Characterization of two beta-tubulin genes from Geotrichum candidum.

Summary The -tubulin genes G1 and G2 from the phytopathogenic hemiascomycete Geotrichum candidum were found to be highly diverged in amino acid sequence from those of other filamentous fungi. G1 and G2 were also divergent from each other, with the coding regions sharing only 66% nucleotide sequence homology and 64% amino acid identity. However, the proteins shared 82% similarity and only 25 of the 161 non-identical amino acid substitutions were non-conservative. The organization of G1 is similar to other fungal -tubulin genes, but G2 has several unusual features; it has 2 amino acid additions in the N-terminal 40 residues and must employ an uncommon 5 splice junction sequence in preference to an overlapping perfect consensus. The amino acid change found to confer benomyl resistance in Neurospora crassa was also present in G2. G1 has four introns which are located similarly to those of -tubulin genes in other fungi. G2, however, has a single intron in a unique location. Translational fusions employing the 5 non-coding regions of the two Geotrichum -tubulin genes were made with the hygromycin phosphotransferase gene and shown to function in Schizosaccharomyces pombe and Trichoderma hamatum. However, G. candidum could not be transformed with these or other tested plasmids commonly used for fungal transformation.     

Interferon γ but not tumor necrosis factor α decreases susceptibility of human renal cell cancer cell lines to lymphokine-activated killer cells

Summary Human renal cell cancer (RCC) cell lines, ACHN and KRC/Y, with or without exposure to cytokines, were examined for their susceptibility to lymphokine-activated killer (LAK) cells. Flow-cytometric analysis demonstrated constitutional expression of class I antigen on both cell lines, which was enhanced by interferon (IFN), IFN and tumor necrosis factor (TNF). A 4-h⁵¹Cr-release cytotoxicity assay demonstrated that pretreatment of both cell lines with IFN or IFN, but not with TNF, decreased their susceptibility to LAK cells. IFN also decreased susceptibility to natural killer cells in a 16-h⁵¹Cr-release cytotoxicity assay. IFN treatment decreased the susceptibility of ACHN cells in a dose-dependent manner. Cold-target competition assay clearly showed that IFN- but not TNF-pretreated cells compete less effectively than do untreated target cells. Pretreatment with IFN, however, increased expression of intercellular adhesion molecule-1 (ICAM-1) to a degree comparable to that with TNF. Northern blot analyses using a 520-base-pair ICAM-1 cDNA as a probe demonstrated that more 3.3-kb mRNA is expressed in IFN- and TNF-pretreated cells. These results suggest that IFN-treated RCC cell lines may reduce their ability to be recognized by LAK cells, and that IFN-induced protection of RCC cell lines against LAK cells may depend upon a mechanism independent of the expression of class I antigens or ICAM-1 on tumor cells.     

Variants within the 5′-flanking regions of bovine milk protein genes: I. κ-casein-encoding gene

In order to identify DNA variants within the 5-flanking region of the bovine -casein (Cn)-encoding gene, this area of the gene from 13 cows belonging to seven breeds (Holstein Friesian, Brown Swiss, German Simmental, Jersey, Galloway, Scottish Highland and Ceylon Dwarf Zebu) was analysed. For each individual, about 1 kb of the 5-flanking region including exon I was amplified by polymerase chain reaction (PCR). The biotinylated PCR product was immobilized on magnetic beads followed by direct bidirectional sequencing using an automated DNA sequencer. Fifteen DNA variants were identified, some of which are located within potential regulatory sites and possibly involved in the expression of the -casein encoding gene.Abbreviations AP2 activator protein 2 - bp base pair(s) - GRE/RC glucocorticoid response element/reverse complement - HNF3 hepatocyte nuclear factor 3 Cn -casein - MGF mammary gland-specific nuclear factor - nt nucleotid(s), OCT1 octamer-binding site 1 - PA polyacrylamide - PCR polymerase chain reaction - PMF pregnancy-specific mammary nuclear factor - kb kilobase(s) or 1000 bp     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.     

