Storage of embryonically transcribed poly(A) RNA and its utilization during metamorphosis of the hydroidHydractinia echinata

Summary The formation of tentacles and stolons during metamorphosis is severely disturbed if inhibitors of mRNA metabolism are applied during certain phases of development. The periods of sensitivity to -amanitin are late gastrulation and the disk stage of metamorphosis. A cordycepin sensitive phase exists during the first hour of metamorphosis. In all drug sensitive phases an enhanced poly(A) synthesis is found indicating increased mRNA metabolism in these stages. Pulse-chase experiments show that planula larvae store a poly(A)-rich RNA population sedimenting between 28–18s. These long living molecules are of embryonic origin, are located in RNP particles and are degraded during metamorphosis. The particles in question appear to be stored mainly in interstitial cells. In early metamorphosis no uridine is incorporated but labelled poly(A) is added to preexisting molecules.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

RNA Synthesis in Division Synchronized Tetrahymena: Macronuclear and Cytoplasmic RNA*

SYNOPSIS. Synthesis of RNA in the macronucleus and appearance of RNA in the cytoplasm were studied in heat synchronized Tetrahymena pyriformis GL and compared to those found under conditions of logarithmic growth (28 C) and during heat shocks (34 C). In macronuclei of logarithmically growing cells precursors were processed to 2 rRNA species (25S and 17S). In addition, another RNA (15S), more homogeneous than the RNA (8-15S) in the cytoplasm, was observed in the macronucleus. Both 17S and 25S rRNA species were found in the cytoplasm, 17S rRNA appearing more rapidly than 25S rRNA. Synthesis of rRNA was suppressed at 34 C in cells subjected to heat synchronization; 8-15S RNA synthesis appeared to be inhibited to a lesser extent. During the time preceding the first synchronized division, the synthesis of rRNAs in the macronucleus slowly recovered. Early in the cycle, almost no newly synthesized rRNAs were extracted. By 30 min after the last heat shock (EH), most of the RNA synthesized was not identified as rRNA. By 60 min after EH, the pattern of RNA synthesis had not returned to that observed in logarithmically growing cells.     

抑郁症易感基因和抗抑郁药物靶点相关[]神经细胞类型及相互作用网络分析

基于抑郁症的全基因组关联分析研究(GWAS),对于获得的单核苷酸多态性位点(SNP)使用Haploreg软件进行基因注释,得到SNP注释的102个易感基因.。使用MAGMA软件对GWAS的汇总统计数据做基因水平的分析,获得了270个校正之后显著的基因,两者合并共得到320个抑郁症易感基因。通过药物数据库Drugbank获取133个抗抑郁药物靶点基因。使用EWCE包对抑郁症易感基因和抗抑郁药物靶点在三套脑组织单细胞测序数据中,分别进行神经细胞类型富集分析。结果发现大脑皮质的GABA神经元(抑制性神经元)和谷氨酸能神经元(兴奋性神经元)是抑郁症易感基因和抗抑郁药物靶点共同的神经元。这两种类型的神经细胞可能是抗抑郁药物与抑郁症易感基因相互作用的神经细胞,另外少突胶质前体细胞可能是抑郁症特有的易感神经细胞。使用Network Calculator软件构建网络并进行进行网络拓扑学参数分析。结果表明抑郁症易感基因与抗抑郁药物靶点组成了一个具有显著的相互连接的网络。本研究从单细胞层面揭示抑郁症的遗传机制,在网络层面为寻找新的抗抑郁药物靶点提供了一定的启示。     

Genetic analysis of the structure and function of RNase P fromE. coli

A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations E. coli Escherichia coli - RNase P ribonuclease P     

Analysis of ribosomal DNA restriction patterns in the genusKluyveromyces

Riboprinting was used to determine the relationships among strains belonging to 15 species of the genusKluyveromyces. The small subunit ribosomal RNA gene (SSU rDNA) was amplified using the Polymerase Chain Reaction (PCR) and subjected to a battery of nine restriction enzymes. Similarity coefficients between strains were calculated based on shared and unique restriction fragments. Cluster analysis revealed three major groups that generally correlated with previously reported relationships based on other molecular data. Variations in SSU rDNA restriction fragments may be used for differentiation of theKluyveromyces strains included in this study.The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

DNA methylation changes in photoperiod-thermo-sensitive male sterile rice PA64S under two different conditions

Epigenetic modification can occur at a high frequency in crop plants and might generate phenotypic variation without changes in DNA sequences. DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by regulating gene expression. Rice Photoperiod-Thermo-Sensitive Genic Male Sterile (PTGMS) lines can transform from sterility to fertility under lower temperatures and short-day (SD) conditions during anther development. So far, little is known about the DNA methylation variation of PTGMS throughout the genome in rice. In this study, we investigated DNA cytosine methylation alterations in the young panicles of PTGMS line PA64S under two different conditions using methylation sensitive amplified polymorphism (MSAP) method. Compared with the DNA methylation level of PA64S under lower temperatures and SD conditions (fertility), higher methylation was observed in PA64S (sterility). The sequences of 25 differentially amplified fragments were successfully obtained and annotated. Three methylated fragments, which are homologous to D2, NAD7 and psaA, were confirmed by bisulfite sequencing and their expression levels were also evaluated by qPCR. Real time quantitative PCR analysis revealed that five of the six selected methylated genes were downregulated in PA64S (sterility). These results suggested that DNA methylation may be involved in the sterility–fertility transition of PA64S under two different environmental conditions.