts

Electrophoretic mobilities of barley mesophyll cell protoplastsmodified by chemical and enzymatic treatments were measuredin media at various pH values to elucidate the contributionof phosphate, carboxylate and amino groups to the surface chargedensity. Existence of these charged groups was confirmed byresults of treatment of protoplasts with glutaraldehyde (foramino groups), acid phosphatase (for phosphate groups) and l-ethyl-3-(3-dimethylaminopropyl)carbodi-imidetogether with glycine methyl ester (for carboxylate groups).The relative amounts of these groups were estimated from thecurves of surface charge density () vs. surface pH (pH_s) ofthe treated protoplasts, in terms of simplified acid-base dissociationcurves. The estimated ratio of the amounts of phosphate, carboxylateand amino groups was approximate 0.5 :0.5 : 0.7 for the native(unmodified) barley mesophyll cell protoplasts, when the totalnegative charge on the native protoplasts was assumed to be1. (Received January 9, 1989; Accepted April 28, 1989)     

Primary structure of a ribonuclease from bullfrog (Rana catesbeiana) liver

A pyrimidine base-specific ribonuclease was purified from bullfrog (Rana catesbeiana) liver by means of CM-cellulose column chromatography and affinity chromatography on heparin-Sepharose CL-6B, which gave single band on SDS-slab electrophoresis. The primary structure of the bullfrog liver RNase was determined. It consisted of 111 amino acid residues, including 8 half-cystine residues. From the sequence, it was concluded that three disulfide bridges in RNase A were conserved in the bullfrog RNase, that a disulfide bridge in RNase A [Cys65-Cys126 (RNase A numbering)] was deleted, and that a new disulfide bridge was created in the C-terminal part of the enzyme. In this frog RNase, the amino acid residues thought to be essential for catalysis in bovine pancreatic RNase A were conserved except for Asp121 (RNase A numbering). The sequence homology of the bullfrog liver RNase with bovine pancreatic RNase A was 30.6%. The sequence of bullfrog liver RNase was very similar to those of lectins obtained from bullfrog egg by Titani et al. [Biochemistry (1988) 26, 2189-2194] and R. japonica egg by Kamiya et al. [Seikagaku (in Japanese) (1989) 60, 733; and personal communication from Kamiya, Y., Oyama, F., Oyama, R., Sakakibara, F., Nitta, K., Kawauchi, H., and Titani, K.]. The sequence homology between the bullfrog liver RNase and the two lectins was 70.2 and 64.8%, respectively.     

Effect of Top Pruning on Stem Carbohydrates Reserves and Foliar 14CO2 Assimilation in Mulberry Trees during Autumn

Removal of shoot tops from mulberry plants in autumn hastenedthe accumulation of starch in the stems even though photosynthesisin the remaining uppermost leaves was supressed. The proportionof the amount of ¹⁴CO₂ assimilate taken into the veins and thecontents of foliar carbohydrates and protein were not affectedby pruning. (Received December 22, 1983; Accepted March 21, 1984)     

Sequence variation, differential expression and chromosomal location of rice chitinase genes

Rice chitinases are encoded by a small multigene family. To clarify the overall organization of rice chitinase genes, we have isolated and characterized the genes Cht-1, Cht-2 and Cht-3. Although all the three genes encode class I chitinase, the nucleotide sequences of the coding regions of Cht-1 and Cht-3 are very similar (90%), while that of Cht-2 is clearly more divergent (78%). Only Cht-2 has a 130 by intron and encodes a C-terminal peptide sequence similar to that known to function as a vacuolar targeting signal. In 5 flanking regions of Cht-1 and Cht-3, but not of Cht-2, conserved sequences (GGCCGGCYGCCCYAG) were found. Related sequences were found also in the 5 flanking regions of another chitinase gene and a -glucanase gene which has also been reported to be stress-induced in rice. RNA blot hybridization analysis demonstrated that the stress-induced expression patterns of the Cht-1 and Cht-3 genes are similar, but quite different from that of Cht-2. However, all three genes are active in unstressed roots. By restriction fragment length polymorphism (RFLP) linkage analysis, Cht-1 and Cht-3 were mapped onto chromosome 6 and shown to be closely linked (0.8 cM). Cht-2 was mapped onto chromosome 5. All these features suggest that the expression patterns of rice class I chitinase genes may be correlated with their levels of sequence divergence and their chromosomal location.