Determination of the RNA-content in samples of isolated chromatin by circular dichroism measurements

A method is presented to determine the RNA content in isolated chromatin. An increased elipticity at 260nm–290nm in the circular dichroism spectrum of chromatin is due to RNA released together with chromatin from the cell nuclei. Mixtures of chicken erythrocyte chromatin and pure 5s-RNA were used to reconstruct such spectra. From the obtained data an RNA content higher than 3–5% in samples of isolated chromatin can be determined.     

γ-射线对大鼠小肠粘膜上皮细胞RNA合成的影响及其机理的研究

本文观察了15戈瑞γ-射线全身照射后,大鼠小肠粘膜上皮细胞核体外转录活性,从染色质结合的RNA聚合酶和可溶性RNA聚合酶活性变化探讨辐射对核转录活性的抑制机理。实验结果表明:(1)照后2小时、8小时和24小时,核转录活性分别下降22.7％、20.8％和28.2％;(2)染色质结合的RNA聚合酶活性变化与细胞核转录活性变化基本平行,提示核转录活性降低与核内染色质损伤有关;(3)照后24小时,核分离的可溶性RNA聚合酶抑制58％,提示辐射至少部分是通过抑制RNA聚合酶而影响细胞核转录活性;(4)在细胞核和分离的RNA聚合酶都观察到RNA聚合酶Ⅱ抑制程度大于RNA聚合酶Ⅰ和酶Ⅲ,酶活性下降主要表现在RNA聚合酶Ⅱ提示不同类RNA聚合酶对辐射的敏感性不同,酶Ⅱ对辐射更敏感。     

大鼠衰老过程中肝细胞核及染色质的体外转录活性

用同位素掺入法研究不同年龄大鼠的肝细胞核及染色质体外转录活性,所得结果表明:(1)老年大鼠肝细胞核的转录起始能力较断乳鼠及青年鼠分别下降68％及56％。(2)大鼠肝细胞核内与染色质结合的RNA聚合酶所致的转录活性随增龄呈近似线性下降,而不与染色质结合的RNA聚合酶所致的转录活性随增龄则无变化。(3)老年大鼠肝染色质体外转录活性较断乳鼠及青年鼠分别下降52％及35％。这些结果提示。老年大鼠肝染色质功能的改变可能是转录活性改变的主要原因。     

Human cytomegalovirus proteins PP65 and IEP72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts

The cellular distribution of the human cytomegalovirus (HCMV)-specific UL83 phosphoprotein (pp65) and UL123 immediate-early protein (IEp72) in lytically infected human embryo fibroblasts was studied by means of indirect immunofluorescence and confocal microscopy. Both proteins were found to have a nuclear localization, but they were concentrated in different compartments within the nuclei. The pp65 was located predominantly in the nucleoli; this was already evident with the parental viral protein, which was targeted to the above nuclear compartment very soon after infection. The nucleolar localization of pp65 was also observed at later stages of the HCMV infectious cycle. After chromatin extraction (in the so-called in situ nuclear matrices), a significant portion of the pp65 remained associated with nucleoli within the first hour after infection, then gradually redistributed in a perinucleolar area, as well as throughout the nucleus, with a granular pattern. A quite different distribution was observed for IEp72 at very early stages after infection of human embryo fibroblasts with HCMV; indeed, this viral protein was found in bright foci, clearly observable in both non-extracted nuclei and in nuclear matrices. At later stages of infection, IEp72 became almost homogeneously distributed within the whole nucleus, while the foci increased in size and were more evenly spread; in several infected cells some of them lay within nucleoli. This peculiar nuclear distribution of IEp72 was preserved in nuclear matrices as well. The entire set of data is discussed in terms of the necessity of integration for HCMV-specific products into the pre-existing nuclear architecture, with the possibility of subsequent adaptation of nuclear compartments to fit the needs of the HCMV replicative cycle.     

A reproducible microanalytical method for the detection of specific RNA sequences by dot-blot hybridization

A rapid, microanalytical procedure for the reproducible isolation of RNA from small cultured cell samples and application to dot-blot hybridization is described. The procedure employs guanidine hydrochloride solubilization of whole cells, disruption by syringing, and selective precipitation of RNA with ethanol. The method can be performed in a single tissue culture tube and obviates the need for removal of nuclei or for organic solvent extractions. Recovery of RNA from small cell samples (10(6) cells) is 51%, while 97% of the DNA and 99% of the protein are eliminated by the procedure. Detection of specific RNA by dot-blot hybridization using a labeled probe demonstrates high reproducibility of recovered RNA and lack of "masking" with up to a 10-fold excess of starting cell material. Applicability of the procedure to detection of virus-specific RNA in cells persistently infected with mouse hepatitis virus is described.     

Rapid increase of mitochondrial uncoupling protein and its mRNA in stimulated brown adipose tissue. Use of a cDNA probe

The administration of thyroxine to neonatal rats stimulates RNA synthesis by neuronal nuclei isolated from the developing rat brain cortex. Glial nuclei are relatively resistant to thyroxine treatment. The activity of neuronal RNA polymerase II is particularly stimulated by the hormone. Thyroxine also affects neuronal chromatin structure as shown by changes in the relative proportion of different subnuclear fractions obtained by gentle micrococcal nuclease digestion of nuclei from hormone-treated rats.     

