Association of a 76 kDa polypeptide with soluble starch synthase I activity in maize (cv B73) endosperm

Soluble starch synthase (SSS) I was purified 361-fold from hand-dissected endosperm tissue of inbred maize (Zea mays, cv. B73) to specific activities ranging between 5 and 9 µmol min⁻¹ mg⁻¹. A key to this purification protocol was the introduction of a size-exclusion chromatography step, a size-based fractionation which provided abundant levels of desalted SSS forms I and II. The native molecular masses calculated for SSS forms I and II were 75.5 kDa and 180 kDa, respectively. SSSI was then further purified by hydrophobic interaction chromatography on Phenyl-Superose and by FPLC on Mono Q. Analysis of column peaks by SDS—PAGE and scanning densitometry revealed that a 76 kDa polypeptide is strongly correlated with SSSI activity. Antibodies were then generated against a 76 kDa polypeptide extracted from starch granules. These antibodies, which were monospecific for the soluble 76 kDa polypeptide, neutralized greater than 90% of SSSI activity, and precipitated the 76 kDa protein. These results establish the 76 kDa protein as an SSSI in the B73 line of inbred maize. An immunologically similar 76 kDa protein also appears to be tightly associated with the starch granule.     

Maintenance of high levels of allelic variation in spite of a severe bottleneck in population size: the brown bear (Ursus arctos) in the Western Carpathians

Genetic variation in an isolated brown bear population of the Western Carpathians was studied by electrophoretic analysis of 51 presumptive allozyme loci. In spite of a severe population bottleneck at the beginning of the 1930s (40 survivors), average heterozygosity (H=5.3%) was within the range commonly found in mammals and the mean number of alleles per polymorphic locus (A_p=3, over five polymorphic loci) was very high. Possible reasons for the maintenance of high allelic variation are discussed.     

Morphogenesis of potato plants in vitro. II. Endogenous levels,distribution, and metabolism of IAA and cytokinins

The levels of endogenous IAA and cytokinins (zeatin, zeatin riboside, isopentenyladenine, and isopentenyladenosine) were determined in potato plants cultured in vitro under red light (R) and blue light (B) on medium with or without hormones. On medium without hormones in B, plants contained much higher cytokinin levels, particularly in leaves and roots, and also slightly elevated IAA levels. Kinetin in the medium in B changed the distribution of cytokinins and significantly increased IAA level in roots. In R, the presence of kinetin led to an increased cytokinin level in the whole plant, while the IAA level was slightly lower. IAA in the medium in B decreased cytokinin level in all plant parts, while the IAA level did not change significantly. In R, the presence of IAA in the medium led to a moderate increase of CK level and to a significant increase in IAA level, especially in roots. Uptake of 1-¹⁴C-IAA and of ³H-zeatin was generally higher in B than in R. Higher percentage of IAA taken up in B was converted to conjugates in the roots. Metabolism of ³H-zeatin was similar in R and B with only slight differences in metabolite amounts.Thus, in all experimental situations in which tuber formation was stimulated, IAA level in roots and stolons rose significantly, stressing the importance of an IAA gradient for tuber formation.     

Strain improvement of Claviceps purpurea by protoplast fusion without introducing auxotrophic markers

Protoplasts of morphologically and biochemically different Claviceps purpurea strains producing ergotoxins were fused without introducing selective auxotrophic markers. Fused strains thus obtained differed significantly in biosynthetic activity and morphology from the prototrophic isolates obtained after fusion of the same parent strains marked by auxotrophy. Comparison of the two types of fused strains showed about tenfold higher alkaloid production in fusants obtained from prototrophic strains. Selected stable prototrophic isolates also showed a significant productivity improvement in comparison with the original parent strains. Correspondence to: M. Didek-Brumec     

In vitro survival of murine morulae after quick freezing in the presence of chemically defined macromolecules and different cryoprotectants

We studied the ability of frozen-thawed mouse morulae to develop in vitro when the cryoprotectant proteins were substituted with one of the following nonorganic macromolecules: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and ficoll. We also determined how these agents interacted with 3 different cryoprotectants: glycerol (GLY), propylene glycol (PG), and ethylene glycol (EG). The influence of both of the above factors was measured on the basis of post-thaw morphological appearance, the percentage of development to the expanded blastocyst stage and the total cell count. Morulae (n=950) were collected from superovulated mice. Those classified as good or excellent were distributed among the 12 different freezing solutions, obtained by combining the 3 cryoprotectants with the 4 macromolecules (the 3 mentioned above, plus a control of 5% fetal calf serum) in phosphate buffered saline (PBS). Embryos frozen in PVA, PVP and ficoll tended to be a little difficult to recover from the straws. Development to the expanded blastocyst stage was significantly lower (P<0.05) in propylene glycol (43.6%) than in ethylene glycol (79.5%) or in glycerol (76.1%). Polyvinyl alcohol provided a higher survival rate when combined with glycerol (90.3) or ethylene glycol (95.0), but when it was combined with propylene glycol, only 56.5% of embryos survived after thawing. A positive interaction was observed between glycerol and PVA and between ethylene glycol and PVA or ficoll. The results indicate that fetal serum could be successfully substituted for any of the 3 chemically defined macromolecules. However, our findings also suggest that the use of PG as a cryoprotectant should be avoided when mouse morulae are frozen using the quick freezing method.     

Effect of oxygen on ubiquinone-10 production by Paracoccus denitrificans

Summary Ubiquinone-10 (Q₁₀) production was measured in batch cultures of Paracoccus denitrificans grown for 8 h at increasing oxygen concentrations (0–21 % O₂ in the sparging gas). Whereas the cellular level of Q₁₀ decreased monotonically from 1.2 to 0.5 mol/g d.w., the total yield of Q₁₀ was maximal at 2.5 % O₂ and amounted to 350 nmol (0.3 mg) per L of culture.     

促髓细胞分化的锌指基因单向PCR扩增直接测序的研究

锌指基因是一种造血调节基因,编码锌指结构蛋白,主要在髓细胞中表达,促进髓细胞分化,在急性早幼粒白血病维甲酸治疗中,促使病情缓解。本文报道了我们从基因分子上研究锌指基因作用中,探索并建立了单向聚合酶链反应（ＰＣＲ）扩增特定单链ＤＮＡ,直接测序的新方法。它能产生质和量均佳的单链ＤＮＡ,无需纯化即可直接用于测序,使复杂的测序研究简便易行,可在２,３天内完成。这种单向ＰＣＲ扩增特定单链ＤＮＡ直接测序的方法,经对锌指基因的ｃＤＮＡ测序,得到验证。此法不仅适用于疾病研究中的ＤＮＡ测序,还可制各单链ＤＮＡ探针,更利于基因结构组成的研究。     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments

The efficiency ofAgrobacterium-based transformation technique in oilseed rape and cauliflower was influenced by cultivar specificity, donor plant age and explant type. Marked differences in demands for plant hormone contents in the regeneration medium were recorded already among different types of nontransformed explants. The highest regeneration capacity was recorded with stem and leaf segments isolated from one-month-old aseptically grown plants. The regeneration was markedly species-dependent. Regeneration of transformed plants from stem segments and thin layers isolated from field-grown oilseed rape plants (at the most 2% of regenerating explants) and from oilseed rape hypocotyls (0.8% of regenerating explants) and cauliflower (1.2% of explant regenerated transformed shoots) was achieved after disarmedAgrobacterium treatment. Hypersensitive reaction of explants could be prevented by using prolongedin vitro precultivation and delayed application of the selective agent.