烟草愈伤组织继代培养与分化期间核酸代谢的比较研究

柳叶烟草愈伤组织在分化和芽原基形成期间,DNA 和RNA 含量均高于继代培养物;在芽原基形成后和幼芽生长期间(12天以后),DNA和RNA 含量持续上升,而同期继代培养物巳进入生长静止期,DNA 和RNA 含量基本不变或略有下降。根据RNA 电泳结果还进一步分析了两种愈伤组织培养物各RNA 组分变化与总RNA 含量变化的关系。分化培养物在芽原基形成时有明显升高的RNase 活性峰和持续上升的RNA 合成速率;而此时期继代培养物的RNase 活性及RNA 合成能力均较低;分化愈伤组织的DNA 合成速率在幼芽生长期间仍维持上升趋势,且显著高于同期继代愈伤组织的合成速率。这些结果表明,烟草愈伤组织分化培养物比继代培养物有更旺盛的核酸代谢能力。     

Changes in the metabolism of nucleic acids during the growth and senescence of tobacco callus

Changes in DNA and RNA metabolism, DNA composition and RNA species in callus of tobacco ( Nicotiana rustica L. cv. Gansu Yellow Flower) were investigated during the growth and senescence. DNA and RNA contents remained almost unchanged during the callus growth period, but started to decrease synchronously at the time that callus senescence was initiated. Synthesis of DNA and RNA, as measured by incorporation of [³H]-labelled precursor, increased during the growth period and did not decrease until late in senescence. The activities of DNase and RNase (pH 4.5) increased during the early senescence period in accordance with the decrease in the levels of DNA and RNA, but appeared to decrease during late senescence. These results suggest that the decrease in the levels of DNA and RNA in senescing tobacco callus may stem from the increase in the hydrolytic activities of DNase and RNase (pH 4.5) in the early stage of senescence, and that the slowdown of synthesis in the late senescence period may also be a cause. DNA and RNA electrophoresis showed that a low-molecular-weight satellite DNA band disappeared after the onset of senescence and that the nuclear main band DNA gradually decreased, whereas the high-molecular-weight satellite DNA seemed to undergo no significant changes during the senescence period tested. Of the RNA species, 4–5S RNA was far more susceptible to damage during senescence than 25S and 18S rRNA. This suggests different susceptibilities of different DNA and RNA components to damage during the senescence of tobacco callus or alternatively a highly sequenced degradation of DNA and RNA molecules.     

A Comparative Study on Protein Metabolism During Subculture and Differentiation of Tobacco Callus

Comparative study on the subcultured callus of tobacco (Nicotiana tabacum L. cv. Willow leaf) has revealed that protein contents and pr otease activities slowly decreased in the callus during differentiation and bud formation. The synthetic rates of fraction Ⅱ protein (water soluble protein and enzymes) and ribosomal hist one, the levels of total ribosomes, especially the levels of polyribosomes which function the protein synthesis, were higher in the differentiating callus than those in the subcultured callus. This indicates that protein synthesis in differentiating callus is greater than that in non-differentiating callus, and that the protein pattern synthesized in differentiating callus may differ from that in non-differentiating callus. During the late period of culture, the protease activities in subcultured callus rapidly increased, and the levels of polyribosomes, protein synthetic rates and protein contents apparently declined, which may be the result of metabolic changes in callus senescence. Meanwhile in the differentiating callus the protein contents, protein synthesis rates and polyribosome levels although somewhat declined accempanying the growth of formed bud, were still much higher than those in the subcultured callus.     

烟草愈伤组织多酚氧化酶研究

柳叶烟草愈伤组织中多酚氧化酶氧化邻苯二酚的活性明显高于氧化对苯二酚的活性。当以邻苯二酚为底物时,烟草愈伤组织多酚氧化酶分别在pH 5.6和pH 7.4有两个活性高峰。KCN、Dieca和m-CLAM对烟草愈伤组织多酚氧化酶活性都有明显抑制效应。根据凝胶电泳分析,继代培养愈伤组织多酚氧化酶同工酶有5条酶带,而巳分化出芽原基的愈伤组织和新分化长出的小叶都有7—8条酶带。继代培养的愈伤组织多酚氧化酶主要存在于除去线粒体的上清液中,线粒体部分也可测出酶活性。继代培养愈伤组织在接种后18天内,多酚氧化酶活性无重大改变。在此期间,Dieca对呼吸的抑制效应也变化不大。分化组织多酚氧化酶活性显著高于继代培养愈伤组织,在芽原基形成后,酶活性明显升高;此时Dieca对呼吸的抑制也由32%上升到47%。     

RNA polymerase activity in isolated nuclei ofNicotiana sanderae callus: Characteristics and modulation during differentiation

Isolated nuclei from differentiating cultures ofNicotiana sanderae showed increased levels of RNA polymerase activity as compared to the nuclei from callus cultures. The RNA synthetic activity was dependent on nucleotide triphosphates and Mg²⁺ and was destroyed by RNase. Maximum activity was obtained in the presence of 50 mM (NH₄)₂ SO₄ and α-amanitin inhibited 40% and 55% of the activity in the nuclei from callus and differentiating tissue respectively. The nuclei from differentiating tissue elicited a 3-fold increase in RNA polymerase I and a 4-fold augmentation in RNA polymerase II activities.     

