Barley aleurone layers secrete a nuclease in response to gibberellic Acid : purification and partial characterization of the associated ribonuclease, deoxyribonuclease, and 3'-nucleotidase activities

Incubation of barley (Hordeum vulgare L. cv Himalaya) half-seeds with gibberellic acid enhances the secretion of ribonuclease and deoxyribonuclease from aleurone tissue (MJ Chrispeels, JE Varner 1967 Plant Physiol 42: 398-406; L Taiz, JE Starks 1977 Plant Physiol 60: 182-189). These activities were over 50-fold greater in medium of half-seeds incubated with gibberellic acid than in control medium. Ribonuclease and deoxyribonuclease activities initially appeared in the medium 24 to 48 hours after hormone induction and increased for up to 96 hours. Both activities had a pH optimum of 6.0 and a temperature optimum of 55°C. When the medium from gibberellic acid-treated half-seeds was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the major ribonuclease and deoxyribonuclease activity bands comigrated. The two enzyme activities remained associated throughout a 2,700-fold purification employing ammonium sulfate fractionation, Heparin-Agarose affinity chromatography, and Reactive Blue 2-Agarose affinity chromatography. Also accompanying the ribonuclease and deoxyribonuclease activities throughout purification was the ability to hydrolyze the 3′-phosphoester linkage of 3′-AMP. The purified protein was composed of a single polypeptide with an apparent molecular weight of 36 kilodaltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is concluded that in response to gibberellic acid, barley aleurone tissue secretes a nuclease having ribonuclease, deoxyribonuclease, and 3′-nucleotidase activities.     

Characterization of a DNA repair domain containing the dihydrofolate reductase gene in Chinese hamster ovary cells

The formation and removal of UV-induced pyrimidine dimers were measured in restriction fragments near and within the essential dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells in order to map the genomic fine structure of DNA repair. Dimer frequencies were determined at 0, 8, and 24 h after irradiating the cells with 20 J/m2 UV light (254 nm). Within 8 h, the cells had removed more than 40% of the dimers from sequences near the 5' end of the gene, somewhat fewer from the 3' end, but only 2% from the 3' flanking region and 10% from a region upstream from the gene. The corresponding extent of repair in the genome as a whole is 5-10% in the 8-h period. Isoschizomeric restriction enzyme analysis was used to detect the level of methylation in the fragments in which repair was measured. We found that the only hypomethylated sites in and around the DHFR gene were in the fragment near its 5' end, which displayed maximal DNA repair efficiency. The size of the region of preferential DNA repair at the DHFR locus appears to be in the range of 50-80 kilobases, and this finding is discussed in relation to genomic domains and the structure of mammalian chromatin.     

绿茶抗氧化剂成分抑制突变作用的初步研究

绿茶水溶性提取物及茶叶中抗氧化剂成份具有明显的抑制AFB_1及Bap诱导的鼠伤寒沙门氏菌回复突变作用。这种抗氧化剂成份还可以抑制AFB_1和Bap诱导的V79细胞基因正向突变,以及AFB_1诱导的V79细胞SCE和染色体畸变。本实验结果提示,绿茶中抗氧化剂成份可能对AFB_1及Bap的致癌性具有抑制作用。本文就茶叶抗氧化剂抑制突变的可能机制进行了初步讨论。     

Proton NMR of aequorin. Structural changes concomitant with calcium-independent light emission

Aequorin, a Ca(II)-sensitive bioluminescent protein from jellyfish, emits light at 469 nm from an excited state of a substituted pyrazine (oxyluciferin) which results from the oxidation of a chromophore molecule that is noncovalently bound to the protein. The chromophore is oxidized when Ca(II) or other activating metal ions are bound by aequorin. In the absence of Ca(II), spontaneous emission of light, referred to as Ca(II)-independent light emission, occurs at a rate less than 10(-6) of that for Ca(II)-induced emission. Proton nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence were used to study structural changes of aequorin accompanying Ca(II)-independent light emission. Time course studies by 1H NMR and CD demonstrate that as a result of Ca(II)-independent light emission, aequorin progressively changes from a rigid, fully active form showing little segmental mobility to a practically unfolded, discharged (i.e., inactive) form in which a number of amino acid residues are significantly mobile. This slow discharged protein (SDP) is distinct in nature and conformation from aequorin which has been discharged by Ca(II), i.e., the blue fluorescent protein. The rate of Ca(II)-independent discharge of aequorin is substantially reduced in the presence of excess Mg(II); the time constant for inactivation at 5 degrees C is 30 days with no Mg(II) present and 70 days with Mg(II) present. The NMR spectra are nearly identical at a given stage of inactivation whether or not Mg(II) is present. Oxyluciferin remains bound to SDP. If it is removed, however, by column chromatography, the resulting apo-SDP partially refolds, and the segmental mobility acquired in the formation of SDP is significantly attenuated particularly for some of the aromatic amino acid residues.     

Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L

Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-³H]±ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.

Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species.

     

Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K _i =ca. 0.5 µM) was considerably less than that of antimycin (K _i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [³H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [³H]DAA, respectively. The labeling of band 7 by [³H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [³H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.     

Pathogenicity of Fusarium oxysporum Isolates from Malaysia and F. oxysporuin f. sp. elaeidis from Africa to Seedlings of Oil Palms (Elaeis guineensis)

Pathogenicity tests with Fusarium oxysporum isolated form Malaysian oil palm were made with oil palms seedlings raised form Malaysian seed as well s with wilt-susceptible seedlings gown from African seed. Oil palm seedlings grown form Malaysian seed were also inoculated with African isolates of F. oxysporum f. sp. elaeidis and F. oxysporum var. redolens. The experiments were made under normal soil moisture conditions and under water stress. F. oxysporum f. sp. elaeidis isolates form Africa were pathogenic to oil palm seedlings from Malaysian seeds but the Malaysian F oxysporum isolates were non-pathogenic to plams grown from Malaysian seed or the wilt-susceptible palms from African seed. Seedlings from Malaysian seed proved to be highly susceptible to the vascular wilt disease caused by F. oxysporum f. sp. elaeidis as 75–90% of the palms were infected. The susceptibility of the palms from Malaysian seed varied with different African isolates tested. The Yaligimba isolate from Zaire which was found to be F. oxysporum var. redolens was the most virulent. Disease was more severe when oil palm seedlings were subjected to a period of water stress. The incidence of death in the seedlings under stress conditions was 45% as compared with only 15% for palms grown under normal conditions.