Genotypic variation in the germination of common bean in response to cold temperature stress

Beans (Phaseolus vulgaris) are regarded as a susceptible crop to suboptimal temperatures. In temperate regions, low temperatures reduce establishment of beans when planted early in the growing season. Seeds of 14 cultivars/lines or beans were germinated in petri dishes at a constant 8, 10, 12, or 18°C or at 12 h alternating temperatures of 10/8, 12/8 or 18/8°C. Differences in germination percentages and rates between cultivars/lines were significant, especially at low temperatures. Cultivars/lines that germinated best and quickly at constant 8°C were Volare, Great Northern (G.N.) Tara, G.N. Belneb # 1, G.N. Spinel, and San Cristobal. Germination percent and rate of Pinto-UI-111 and Canadian Wonder increased significantly when temperatures were increased by 2 to 4°C for 12 h per 24 h, compared with a constant 8°C. Whereas, germination of G.N. Belneb # 1 was reduced. Polyacrylamide gel electrophoresis was used to study the effect of cold treatment on polypeptide patterns of seven cultivars/lines. Seeds were germinated at 18°C constant for 96 h or at 18°C for 48 h followed by 48 h at 2 or 8°C. During cold treatment the synthesis of some polypeptides increased. Volare, G.N. Tara Pinto-UI-111 and Canadian Wonder showed changes in polypeptide patterns, while Alubia-33-1, Michigan 84100 and BAT-1225 showed no changes in polypeptide patterns if compared to the control (96 h at 18°C in the dark). This suggests a likely essential role of these proteins in the development of chilling tolerance.     

Comparative studies of Feulgen hydrolysis for DNA

Summary We compared the Feulgen hydrolysis curves (37° C, 5 M HCl) of human blood lymphocytes fixed by the following four methods: 96% ethanol, 60 min at 20° C; ethanol-acetone, 11, 120 min at 4° C; ethanolglacial acetic acid mixture (31), containing 2% of formaldehyde (EAF), 90 min at 20° C; and ethanol-glacial acetic acid (31), 60 min followed by 5% chrome alum solution for 360 min at 20° C. The best results were obtained with EAF-fixation, with respect to the highest amount of DNA-Schiff complex at the peak point of the curve, the longest plateau region and the smallest scattering of DNA-Schiff amount values along the plateau. With other types of fixation the addition of polyethylene glycol (PEG) 6,000 to the hydrolysis solution resulted in modification of the shape of the hydrolysis curve so that it became nearly similar to the EAF-curve. The effect of PEG₆₀₀₀ on the EAF-curve was minimal. Slides fixed by ethanol were used for a comparison of polyethylene glycols with m.w. 1,500, 6,000 and 20,000. The longest plateau was obtained with PEG₆₀₀₀. The only effect of PEG₁₅₀₀ was a dramatic increase of DNA-Schiff amount at the peak point. PEG₂₀₀₀₀ had no significant effect on the shape of the hydrolysis curve. The results are discussed in terms of Kjellstrand's chain with a stable structure model of Feulgen hydrolysis.     

Protease inhibitor localization in control and streptozotocin-diabetic skeletal muscles

Summary ₁-Antitrypsin and ₁-inhibitor-3 were localized for the first time inside skeletal muscle cells. Their content, especially that of ₁-inhibitor-3, was greatly reduced following streptozotocin-induced diabetes. ₁-Antitrypsin and ₁-inhibitor-3 were also observed in the vascular components and interstitial space surrounding both control and diabetic soleus muscles as revealed by immunofluorescence. In diabetic muscles, the non-myofibre locale of ₁-inhibitor-3 was reduced, and to a lesser extent, ₁-antitrypsin. Both myofibre and extracellular patterns were reversed to control levels by insulin replacement.     

On the sufficiency of the velocity field for perception of heading

All models of self-motion from optical flow assume the instantaneous velocity field as input. We tested this assumption for human observers using random-dot displays that simulated translational and circular paths of movement by manipulating the lifetime and displacement of individual dots. For translational movement, observers were equally accurate in judging direction of heading from a velocity field with a two-frame dot life and a direction field in which the magnitudes of displacement were randomized while the radial pattern of directions was preserved, but at chance with a speed field in which the directions were randomized, preserving only magnitude. Accuracy declined with increasing noise in vector directions, but remained below 2.6° with a 90° noise envelope. Thus, the visual system uses the radial morphology of vector directions to determine translational heading and can tolerate large amounts of noise in this pattern. For circular movement, observers were equally accurate with a 2-frame velocity field, 3-frame acceleration displays, and 2-frame and 3-frame direction fields, consistent with the use of the pattern of vector directions to locate the center of rotation. The results indicate that successive independent velocity fields are sufficient for perception of translational and circular heading.     

Thermal Stability of Glucose Oxidase from Penicillium adametzii

The thermal stability of glucose oxidase was studied at temperatures between 50 and 70°C by kinetic and spectroscopic (circular dichroism) methods. The stability of glucose oxidase was shown to depend on the medium pH, protein concentration, and the presence of protectors in the solution. At low protein concentrations (<15 g/ml) and pH > 5.5, the rate constants k _in, s^–1, for thermal inactivation of glucose oxidase were high. Circular dichroic spectra suggested an essential role of structures in stabilizing the protein globule. At a concentration of 15 g protein/ml, the activation energy E _Aof thermal inactivation of glucose oxidase in aqueous solution was estimated at 79.1 kcal/mol. Other thermodynamic activation parameters estimated at 60°C had the following values: H= 78.4 kcal/mol, G= 25.5 kcal/mol, and S= 161.9 entropy units. The thermal inactivation of glucose oxidase was inhibited by KCl, polyethylene glycols, and polyols. Among polyols, the best was sorbitol, which stabilized glucose oxidase without affecting its activity. Ethanol, phenol, and citrate exerted destabilizing effects.     

