Rapid identification of 1-aminocyclopropane-1-carboxylate (ACC) synthase genotypes in cultivars of Japanese pear (Pyrus pyrifolia Nakai) using CAPS markers

In Japanese pear (Pyrus pyrifolia Nakai), fruit storage potential is closely related to the amount of ethylene produced. We have developed a rapid and accurate method for analyzing genes involved in high ethylene production during fruit ripening in Japanese pear. This involves cleaved-amplified polymorphic sequences (CAPS) of two 1-aminocyclopropane-1-carboxylate (ACC) synthase genes (PPACS1 and PPACS2). Two CAPS markers (A for PPACS1 and B for PPACS2), associated with the amount of ethylene produced, were identified. Marker A was associated with high ethylene producers and marker B with moderate ethylene producers. The absence of these two markers enabled the identification of low ethylene producers. Using these markers, we have identified ethylene genotypes for 40 Japanese pear cultivars and two Chinese pear (P. bretschneideri) cultivars that are commercially important and used in breeding programs. Furthermore, we performed linkage analysis of these two genes in the F(2) population, which revealed that the recombination frequency between the two markers was 20.8 +/- 3.6%. This information is critical to the selection of parents and in breeding strategies to improve storage ability of Japanese pears.     

Polymerization and calcium binding of the tubulin-colchicine complex in the GDP state

The tubulin-colchicine complex instead of tubulin was used in an imidazole buffer throughout experiments. The interaction with calcium was examined, especially in the GDP state. The high affinity sites of calcium took part in the polymerization of the complex in the GTP state, while the low ones participated in the depolymerization. The complex had 2 high affinity sites with the dissociation constant of 11.5 x 10(-6) M, and 16 low affinity sites with the dissociation constant of 2.27 x 10(-4) M in the GTP state. In the case of GDP state, the dissociation constant of the high affinity site was 7.2 x 10(-6) M, and the low affinity site was not observed. The ultracentrifugal experiment indicated a little compact structure in the GTP state compared with the GDP state. This agreed with the results of calcium binding.     

Transformation of Aspergillus aculeatus using the drug resistance gene of Aspergillus oryzae and the pyrG gene of Aspergillus nidulans

Transformation systems for Aspergillus aculeatus has been developed, based on the use of the pyrithiamine resistance gene of Aspergillus oryzae and the orotidine-5'-monophosphate decarboxylase gene (pyrG) of Aspergillus nidulans. An A. aculeatus mutant which can be transformed effectively by the A. nidulans pyrG gene was isolated as a transformation host. This is the first report of transformation of A. aculeatus.     

The crystal structure of the ttCsaA protein: an export-related chaperone from Thermus thermophilus

The CsaA protein was first characterized in Bacillus subtilis as a molecular chaperone with export-related activities. Here we report the 2.0 Angstrom-resolution crystal structure of the Thermus thermophilus CsaA protein, designated ttCsaA. Atomic structure and experiments in solution revealed a homodimer as the functional unit. The structure of the ttCsaA monomer is reminiscent of the well known oligonucleotide-binding fold, with the addition of extensions at the N- and C-termini that form an extensive dimer interface. The two identical, large, hydrophobic cavities on the protein surface are likely to constitute the substrate binding sites. The CsaA proteins share essential sequence similarity with the tRNA-binding protein Trbp111. Structure-based sequence analysis suggests a close structural resemblance between these proteins, which may extend to the architecture of the binding sites at the atomic level. These results raise the intriguing possibility that CsaA proteins possess a second, tRNA-binding activity in addition to their export-related function.     

The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method

A temperature-jump (T-jump) time-resolved X-ray crystallographic technique using the Laue method was developed to detect small, localized structural changes of proteins in crystals exposed to a temperature increase induced by laser irradiation. In a chimeric protein between thermophilic and mesophilic 3-isopropylmalate dehydrogenases (2T2M6T), the initial structural change upon T-jump to a denaturing temperature (approximately 90 degrees C) was found to be localized at a region which includes a beta-turn and a loop located between the two domains of the enzyme. A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this beta-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of T:(m) by approximately 10 degrees C) and no T-jump-induced structural change in this region was detected by our method. These results indicate that thermal unfolding of the original chimeric enzyme, 2T2M6T, is triggered in this beta-turn region.     

