Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale ( Balaenoptera bonaerensis )

Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against alpha-tubulin 4-6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.     

Hyperthermophilic DNA methyltransferase M.PabI from the archaeon Pyrococcus abyssi

Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5'-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5'-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95 degrees C and retained at least half the activity after 9 min at 95 degrees C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of kcat, Km, DNA, and Km, AdoMet. The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn2+ but not by Mg2+, Ca2+, or Mn2+. These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.     

The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols

A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.     

Coordinated regulation of dorsal bone morphogenetic protein 4 and ventral Sonic hedgehog signaling specifies the dorso‐ventral polarity in the optic vesicle and governs ocular morphogenesis through fibroblast growth factor 8 upregulation

Dorsal and ventral specification in the early optic vesicle plays a crucial role in vertebrate ocular morphogenesis, and proper dorsal‐ventral polarity in the optic vesicle ensures that distinct structures develop in separate domains within the eye primordium. The polarity is determined progressively during development by coordinated regulation of extraocular dorsal and ventral factors. In the present study, we cultured discrete portions of embryonic chick brains by preparing anterior cephalon, anterior dorsal cephalon and anterior ventral cephalon, and clearly demonstrate that bone morphogenetic protein 4 (BMP4) and Sonic hedgehog (Shh) constitute a dorsal‐ventral signaling system together with fibroblast growth factor 8 (FGF8). BMP4 and Shh upregulate Tbx5 and Pax2, as reported previously, and at the same time Shh downregulates Tbx5, while BMP4 affects Pax2 expression to downregulate similarly. Shh induces Fgf8 expression in the ventral optic vesicle. This, in turn, determines the distinct boundary of the retinal pigmented epithelium and the neural retina by suppressing Mitf expression. The lens develops only when signals from both the dorsal and ventral regions come across together. Inverted deposition of Shh and BMP4 signals in organ‐cultured optic vesicle completely re‐organized ocular structures to be inverted. Based on these observations we propose a novel model in which the two signals govern the whole of ocular development when they encounter each other in the ocular morphogenic domain.     

Comparison of promoter activities of heterologous and homologous promoters in Bacillus subtilis

Summary We have constructed promoter probe vectors with the Escherichia coli galactokinase monitoring system that can be used in Bacillus subtilis. In vivo studies with these vectors demonstrated that the E. coli trp and tac(trp::lac) promoter regions could be utilized in B. subtilis. These promoter regions and the promoter region for the erythromycin resistance gene originating from Staphylococcus aureus were preferentially utilized during the stationary growth phase of B. subtilis, whereas the B. subtilis P21K and P29K promoters were utilized during the exponential growth phase and decreased rapidly during the stationary phase. The apparent strength of these promoters of E. coli in B. subtilis, in terms of galactokinase units, was comparable with those of the B. subtilis promoters.     

Phylogenetic relationships of Australian skinks of the Mabuya group (Reptilia: Scincidae) inferred from mitochondrial DNA sequences

Portions of two mitochondrial genes (12S and 16S ribosomal RNAs) were sequenced to analyze the phylogenetic relationships of the Mabuya group from the Australian region (Corucia, Egernia and Tiliqua). Results indicated the monophyly of these genera and their divergence from Asian and African members of this group. This suggests that the diversity of the Mabuya group in the Australian region has increased through an endemic radiation, not through multiple colonizations from outside. Among the genera from this region, Corucia and Tiliqua were closest to each other. This result contradicts with those of the previous hypotheses on the basis of morphological and immunological data that, respectively, suggested closest affinities between Corucia and Egernia, and Egernia and Tiliqua. We suppose that the morphological characters exclusively joining Corucia and Egernia are actually in plesiomorphic state.     

Expression of foreign type I ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) stimulates photosynthesis in cyanobacterium Synechococcus PCC7942 cells

A reporter gene assay revealed that promoters derived from Synechococcus PCC7942 (S.7942) psbAI and Synechocystis PCC6803 (S.6803) psbAII were suitable for the expression of foreign ribulose-bisphosphate carboxylase (RuBisCO; EC 4.1.1.39) in S.7942 cells. Transformational vectors with a promoter and a foreign RuBisCO gene, cvrbc originated from Allochromatium vinosum, were constructed on a binary vector, pUC303, and introduced to S.7942 cells. When the cvrbc was expressed with the S.7942 psbAI promoter, the total RuBisCO activity increased 2.5- to 4-fold than that of the wild type cell. The S.6803 psbAII promoter increased the activity of the transformant 1.5–2 times of that of wild type cell. There was a significant increase in the rate of photosynthesis depending on the increase of RuBisCO activity. The maximum rate of photosynthesis of the transformant cell was 1.63 times higher than that of the wild type under the illumination of 400 μmol m⁻² s⁻¹, at 20 mM bicarbonate and at 30 °C. Although the photosynthesis of the higher plant is limited by the ability of photosystems under high irradiance and the high CO₂ concentration, that of the S.7942 cell is limited by the RuBisCO activity, even at high CO₂ concentrations and under high irradiance.     

Serum-free culture of an embryonic cell line from <Emphasis Type="Italic">Bombyx mori</Emphasis> and reinforcement of susceptibility of a recombinant BmNPV by cooling

We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67 ± 5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.     

Application of fuzzy reasoning to control of glucose and ethanol concentrations in baker's yeast culture

Fuzzy reasoning was applied to control both ethanol and glucose concentrations in fed-batch cultures of baker's yeast. This fuzzy controller consisted of three membership functions (concentrations of dissolved oxygen (DO), ethanol and glucose) and 18 production rules. Fuzzy inference was carried out by IF {A is a and B is b,...#x007D;, THEN {C is c} from the on-line measured concentrations of DO, ethanol and glucose. When medium concentrations of ethanol and glucose in fed-batch culture of baker's yeast were set at 2 g/l and 0.2 g/l, both ethanol and glucose concentrations were controlled at 2.67±0.35 g/l and 0.27±0.25 g/l, respectively, ethanol production was reduced from 26 g/l to 34 g/l, cell yield increased from 0.38 to 0.53 g dry cell/g consumed glucose and ethanol yield decreased from 0.50 to 0.14 g ethanol/g consumed glucose, respectively, as compared with those of the glucose only control at 0.2 g/l.