Influence of the antimitotic naphthoquinone on the cleavage of the eggs of the mollusc,Mytilus edulis L.

Summary 1. A brief account is given of the cleavage ofMytilus edulis L.2.Naphthoquinone in concentrations above 1 million produces cytolysis of the newly fertilised eggs. It causes some blockage of cleavage at the single cell stage in concentrations down to 140 M. The main area of its antimitotic activity is in the range between 1 M and 16 M. Resistance to the substance increases as cleavage proceeds. 3. Some abnormalities of cleavage are found at all antimitotic concentrations. The types of abnormality are described. They do not appear to fit easily into the hypothesis of Huber (1947) that the effect of this substance is to enhance the contraction and inhibit the expansion of the cell cortex.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

ε-caprolactam,a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase

We found that -caprolactam is a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase. When Rhodococcus rhodochrous J1 cells were cultivated at 28°C for 120 h in a nutrient medium supplemented with 0.5% (w/v) -caprolactam, an enormous amount of nitrilase was formed in the cells which corresponded to approximately 30% of all soluble protein. The level of -caprolactam in the culture broth barely decreased in the course of cultivation. -Butyrolactam and -valerolactam also caused effective induction. The induction of nitrilase formation by -caprolactam was also observed in some other Rhodococcus strains.     

Differences in the heat-shock response between thermotolerant and thermosusceptible cultivars of hexaploid wheat

Summary Heat-shock protein (HSP) gene expression in two wheat lines cv Mustang (heat-tolerant) and cv Sturdy (heat-susceptible) were analyzed to determine if wheat genotypes differing in heat tolerance also differ in in-vitro HSP synthesis (translatable HSP mRNAs) and steady-state levels of HSP mRNA. Several sets of mRNA were isolated from seedling leaf tissues which had been heat-stressed at 37 °C for various time intervals. These mRNAs were hybridized with HSP cDNA or genomic DNA probes (HSP17, 26, 70, 98, and ubiquitin). Protein profiles were compared using in-vitro translation and 2-D gels. The Northern slot-blot data from the heat-stress treatment provide evidence that the heat-tolerant cv Mustang synthesized low molecular weight (LMW) HSP mRNA earlier during exposure to heat shock and at a higher level than did the heat-susceptible cv Sturdy. This was especially true for the chloroplast-localized HSP. The protein profiles shown by 2-D gel analysis revealed that there were not only quantitative differences of individual HSPs between the two wheat lines, but also some unique HSPs which were only found in the Mustang HSP profiles. The high level of RFLP between the two wheat lines was revealed by Southern blot hybridization utilizing a HSP17 probe. These data provide a molecular basis for further genetic analysis of the role of HSP genes in thermal tolerance in wheat.     

Slow growth phenotype - A possible approach to improved plasmid maintenance in Saccharomyces cerevisiae

Summary Transformation-induced slow growth phenotype (SGP) in yeast is repressed in the presence of 2m plasmids. A full 2m-sequence-based recombinant plasmid (pJB502) was found to be more stable in a 2m-free- [cir] strain of Saccharomyces cerevisiae than in a cir⁺ strain. This could not be attributed to differences in growth rate calculated from kinetic analysis of plasmid loss, but transformed [cir] isolates, which had lost the recombinant plasmid, exhibited varying degrees of SGP in batch culture. One of these isolates was outcompeted in chemostat culture by the recombinant-plasmid-containing strain, suggesting that improved plasmid maintenance can result from SGP in cir hosts.     