Community dynamic models of two dioecious tree species

The effects of dioecy on community dynamics were examined by using transition matrix models for two dioecious tree species, one a superior competitor with a narrow dispersal range and the other an inferior competitor with a wide dispersal range. The models are based on tree-by-tree replacements in each identical microsite occupied by either male or female canopy trees of the superior competitor and canopy trees of the inferior competitor. Coexistence of the two species is possible not only because of a trade-off between competitive and dispersal abilities but also because of the existence of a competitor gap, which the superior competitor cannot occupy. The competitor gap is created under the male trees of the superior competitor. The inferior competitor occupies the competitor gap because of its wide dispersal range. The relative abundance of the two species depends on the dispersal ability and sex ratios of the superior competitor. The decreasing dispersal ability and the female abundance of the superior competitor increase the competitor gap, which allows the regeneration of the inferior competitor.An erratum to this article can be found at     

Cloning and sequencing of an endoglucanase gene from Scopulariopsis brevicaulis TOF-1212, and its expression in Saccharomyces cerevisiae

The egI gene, encoding a major endoglucanase (EGI) of Scopulariopsis brevicaulis TOF-1212, was cloned and sequenced. The eglgene consisted of 868 bp with one intron and encoded a protein of 229 amino acids with a calculated molecular mass of 22,392 daltons. The EGI was assigned to a family 45 of glycosyl hydrolases and showed high similarity with other fungal endoglucanases, especially with those of Humicola grisea and Fusarium oxysporum, on the basis of hydrophobic cluster analysis. The egI gene was expressed under the promoter of the phosphoglycerate kinase gene (PGK) in Saccharomyces cerevisiae. The transformed cells were able to secrete the enzyme efficiently in an active form.     

Cerebrosides in grapevine leaves: distinct composition of sphingoid bases among the grapevine species having different tolerances to freezing temperature

Cerebrosides from leaves of three grapevine species were analyzed in detail. The relative proportions of 8-E/Z isomers of 4-hydroxy-8-sphingenines [i.e. 8-E/Z t18:1(8E) and (8Z)] differed amongst the species in respect to freezing tolerance. This suggests that the occurrence of high levels of t18:1(8Z) in cerebrosides is correlated with freezing tolerance in these species.     

Papilin in development; a pericellular protein with a homology to the ADAMTS metalloproteinases

Papilin is an extracellular matrix glycoprotein that we have found to be involved in, (1) thin matrix layers during gastrulation, (2) matrix associated with wandering, phagocytic hemocytes, (3) basement membranes and (4) space-filling matrix during Drosophila development. Determination of its cDNA sequence led to the identification of Caenorhabditis and mammalian papilins. A distinctly conserved 'papilin cassette' of domains at the amino-end of papilins is also the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteinases; this cassette contains one thrombospondin type 1 (TSR) domain, a specific cysteine-rich domain and several partial TSR domains. In vitro, papilin non-competitively inhibits procollagen N-proteinase, an ADAMTS metalloproteinase. Inhibiting papilin synthesis in Drosophila or Caenorhabditis causes defective cell arrangements and embryonic death. Ectopic expression of papilin in Drosophila causes lethal abnormalities in muscle, Malpighian tubule and trachea formation. We suggest that papilin influences cell rearrangements and may modulate metalloproteinases during organogenesis.     

Grb7 signal transduction protein mediates metastatic progression of esophageal carcinoma

We have previously reported the association of tumor cell invasion with expression of growth factor receptor-bound protein 7 (Grb7). This molecule contains a Src homology 2 (SH2) domain and shares structural homology with a cell migration molecule designated Mig-10 found in Caenorhabditis elegans. In the present study, Grb7 expression was analyzed in human esophageal carcinomas with or without metastatic spread. The Grb7 protein was overexpressed in 14 of 31 esophageal carcinomas as compared to the adjacent normal mucosa (45%) and this finding was significantly correlated with the presence of lymph node metastases. We also identified that Grb7 protein in esophageal carcinoma cells was phosphorylated on tyrosine by epidermal growth factor as well as attachment to extracellular matrix proteins including fibronectin. Such fibronectin-dependent phosphorylation of Grb7 was regulated by integrin signaling that leads to the interaction with focal adhesion kinase protein. Furthermore, ectopic expression of a Grb7-SH2 dominant-negative fragment inhibited the fibronectin-dependent phosphorylation of endogenous Grb7, and reduced migration of esophageal carcinoma cells into fibronectin. Our results suggest a role of Grb7 mediated signal transduction in generation of an invasive cell phenotype against extracellular matrix, and thus contributes to metastatic progression of human esophageal carcinoma.