Relation of special RNA to sensitivity of yeast strains to auxin

RNA functional in auxin action was studied in the followingfour strains of yeasts differing in their sensitivity to auxin:KV2, diploid strain of Saccharomyces ellipsoideus irresponsiveto auxin but made responsive by gibberellic acid treatment;N55, auxinresponsive mutant derived from KV2; A2-0, diploidstrain of 5. cerevisiae irresponsive even when gibberellic acidis given; and A2–N102, responsive mutant derived fromA2–0. The RNA fraction extracted from N55 and partitionedin a phenol layer sensitized KV2 to auxin. This kind of RNAwas not detected in KV2 cells. The functional RNA from N55 wasof low molecular weight, as was functional RNA isolated fromgibberellic acid-treated Jerusalem artichoke tuber. Gibberellicacid-treated KV2 cells have been known to contain RNA whichfunctions similarly to the RNA mentioned above. Both A2–0and A2–N102 contained RNA which made KV2 cells responsiveto auxin. Some factors other than functional RNA may determinethe difference in the sensitivity to auxin between A2–0and A2–N102. (Received August 20, 1968; )     

Effects of Growth Regulators on Ribonucleic Acid Metabolism of Barley Leaf Segments

Illumination or gibberellic acid treatment of etiolated barley leaf segments stimulates unrolling and results in an increased level of RNA. In contrast, segments treated with abscisic acid do not unroll and have a lower content of RNA. Gibberellic acid treatment enhanced the capacity of segments to incorporate radioactivity from ³²P-orthophosphate into all the RNA components detected by gel electrophoresis; abscisic acid greatly restricted the incorporation of precursors into all the RNA fractions. In conjunction with a changed capacity for RNA synthesis it was observed that abscisic acid-treated segments had a lowered soluble DNA-dependent RNA polymerase level in comparison to gibberellic acid-treated or illuminated segments. However, the influence of growth regulators on RNA polymerase content of the segments was associated with general effects on protein level rather than a specific effect on the synthesis of polymerase enzyme.     

Isolation of rapidly labeled RNA from mouse L-cells using cupric sulfate.

Cultured mouse L-cells, pulse labeled for 5 min with 3H-uridine, were gently suspended in 0.5mM cupric sulfate and 0.5% sodium dodecyl sulfate at 4 degrees C and treated with cold phenol. Only the RNA, containing less than 1% DNA, was extracted by this procedure. The rapidly labeled ribosomal RNA precursors (44S, 34S) and cytoplasmic 8S RNA showed specific activities higher than that of tRNA and were present in the RNA fraction insoluble in 2M NaCl.     

Association of protein C23 with rapidly labeled nucleolar RNA

The association of nucleolar phosphoprotein C23 with preribosomal ribonucleoprotein (RNP) particles was examined in Novikoff hepatoma nucleoli. RNA was labeled with [3H]uridine for various times in cell suspensions, and RNP particles were extracted from isolated nucleoli and fractionated by sucrose gradient ultracentrifugation. The majority of protein C23 cosedimented with fractions containing rapidly labeled RNA (RL fraction). To determine whether there was a direct association of RNA with protein C23, the RL fraction was exposed to ultraviolet (UV) light (254 nm) for short periods of time. After 2 min of exposure there was a 50% decrease in C23 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, with no significant further decrease at longer times. When UV-treated fractions were subjected to phenol/chloroform extractions, as much as 30% of the labeled RNA was found in the phenol (protein) layer, indicating that RNA became cross-linked to protein. Similarly, there was an increase in protein C23 extracted into the water layer after irradiation. By SDS-PAGE analyses the cross-linked species migrated more slowly than protein C23, appearing as a smear detected either by [3H]uridine radioactivity or by anti-C23 antibody. With anti-C23 antibodies, up to 25% of the labeled RNA was precipitated from the RL fraction. Dot-blot hybridizations, using cloned rDNA fragments as probes, indicated that the RNA in the RL fraction and the immunoprecipitated RNA contained sequences from 18S and 28S ribosomal RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide status and protein synthesis in vivo in the apices of juvenile and mature Sequoiadendron giganteum during budbreak

Adenine and guanine nucleotide contents of isolated apices collected from a juvenile and a mature clone of Sequoiadendron giganteum (Lindl.) Buchholz during budbreak were determined. GDP and GTP contents were significantly higher in the juvenile clone apex than in the mature ones, whereas there was no difference in ATP concentration between the two materials. In vivo, induction of protein synthesis was similar in the two clones after 10 min of [³⁵S]-methionine labeling. The increase of [³⁵S]-methionine-tRNAs and labeled proteins continued up to 30 min for the juvenile clone. They markedly declined for the mature clone after 10 min. Only the diminution of this in vivo protein synthesis was well correlated with a decrease in GTP content.     

Comparative study of lipoteichoic acid from Streptococcus pyogenes type 29 isolated by different methods

A comparative study of lipoteichoic acid preparations extracted from Streptococcus pyogenes with cold and hot phenol and trichloracetic acid was made. The most delicate way for isolation of lipoteichoic acid from the given microorganisms is cold phenol extraction.     