Timing of diapause induction outside the natural distribution range of a species: an outdoor experiment with the bean bug Riptortus clavatus

The phytophagous bug Riptortus clavatus (Thunberg) (Heteroptera: Alydidae) produces two or three generations per year in Central Japan and overwinters in the adult stage. In bugs from the Kyoto population (35°00 N, 135°45 E), we studied (1) the effects of day-length on the nymphal and preoviposition periods under constant photoperiod at 20.5 °C, and (2) photoperiodic induction of adult diapause at 20.5 °C and under a combination of constant photoperiod and natural daily rhythm of temperature in the forest-steppe zone of Russia (50°38 N, 35°58 E). Then, we examined (3) the timing of diapause induction under quasi-natural conditions in the same region, far outside the species' natural geographical range. At 20.5 °C, the nymphal period in both males and females was significantly longer under regimes with shorter photophases than under those with longer photophases. The preoviposition period in females was significantly longer under the near-critical long-day regime L14:D10 than under typical long-day regimes (L15:D9, L16:D8, and L17:D7). The critical day-length for diapause induction was shorter under conditions of natural daily rhythm of temperature than those reported at constant 20, 25, and 30 °C. Under quasi-natural conditions in the forest-steppe zone, R. clavatus entered diapause in September, much later than the local populations of true bugs studied to date. This experiment showed that R. clavatus was maladapted to new environmental conditions: diapause was induced too late with the result that all or most nymphs hatched in late August or early September will die.     

Energy-rich glyceric acid oxygen esters: Implications for the origin of glycolysis

The apparent Gibbs free energy change of hydrolysis (G°', pH 7) of the 2- and 3-O-glyceroyl esters of 2- and 3-O-L-glyceroyl-L-glyceric acid methyl ester were measured at 25 °C. The 2- and 3-glyceroyl esters were found to be energy-rich with G°' values of –9.1 kcal mol^–1 and –7.8 kcal mol, respectively. This result indicates that the analogous 2- and 3-glyceroyl esters of polyglyceric acid are also energy-rich and, therefore, could have acted as an energy source for primitive phosphoanhydride synthesis.     

Comparative study on the chloroplast,mitochondrial and nuclear genome differentiation in two cultivated rice species,Oryza sativa and O. glaberrima,by RFLP analyses

Summary Restriction fragment length polymorphisms of chloroplast (ct), mitochondrial (mt) and nuclear DNA were investigated using eight cultivars of Oryza sativa and two cultivars of O. glaberrima. Relative variability in the nuclear and cytoplasmic genomes was estimated by a common measure, genetic distance. Based on the average genetic distances among ten cultivars for each genome, the evolutionary variabilities of the mitochondrial and nuclear genomes were found to be almost the same, whereas the variability of the chloroplast genome was less than half that of the other two genomes. Cluster analyses on ct and mt DNA variations revealed that chloroplast and mitochondrial genomes were conservative within a taxon and that their differentiations were well-paralleled with respect to each other. For nuclear DNA variation, an array of different degrees of differentiation was observed in O. sativa, in contrast with little variation in O. glaberrima. As a whole, differentiation between O. sativa and O. glaberrima was clearly observed in all three genomes. In O. sativa, no notable difference was found between the cultivars Japonica and Javanica, whereas a large differentiation was noticed between Japonica (including Javanica) and Indica. In all three genomes, the average genetic distances within Indica were much larger than those within Japonica (including Javanica), and almost similar between Japonica (including Javanica) and Indica. These facts indicate that differentiation in O. sativa was due mainly to Indica.     

Estimation of Standing Stock and Decomposition Rates of Labile Organic Matter in Waters of Novorossiisk Bay,Black Sea

The annual dynamics of the decomposition rate, standing stock, and residence time of labile organic matter as an index of full self-purification were investigated in Novorossiisk Bay, Black Sea. The results are suggestive of fairly effective processes of biological self-purification in polluted waters of the bay. The decomposition rate was highest (0.3–0.7 mgO₂/l per day) during the summer, and it decreased by 4–8 times in winter. The residence time of labile organic matter was 97–104 days in winter and 8–11 days in summer. Oxygen consumption rates measured in different areas of the bay conformed to their trophic status and were not above the normal level for summer.     

A continuous migration model with stable demography

A probability model of a population undergoing migration, mutation, and mating in a geographic continuum R is constructed, and an integrodifferential equation is derived for the probability of genetic identity. The equation is solved in one case, and asymptotic analysis done in others. Individuals at x, y R in the model mate with probability V(x, y) dt in any time interval (t, t + dt). In two dimensions, if V(x,y) = V(x–y) where V(x) V(x/)/ ² approaches a delta function, the equilibrium probability of identity vanishes as 0. The asymptotic rate at which this occurs is discussed for mutation rates u u _o > 0 and for Cu , > 0, and u 0.Partially supported by NSF grant MCS79-03472Research was partially supported by Task Agreement No. DE-AT06-76EV71005 under Contract No. DE-AM06-76RL02225 between the U.S. Dept. Energy and the University of Washington     

Macroscopic Aggregation of Tobacco Mosaic Virus Coat Protein

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry melting curves, TMV CP in the form of a trimer–pentamer mixture (4S-protein) has very low thermal stability, with a transition temperature at about 40°C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial -helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.