Comparative Analysis of argK-tox Clusters and Their Flanking Regions in Phaseolotoxin-Producing Pseudomonas syringae Pathovars

DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181–10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064–11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named “tox islands.” The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437–457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

ent-Eudesmane sesquiterpenoids, galloyl esters of the oak lactone precursor, and a 3-O-methylellagic acid glycoside from the wood of Platycarya strobilacea

ent-Eudesmane sesquiterpenoids, 8,11-dihydroxy-2,4-cycloeudesmane, 11-hydroxy-2,4-cycloeudesman-8-one and 2,4-cyclo-7(11)-eudesmen-8-one, were isolated from the wood of Platycarya strobilacea, which has been used as an aromatic tree since at least the 18th century. On charring the wood, 2,4-cyclo-7(11)-eudesmen-8-one was detected in the smoke. In the charred wood, the concentrations of ellagitannins, such as galloyl pedunculagin, dramatically decreased, whereas concentrations of pentagalloyl glucose, and other gallotannins were relatively stable. In addition, two other compounds, the 6′-O-m- and p-digalloyl oak lactone precursor and the 3-O-methylellagic acid 4′-O-(4″-O-galloyl)-xylopyranoside, were isolated from the charred wood along with m- and p-digallic acid.     

Role of AtPolζ, AtRev1, and AtPolη in UV light-induced mutagenesis in Arabidopsis

Translesion synthesis (TLS) is a DNA damage tolerance mechanism in which DNA lesions are bypassed by specific polymerases. To investigate the role of TLS activities in ultraviolet light-induced somatic mutations, we analyzed Arabidopsis (Arabidopsis thaliana) disruptants of AtREV3, AtREV1, and/or AtPOLH genes that encode TLS-type polymerases. The mutation frequency in rev3-1 or rev1-1 mutants decreased compared with that in the wild type, suggesting that AtPolζ and AtRev1 perform mutagenic bypass events, whereas the mutation frequency in the polh-1 mutant increased, suggesting that AtPolη performs nonmutagenic bypass events with respect to ultraviolet light-induced lesions. The rev3-1 rev1-1 double mutant showed almost the same mutation frequency as the rev1-1 single mutant. The increased mutation frequency found in polh-1 was completely suppressed in the rev3-1 polh-1 double mutant, indicating that AtPolζ is responsible for the increased mutations found in polh-1. In summary, these results suggest that AtPolζ and AtRev1 are involved in the same (error-prone) TLS pathway that is independent from the other (error-free) TLS pathway mediated by AtPolη.     

Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.     

Peroxisome biogenesis and molecular defects in peroxisome assembly disorders

Peroxisome assembly in mammals requires more than 14 genes. So far, we have isolated seven complementation groups (CGs) of peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, Z65, Z24/ZP107, ZP92, ZP105/ZP139, ZP109, ZP110, ZP114. Two peroxin cDNAs, PEX2 and PEX6, were first cloned by genetic phenotype-complementation assay using Z65 and ZP92, respectively, and were shown to be responsible for peroxisome biogenesis disorders (PBD) such as Zellweger syndrome, of CG-F (the same as CG-X in U.S.A.) and CG-C (the same as CG-IV), respectively. Pex2p is a RING zinc finger membrane protein of peroxisomes and Pex6p is a member of the AAA ATPase family. We likewise isolated PEX12 encoding a peroxisomal integral membrane protein in the RING family, by functional complementation of ZP109, demonstrating PEX12 to be responsible for CG-III PBD. We also cloned PEX1 by screening of human liver cDNA library, using ZP107. PEX1 mutation was delineated to be the genetic cause of PBD in the most highest incidence group, CG-E (the same a CG-I). Moreover, we recently found that Pex5p, using PEX5-defective ZP105 and ZP139. Thus, CHO cell mutants defective in peroxisome biogenesis are indeed shown to be very useful for the studies of peroxisome assembly and delineating pathogenic genes in PBD. Furthermore, we have isolated novel CGs of CHO mutants, ZP119 and ZP126.     

Cytotaxonomic study of ferns of Yunnan,southwestern China

A cytotaxonomic study was made of 90 fern species of Yunnan, southwestern China, based on collections from northwestern, central, and southwestern Yunnan and a few Cheng's collections. The results verified most of the formerly reported basic chromosome numbers of Chinese genera, and recorded for the first time the basic numbers ofGymnogrammitis andSorolepidium and Chinese members of several other genera. Cytotaxonomy of some problematical genera was discussed. Biogeographical relationships between Japanese, Chinese, and Himalayan ferns were compared with special reference to local cyto-reproductive variations inConiogramme, Deparia, Onychium, andPteris cretica. The present evidence indicates that most triploid species examined are agamosporous, as general in filicalean ferns, and also suggests the sexual 32-spored sporogenesis inHypodematium crenatum andSorolepidium graciale as in Lindsaeaceae.     

A newly isolatedBacillus stearotheromophilus K1041 and its transformation by electroporation

Summary Optimal conditions for the plasmid transformation of a newly isolatedBacillus stearothermophilus K1041 by electroporation were investigated. The optimal conditions allowed a transformation efficiency of 5.8×10⁵ transformants per μg plasmid pUB110.     

Effect of metamorphosis on the major hemolymph proteins of the silkworm

During the metamorphosis of the silkworm, Bombyx mori, three major hemolymph proteins (MHPs) (molecular weights 17,000, 25,000, 27,000) were detected and found to be distributed in the hemolymph and in the tissues of several organs, such as the fat body, midgut, ovary, testis, and even eggs. The MHPs in eggs gradually decreased and disappeared during embryogenesis. The formation, distribution, and utilization of MHPs in tissues other than the gonad, however, were not affected by sex. Radioisotope experiments in vivo revealed that the MHPs were synthesized at an early period of the fifth larval instar. The synthesis of at least two of them occurred in the fat body. MHPs in the hemolymph entered the tissues at the onset of the larval-pupal transformation. On the basis of their appearance, distribution, and depletion, the MHPs may be classified as reserve proteins which are synthesized in the larval stage and utilized later in the developmental stages.