凡纳滨对虾低温差异表达miRNA的分析鉴定及靶基因的验证

为了解析miRNA及靶基因在凡纳滨对虾低温适应过程中的分子调控机制, 研究开展了凡纳滨对虾常温(28℃)及低温锻炼(16℃, 6d)下肝胰腺小RNA文库的测序和分析。从常温对虾的小RNA文库测序获得18—32 nt的高质量序列10690259条, 鉴定出已知的成熟miRNAs 57条; 而从低温锻炼对虾的小RNA文库获得18—32 nt的高质量序列序列8587144条, 鉴定出已知的成熟miRNAs 48条。分析获得25个在低温锻炼下呈显著差异表达的miRNAs。运用qRT-PCR验证了3条低温下差异极显著的miRNAs的表达模式。结果显示, 3条miRNAs的表达模式与高通量测序结果基本一致。预测低温差异表达miRNAs的靶基因, 并与转录组分析获得的低温显著差异表达基因进行比对, 从二者重合的基因集中挑选出4个基因, 即nuclear export mediator factor Nemf-lik、synapse-associated protein、seleno proteins 以及DEAD-box RNA helicase Variant 1, 运用qRT-PCR验证其在不同条件低温锻炼凡纳滨对虾肝胰腺中的表达规律, 为解析miRNAs及其靶基因在对虾低温适应过程中的分子调控机制提供了基础数据。     

植物叶绿体4.5S核糖体rRNA作为分子杂交探针的研究

我们提取纯化了芹菜,菠菜和蕃茄叶绿体核糖体4.5SRNA(4.5SrRNA)并在其5’端标记~(32)P,作为探针与菠菜,蕃茄和芹菜叶绿体DNA(ctDNA)进行分子杂交。结果不仅证明4.5SrRNA可作为公用分子杂交探针,而且也说明不同植物4.5SrRNA序列有相当大的同源性。     

Hybridization properties of nucleolar ribonucleic acid.

Rat liver nuclei were fractionated into chromatin and nucleolar fractions. Chromatin DNA, which does not form hybrids with rRNA, was, nevertheless, able to hybridize with 32P-labelled total nucleolar RNA. The optimal temperature for this hybridization was 55 degrees C when the reaction was carried out in 2 X SSC (0.3 MnaCl + 0.3 M-sodium citrate). The hybrids formed were specific, as judged by analysis of thermal elution profiles. The low Tm (73 degreesC) observed could be explained by the low amount of DNA in the filters. The lenth of the hybridized sequences was extimated as 54 mucleotide pairs. Contamination to nucleolar RNA by nucleoplasmic RNA was ruled out by showing the former was able to form more hybrids than the latter. Competition experiments showed that hybridization of nucleolar RNA, although not competed with by rRNA, suffered pronounced competition from total microsomal RNA, even though the levels of competition obtained did not equal thsoe with cold nucleolar RNA as competitor.     

Effect of interferon on polymerization of single-stranded and double-stranded mengovirus ribonucleic acid

Gordon, Irving (University of Southern California, Los Angeles), Sara S. Chenault, Douglas Stevenson, and Jean D. Acton. Effect of interferon on polymerization of single-stranded and double-stranded mengovirus ribonucleic acid. J. Bacteriol. 91:1230-1238. 1966.-The effect of interferon on actinomycin-resistant mengovirus ribonucleic acid (RNA) replication in L cells was investigated to determine whether defective or partially polymerized RNA products were made and whether synthesis of any specific class of virus RNA was prevented. RNA labeled with uridine-C(14) was extracted in hot and cold phenol and analyzed by zonal sucrose density centrifugation. Both single- and double-stranded infectious RNA peaks were identified. Interferon treatment caused almost complete depression of uridine-C(14) incorporation throughout linear sucrose gradients except in the 4S region, and no infectivity was detectable in any fraction. These inhibitory effects are attributable to the action of interferon, because they were reversed when cultures were treated with actinomycin D simultaneously with interferon. The results, with those of other investigators, indicate that the step at which interferon interrupts virus multiplication is between the events immediately after uncoating and the formation of template "minus" strands; under the conditions of our experiments, no partially polymerized virus RNA products were made.     

Variations of DNA methylation in Eucalyptus urophylla×Eucalyptus grandis shoot tips and apical meristems of different physiological ages

Global DNA methylation was assessed by high-performance liquid chromatography (HPLC) for the first time in Eucalyptus urophylla×Eucalyptus grandis shoot tips comparing three outdoor and one in vitro sources of related genotypes differing in their physiological age. The DNA methylation levels found were consistent with those reported for other Angiosperms using the same HPLC technology. Notwithstanding noticeable time-related fluctuations within each source of plant material, methylation rate was overall higher for the mature clone (13.7%) than for the rejuvenated line of the same clone (12.6%) and for the juvenile offspring seedlings (11.8%). The in vitro microshoots of the mature clone were less methylated (11.3%) than the other outdoor origins, but the difference with the juvenile seedlings was not significant. Immunofluorescence investigations on shoot apices established that the mature source could be distinguished from the rejuvenated and juvenile origins by a higher density of cells with methylated nuclei in leaf primordia. Shoot apical meristems (SAMs) from the mature clone also showed a greater proportion and more methylated cells than SAMs from the rejuvenated and juvenile origins. The nuclei of these latter were characterized by fewer and more dispersed labeled spots than for the mature source. Our findings establish that physiological ageing induced quantitative and qualitative variations of DNA methylation at shoot tip, SAM and even cellular levels. Overall this DNA methylation increased with maturation and conversely decreased with rejuvenation to reach the lower scores and to show the immunolabeling patterns that characterized juvenile material nuclei.     

