Ultrastructural and biochemical comparisons of nuclear matrices prepared by high salt or LIS extraction

Summary We have directly compared two independently published methods for isolating operationally defined nuclear matrices by studying EM ultrastructure, protein composition and distribution of replicating DNA. Nuclear matrices prepared by extraction with 2 M NaCI consisted of fibrous pore complex lamina, residual fibrillar and granular components of nucleoli and interchromatin granules, and an extensive anastomosing internal fibrous network. These matrices were enriched in high molecular weight nonhistone proteins but were virtually devoid of histones. Consistent with previously published data, newly-replicated DNA was resistant to this high salt extraction. Nuclear matrices prepared by extraction of nuclei with 25 mM lithium 3,5-diiodosalicylate, LIS, also contained fibrous pore complex lamina, but lacked morphologically distinct residual nucleoli and were markedly depleted in internal structure. The reduced amounts and complexity of proteins associated with the LIS matrix were consistent with the ultrastructural data. Moreover, much less newly-replicated DNA was recovered in LIS matrices. The data show that LIS dissociates nuclear ultrastructure and extracts both protein and DNA in proportion to the concentration used, regardless of whether nuclei or high salt nuclear matrices are used as starting material. While the data suggest that LIS may not necessarily be an optimal reagent for preparing nuclear matrices containing internal structural elements from all tissue sources, it may be useful for selectively solubilizing and analyzing components of the nuclear matrix.Abbreviations EM electron microscopy - HS high salt, 2 M NaCl - LIS lithium 3,5-diiodosalicylate - DEPC diethyl pyrocarbonate - PMSF phenylmethylsulfonyl fluoride - SBTI soybean trypsin inhibitor - VRC vanadium ribonucleoside complex - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - EDTA ethylenediaminetetraacetic acid - PAGE polyacrylamide gel electrophoresis, Type A and Type B structures were isolated as described in Experimental Procedures by methods A and B, respectively     

Prereplicative increase of nuclear matrix-bound DNA polymerase-α and primase activities in HeLa S3 cells following dilution of long-term cultures

We investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85% of the cells were in the G₁ phase of the cycle, whereas 8% were in the S phase. After dilution with fresh medium, 18–22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10% of nuclear activity. As judged by [³H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activitites was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures. J. Cell. Biochem. 71:11–20, 1998. © 1998 Wiley-Liss, Inc.     

Determination of the in vivo structural DNA loop organization in the genomic region of the rat albumin locus by means of a topological approach

Nuclear DNA of metazoans is organized in supercoiled loops anchored to a proteinaceous substructure known as the nuclear matrix (NM). DNA is anchored to the NM by non-coding sequences known as matrix attachment regions (MARs). There are no consensus sequences for identification of MARs and not all potential MARs are actually bound to the NM constituting loop attachment regions (LARs). Fundamental processes of nuclear physiology occur at macromolecular complexes organized on the NM; thus, the topological organization of DNA loops must be important. Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence. The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced. Therefore, it is possible to determine the specific DNA loop configuration without previous characterization of the LARs involved. We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase ineukaryotes, and essential for DNA replication. By applying serial extractions to mammaliancells synchronized by release from quiescence, we reveal dynamic changes to thesub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase,identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix.The data distinguish 3 states that correspond to loose association with chromatin prior toDNA replication, transient highly stable binding to the nuclear-matrix coincident withinitiation, and a post-initiation phase when MCM2 remains tightly associated withchromatin but not the nuclear-matrix. The data suggests that functional MCM complexloading takes place at the nuclear-matrix.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.     

Nucleotide sequences and stability of a Nicotiana nuclear DNA segment possessing autonomously replicating ability in yeast

The nucleotide sequence of a tobacco (Nicotiana tabacum) chromosomal DNA segment(t3-ars) capable of replication in yeast (ars: autonomously replicating sequences) is presented. The subcloned region (618 bp) contained 11 bp consensus (5 A/TTTTATP_uTTTA/T 3) essential for several yeast ars, and 73% A and T. Unique 70 bp repetitive sequences resided next to this sequence. Thirty-two bp AT repeats were also seen in the neighbourhood of the repetitive sequence. The hybrid plasmid containing t3-ars was mitotically stabilized by the help of yeast centromere (CEN4).     

利用靶定基质蛋白基因m的小干扰RNA抑制2009甲型H1N1流感病毒的复制

目的:设计、构建并筛选针对流感病毒基质蛋白基因m的小干扰RNA(siRNA),检测其对2009甲型H1N1流感病毒复制的抑制效果。方法:设计3条针对流感病毒m基因的siRNA,并克隆到短发夹型(shRNA)siRNA表达载体pSilencer2.1-U6-hygro上;经测序证明构建成功后,将表达3种siRNA的质粒和阴性对照质粒psiRNA-control分别转染MDCK细胞,用潮霉素筛选稳定表达细胞株,用H1N1流感病毒感染细胞,通过Real-time PCR和Western印迹检测干扰效果。结果:构建了3个针对流感病毒m基因的siRNA表达质粒,3种siRNA均使m基因的mRNA水平降低,其中M1-306的抑制效率达60%;3种siRNA均使流感病毒NA蛋白的表达量降低,M2-25、M1-105的抑制效率明显,M1-306略低。结论:针对m基因的siRNA可以有效抑制流感病毒在MDCK细胞中的复制。     

Rhythmic contraction and intracellular Ca2 + oscillatory rhythm in spontaneously beating cultured cardiac myocytes

Cultured cardiac myocytes from neonatal rats show spontaneous and rhythmic contractions. The intracellular concentration of free Ca^2 + also changes rhythmically, associated with the rhythmic contraction of myocytes (Ca^2 + oscillation). This study aims to elucidate whether spontaneous rhythmic contraction affects the dynamics of intracellular Ca^2 + oscillation in cultured cardiac myocytes. In cultures at four days in vitro (4 DIV), spontaneous Ca^2 + oscillation was synchronized among myocytes. Treatment of cultures with an uncoupler of E - C coupling resulted in a cessation of the spontaneous contraction of cardiac myocytes, but did not abolish the Ca^2 + oscillation. The intercellular synchronization of intracellular Ca^2 + oscillation persisted, and both the intervals and the fluctuation of the oscillation tended to increase after the termination of rhythmic contraction. The present study demonstrated that mechanical factors associated with rhythmic contraction did not affect the intercellular synchronization of intracellular Ca^2 + oscillation, but possibly contributed to the stability of the oscillatory rhythm.