In budding yeast, exit from mitosis is achieved by inactivation of Cdc28͞Clb2 activity. Although it is not clear at present how mitotic exit is triggered, a growing body of evidence suggests that the Tem1 GTPase plays a critical role in mediating this pathway and that Bfa1 and Bub2 constitute a two-component GTPase-activating protein to negatively regulate Tem1. Here, we have demonstrated that introduction of bfa1⌬ suppresses the growth defects associated with the cdc5-1 mutation significantly better than that of bub2⌬, suggesting that Bfa1 may have a previously uncharacterized role in this pathway. Overexpression of BFA1 efficiently arrested the cell cycle at postanaphase even in the absence of BUB2, whereas overexpression of BUB2 weakly induced mitotic arrest only in the presence of BFA1. Coimmunoprecipitation and in vitro binding studies indicate that Bfa1 binds strongly to Tem1 independently of Bub2. Provision of GDP؉AlF 4 ؊ , which mimics the GTPase transition state, enhanced the Bub2-Tem1 interaction both in vitro and in vivo. Interestingly, introduction of bfa1⌬, but not bub2⌬, greatly increased the interaction between Tem1 and Cdc15, a step that is thought to be critical for activating the mitotic exit network. Our data suggest that, in addition to its role as a putative, two-component GTPase-activating protein with Bub2, Bfa1 also can play a role in the regulation of mitotic exit by directly inhibiting the interaction between Tem1 and Cdc15 even in the absence of Bub2.Cdc15 ͉ two-component GAP ͉ budding yeast

Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn 2+ -binding coordination. To elucidate the role of the Zn 2+ , we disrupted the Zn 2+ -binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn 2+ -binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50°C. The mutations also abolished the Zn 2+ -induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn 2+ at 60°C, whereas the mutant enzymes exhibited no response to Zn 2+ . Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn 2+ on lipase L1 at elevated temperatures.

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The E7 protein produced by high-risk human papillomavirus (HPV) induces a degradation of the retinoblastoma tumor suppressor RB through direct interaction, which suggests that an inhibitor for the interaction can be a potential anticancer drug. A surface plasmon resonance (SPR) imaging-based protein array chip was developed for the high-throughput screening of inhibitor molecules targeting RB-E7 interaction. The glutathione S-transferase-fused E7 protein (GST-E7) was first layered onto a glutathionylated gold chip surface that had been designed to specifically bind to GST-fused proteins. Subsequently, a microarrayer was used to spot the hexa-histidine-tagged RB proteins (His(6)-RB) onto the GST-E7-layered gold chip surface, and the resulting SPR image was analyzed. Upon increased His(6)-RB concentration in the spotting solution, the SPR signal intensity increased proportionally, indicating that His(6)-RB bound to GST-E7 in a concentration-dependent manner. The His(6)-RB/GST-E7 interaction was challenged by spotting the His(6)-RB solution in the presence of a RB binding peptide (PepC) derived from a motif on E7. The SPR imaging data showed that PepC inhibited the His(6)-RB/GST-E7 interaction in a concentration-dependent manner. Our results show that the SPR imaging-based protein array chip can be applied to screen small molecule inhibitors that target protein-protein interaction.

Diversity of A mating type in Lentinula edodes has been assessed by analysis of A mating loci in 127 strains collected from East Asia. It was discovered that hypervariable sequence region with an approximate length of 1 kb in the A mating locus, spanning 5' region of HD2-intergenic region-5' region of HD1, could represent individual A mating type as evidenced by comprehensive mating analysis. The sequence analysis revealed 27 A mating type alleles from 96 cultivated strains and 48 alleles from 31 wild strains. Twelve of them commonly appeared, leaving 63 unique A mating type alleles. It was also revealed that only A few A mating type alleles such as A1, A4, A5, and A7 were prevalent in the cultivated strains, accounting for 62.5% of all A mating types. This implies preferred selection of certain A mating types in the process of strain development and suggests potential role of A mating genes in the expression of genes governing mushroom quality. Dominant expression of an A mating gene HD1 was observed from A1 mating locus, the most prevalent A allele, in A1-containing dikaryons. However, connections between HD1 expression and A1 preference in the cultivated strains remain to be verified. The A mating type was highly diverse in the wild strains. Thirty-six unique A alleles were discovered from relatively small and confined area of mountainous region in Korean peninsula. The number will further increase because no A allele has been recurrently observed in the wild strains and thus newly discovered strain will have good chances to contain new A allele. The high diversity in small area also suggests that the A mating locus has evolved rapidly and thus its diversity will further increase.

A Gram-negative bacterium, designated strain EMB117 T , was isolated from a municipal wastewater treatment plant and characterized by polyphasic taxonomy. The cells were non-spore-forming rods that showed gliding motility. Optimal growth occurred at 25-30 6C and pH 7.0-8.0. Strain EMB117 T contained phosphatidylethanolamine as the predominant polar lipid, and the major fatty acids were iso-C 15 : 0 , iso-C 17 : 0 3-OH, iso-C 15 : 0 3-OH and summed feature 3 (C 16 : 1 v7c and/or iso-C 15 : 0 2-OH). The G+C content of the genomic DNA was 33.5 mol% and the major isoprenoid quinone was MK-6. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain EMB117 T belonged to the genus Flavobacterium and was most closely related to Flavobacterium johnsoniae DSM 425 T (97.8 % sequence similarity). The DNA-DNA relatedness between strain EMB117 T and F. johnsoniae ATCC 17061 T was about 18 %. On the basis of the phenotypic, chemotaxonomic and molecular data, strain EMB117 T represents a novel species within the genus Flavobacterium, for which the name Flavobacterium defluvii sp. nov. is proposed. The type strain is EMB117 T (=KCTC 12612 T =DSM 17963 T ).

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