Organs of gibberellin synthesis in light-grown sunflower plants

The sites of gibberellin (GA) synthesis in light grown sunflower plants were studied. The results of organ excision and the exogenous application of indole acetic acid and gibberellic acid indicated that gibberellin synthesis occurred in the young leaves of the apical bud. This was substantiated using a combination of diffusion and extraction techniques. Diffusion of sunflower apical buds on agar for 20 hours revealed a level of gibberellin greater than that obtained by solvent extraction of a similar number of apices, indicating that synthesis of gibberellin was occurring in those apices during the diffusion period. The gibberellin level of apices extracted following a 20 hour diffusion period was the same as that obtained from buds extracted immediately following excision from the plant, again suggesting that apical buds are sites of gibberellin synthesis. A similar experiment was conducted with young internodal sections, the results indicating that they were not sites of gibberellin synthesis.     

Polyadenylated RNA from Vicia faba meristematic root cells. Localization and size estimation of the poly (A) segment.

After incubating root apices from two-day-old bean seedlings with [3H] adenine the RNA was extracted from whole cells or polysomes, and the poly (A) sequences were isolated by nuclease digestion followed by poly(U)-Sepharose chromatography. The alterations of the RNA molecules due to the various treatments were monitored by sucrose density gradients. It was found that sequential extraction first at pH 7.6 then at pH 9.0 did not result in a separation between RNA poor in poly(A) sequences and poly(A)-rich RNA. Furthermore chromatography analysis of hydrolysates from nuclease-resistant RNA extracted either at pH 7.6 or pH 9.0 revealed that AMP constituted nearly 95% of the bases and that the poly(A) sequences, about 200 bases, were located at the 3' terminus of the polyadenylated RNA. No size difference was found for the poly(A) segment between the pH-7.6-extracted RNA and that extracted at pH 9.0.     

肾上腺皮质激素对心房钠尿肽基因表达的促进作用

给完整的及切除肾上腺的雌性 Wistar 大鼠分別注射地塞米松、去氧皮质酮或地塞米松加去氧皮质酮;冷酚法提取心房总 RNA,用α-~(32P)标记的大鼠心房肽 cDNA 探针与之杂交。完整大鼠接受地塞米松和切除肾上腺后接受地塞米松加去氧皮质酮的大鼠,心房肽基因转录产物增加2倍,其余组无显著变化。结果提示糖皮质激素可促进心房肽基因表达,但此作用依赖于盐皮质激素的同时存在,单纯盐皮质激素不能增强该基因的表达。     

The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata

RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit of the trypanosomatid flagellate Crithidia fasciculata and analyzed by polyacrylamide gel electrophoresis at 4 °C consisted of one species with a molecular weight of 1.3 × 10⁶ (relative to ribosomal RNA from E. coli MRE 600). When extracted with hot phenol (65 °C), the large ribosomal subunit gave rise to two components with molecular weights of 0.72 and 0.56 × 10⁶. On heating for 60 s, followed by rapid cooling, the single cold-phenol-extracted 1.30 × 10⁶-dalton species completely dissociated into two components of molecular weights 0.72 and 0.56 × 10⁶, present in equimolar amounts. When analyzed by polyacrylamide-agarose gel electrophoresis in the presence of SDS, RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit consisted of three components of molecular weights 1.3, 0.72, and 0.56 × 10⁶, present in apparently equimolar amounts. RNA from the small cytoplasmic ribosomal subunit consisted of one species with a molecular weight of 0.84 × 10⁶, independent of extraction or analytical conditions. It is proposed that under high salt and low temperature conditions, the large ribosomal RNA molecule is held together by its secondary structure, and that denaturing extraction or analytical conditions reveal an otherwise “hidden” lesion present in the molecule in vivo.     

The Role of Membrane-Bound Enzymes in an Early Response of Aleurone Tissue to Gibberellic Acid

Treatment of aleurone layers of barley seed with gibberellicacid increases the observable phosphorylcholine glyceride transferaseactivity in a membrane fraction prepared from extracts of thealeurone cells. This gibberellic acid-dependent increase inglyceride transferase activity requires neither RNA synthesisnor protein synthesis. Membrane fractions prepared from mixturesof extracts of gibberellic acid-treated layers and control layershave a specific activity of glyceride transferase higher thanexpected on the basis of simple addition of the activities fromthe two sources. Therefore, some kind of activation is occurring.