Synthesis and repair of RNA-containing supercoiled plasmid ColE1 DNA in Escherichia coli

Supercoiled plasmid molecules sensitive to nicking by RNase or alkali have been shown to accumulate during replication of colicinogenic factor E1 (ColE1) in Escherichia coli in the presence of chloramphenicol. The possibility that this sensitivity is due to the covalent integration of RNA molecules during the synthesis of plasmid DNA is supported by the demonstration that (a) strands of supercoiled ColE1 newly replicated in the presence of chloramphenicol exhibit sensitivity to RNase and alkali treatment, while (b) RNase- and alkali-resistant circular strands of plasmid DNA synthesized either before or after the addition of chloramphenicol remain resistant during subsequent replication of the plasmid in the presence of chloramphenicol. Furthermore, newly made plasmid DNA strands cannot act as templates for further rounds of replication if they possess an RNA segment. The existence of a repair mechanism for the removal of the RNA segment from supercoiled ColE1 DNA molecules was demonstrated by pulse-chase experiments. It was observed that the proportion of RNase-sensitive molecules is considerably higher in pulse-labeled as compared to continuously labeled ColE1 DNA synthesized in the presence of chloramphenicol, and the proportion of pulse-labeled ColE1 DNA that is RNase sensitive is greatly reduced during a chase period. Removal of the RNA segment is also carried out effectively at the restrictive temperature in temperature-sensitive DNA polymerase I mutants. In a survey of other bacterial mutants defective in the repair of damaged DNA, a substantial increase in the rate of accumulation of RNase-and alkali-sensitive supercoiled ColE1 DNA in the presence of chloramphenicol was observed in recBC and uvrA mutants in comparison with the wild-type strains.     

Amino acids essential for RNase H activity of hepadnaviruses are also required for efficient elongation of minus-strand viral DNA.

The hepadnavirus P gene contains amino acid sequences which share homology with all known RNases H. In this study, we made four mutants in which single amino acids of the duck hepatitis B virus (DHBV) RNase H region were altered. In two of them, amino acids at locations comprising the putative catalytic site were changed, while the remaining mutants had alterations at amino acids conserved among hepadnaviruses. Transfection of these mutant genomes into permissive cells resulted in synthesis of several discrete viral nucleic acid species, ranging in apparent sizes from approximately 500 to 3,000 bp, numbered I, II, III, IV, and V. While the locations of the species were similar in all mutants, the proportions of the species varied among the mutants. Analysis of the nucleic acid species revealed that they were hybrid molecules of RNA and minus-strand DNA, indicating that the RNase H activity was missing or greatly reduced in these mutants. Primer extension experiments showed that the mutant viruses initiated minus-strand viral DNA synthesis normally. The 3' termini of minus-strand DNA in species II, III, and IV were mapped just downstream of nucleotides 1659, 1220, and 721, respectively. Species V contained essentially full-length minus-strand viral DNA. A parallel amino acid change in the putative catalytic site of the HBV RNase H domain resulted in accumulation of low-molecular-weight hybrid molecules consisting of RNA and minus-strand DNA and similar in size and pattern to those seen with DHBV. These studies demonstrate experimentally the involvement of the C-terminal portion of the P gene in RNase H activity in both DHBV and human hepatitis B virus and indicate that the amino acids essential for RNase H activity of hepadnavirus P protein are also important for the efficient elongation of minus-strand viral DNA.     

Growth hormone modulates the degradative capacity of muscle nucleases but not of cathepsin D in post-weaning mice

We determined whether recombinant human growth hormone (rhGH) administration might modulate the enzyme degradative capacity of the muscle lysosomal system and influence muscle growth. Muscle cathepsin D, acid RNase and DNase II activities are determined in the gastrocnemius muscle of rhGH-treated post-weaning female BALB/c mice. Linear regressions were used to analyze the relationships of each enzyme with their respective substrate. GH induced a depletion-recovery response of muscle growth through a mechanism which is similar to catch-up growth. In these conditions, cathepsin D activity decreased with age in all animals (GH: 40%; saline: 79%), showing a substantial developmental decline that could reflect changes in the rate of protein breakdown. However, the degradative capacity of cathepsin D was paradoxically unmodified in rhGH-mice compared with saline mice (according to the enzyme vs. substrate linear regression slope), in spite of the increase in enzyme activity elicited by GH. This suggests that the muscle protein breakdown is not increased by GH-treatment in post-weaning mice. The enhancement of muscle protein deposition as indicated by the augmented muscle cell size (protein:DNA ratio) of rhGH-mice (increased 178% from 25 to 50 days) vs. saline, can be attributed to a higher muscle K(RNA). In contrast, acid RNase and DNase II activities directly participate in muscle RNA and DNA degradation. Both nucleases were inhibited by GH treatment (a decrease of 48% and 63%, respectively, vs. saline at 50 days). The decrease in RNase activity suggests an inverse relation between the rate of protein synthesis (high) and acid RNase activity (low), leading to spare muscle RNA for synthesizing protein during catch-up growth. Also, low DNase II activity could contribute to inhibiting of muscle DNA degradation, facilitating muscle growth. Thus, GH seems to act as a direct modulator of the degradative capacity of skeletal muscle nucleases but not of cathepsin D, influencing DNA and RNA degradation during the depletion-recovery response to GH of gastrocnemius muscle in female post-weaning mice.