The zipcode-binding protein ZBP1 influences the subcellular location of the Ro 60-kDa autoantigen and the noncoding Y3 RNA

The Ro 60-kDa autoantigen, a ring-shaped RNA-binding protein, traffics between the nucleus and cytoplasm in vertebrate cells. In some vertebrate nuclei, Ro binds misfolded noncoding RNAs and may function in quality control. In the cytoplasm, Ro binds noncoding RNAs called Y RNAs. Y RNA binding blocks a nuclear accumulation signal, retaining Ro in the cytoplasm. Following UV irradiation, this signal becomes accessible, allowing Ro to accumulate in nuclei. To investigate how other cellular components influence the function and subcellular location of Ro, we identified several proteins that copurify with the mouse Ro protein. Here, we report that the zipcode-binding protein ZBP1 influences the subcellular localization of both Ro and the Y3 RNA. Binding of ZBP1 to the Ro/Y3 complex increases after UV irradiation and requires the Y3 RNA. Despite the lack of an identifiable CRM1-dependent export signal, nuclear export of Ro is sensitive to the CRM1 inhibitor leptomycin B. In agreement with a previous report, we find that ZBP1 export is partly dependent on CRM1. Both Ro and Y3 RNA accumulate in nuclei when ZBP1 is depleted. Our data indicate that ZBP1 may function as an adapter to export the Ro/Y3 RNA complex from nuclei.     

Effect of phytohormones on nuclear RNA synthesis in germinating seeds ofTrigonella foenumgraeceum and its callus

Treatment ofTrigonella foenumgraeceum (fenugreek) seedlings with naphthalene acetic acid plus gibberellic acid enhanced the RNA synthesising capacity of nuclei isolated from the hypocotyl and cotyledonary regions. This increase was more pronounced in the nuclei from the hypocotyl region than from the cotyledonary region.In vitro addition of these phytohormones did not stimulate RNA synthesis by nuclei. The RNA synthesis by mitochondria was not affected by preincubating the seedlings with the hormones. The nuclei isolated from callus cultures of fenugreek hypocotyl treated with the hormone also showed increased RNA synthesis.     

Effect of a partial trisomy in the mouse upon cellular and nuclear RNA

In a previous paper (Rake and Edwards, 1987) it was shown that the majority of the chromatin from trisomic mouse cells has nucleosomes with a smaller repeat length of DNA than the nucleosome repeat length of normal cells. Here it is shown that the RNA content of the total cell and of the nuclei is the same in all tissues studied, in both normal and trisomic cells. However, the amount per unit time or rate of RNA synthesis is depressed in the trisomic liver and brain nuclei. The depression of RNA synthesis could not be specified to the small trisomic section of the chromatin but instead must reflect the overall nuclear activity. These results, along with those of Devlinet al. (1988), indicate that the trisomic condition alters a substantial part of nuclear organization and activity, not just the small trisomic part.     

RNA二级结构的最小自由能算法

RNA(即tRNA,rRNA,mRNA和SnRNA)有两大主要功能：一是某些病毒的遗传物质;二是参与蛋白质的合成,这些与细胞分化、代谢、记忆的储存等有重要关系,这些功能与RNA二级结构的稳定性。自由能密切相关．常用的计算自由能的方法有热力学微扰法及热力学微积分法等．本文以寻找最小自由能二级结构为目的,给出了RNA二级结构的最小自由能算法,该算法的时间复杂性不超过O(n^4)。     

Binding of glucocorticoid-receptor complexes with the acceptor zones of nuclei and effect of glucocorticoids on RNA synthesis in hormone-sensitive and hormone-resistant cell populations

The binding of free radioactive glucocorticoid and the glucocorticoid-receptor complex to rat liver nuclei was studied in vitro. The binding is non-saturated and independent of preliminary injection of the "cold" hormone. In the course of DNA hydrolysis the amount of the radioactive hormone bound to the chromatin moiety in vivo remains practically unchanged relatively to the initial radioactivity of the protein. The liberation of the nuclei into a cell-free medium and the effect of DNAase I on the nuclei are associated with the redistribution of the hormone-receptor complex in the chromatin molecule and with the appearance of new, previously masked acceptor zones of the hormone binding. During the first 1-2 hours following the hormone injection the endogenous RNA-synthesizing activity of the nuclei is decreased. The increase of RNA synthesis in liver nuclei occurs not earlier than 3 hours after the injection. In Zajdela hepatoma nuclei the repression of RNA synthesis persists as long as 3 hours after the injection of dexamethasone. When RNA synthesis is determined in the nuclei in the presence of exogenous RNA-polymerase of E. coli in vitro, the increase in nuclear RNA synthesis can be observed beginning with the 30th min after the hormone injection. It is assumed that this effect is due to conformational changes in the chromatin structure, which are concomitant with the initial steps of association of the hormone-receptor complex.     

Specific radial positions of centromeres of human chromosomes X, 1, and 19 remain unchanged in chromatin-depleted nuclei of primary human fibroblasts: evidence for the organizing role of the nuclear matrix

Radial positions of centromeres of human chromosomes X, 1, and 19 were determined in the nuclei of primary fibroblasts before and after removal of 60%-80% of chromatin. It has been demonstrated that the specific radial positions of these centromeres (more central for the chromosome 19 centromere and more peripheral for the centromeres of chromosomes 1 and X) remain unchanged in chromatin-depleted nuclei. Additional digestion of nuclear RNA did not influence this specific distribution. These results strongly suggest that the characteristic organization of interphase chromosomes is supported by the proteinous nuclear matrix and is not maintained by simple repulsing of negatively charged chromosomes.