Isolation,fractionation and biochemical analysis of the metaphase chromosomes of Microtus agrestis

A method has been developed for isolating metaphase chromosomes from Microtus agrestis fibroblasts in relatively large quantities with recovery of about 50% of the chromosomes present in the metaphase cells. The method employs pressure homogenisation to release the chromosomes from the cells. The average chemical composition of the Microtus chromosome preparations is 24.6% DNA, 19.9% RNA and 55.5% protein. The isolated chromosomes were fractionated by sedimentation velocity in a density gradient into three size groups in one of which 75–80% of the chromosomes were the large sex-chromosomes. The relative composition of this fraction containing most of the heterochromatin of the cell was DNA: 100, RNA: 59, acid-soluble protein: 54, acid-insoluble protein: 178. — Disc electrophoresis studies revealed no significant difference in the histone patterns between the euchromatic and heterochromatic chromosomes of the three chromosome size-groups. Metaphase chromosomes appear to have a lower lysine-rich histone content than interphase nuclei.     

RNS- und Protein-Synthese in Puffs isolierter Speicheldrüsen-Chromosomen von Chironomus

Isolated salivary gland chromosomes of Chironomus tentans incubated in a simple salt or sucrose solution are able to synthesize not only RNA but also protein in their puffs. The migration of radioactively labelled material from isolated nucleoli to isolated chromosomes suggests that not all protein in the puffs is synthesized by the puffs themselves but that part of the puff protein stems from the nucleoli. It is concluded that besides bound RNA polymerase a puff contains ribosomes attached to the growing mRNA molecules.     

Membrane-bound mitochondrial DNA: isolation, transcription and protein composition.

Mitochondrial membrane-bound DNA complex from bovine heart mitochondria lysed in the presence of Triton X-100 was isolated by differential centrifugation. The yield of "nucleoid" is about 30 microgram protein/mg mitochondrial protein. It contains about 3-5 microgram DNA/mg protein and varying amounts of RNA. The heart mitochondrial nucleoid actively synthesizes RNA. The nucleoid fraction contains about sixteen different proteins as evidenced by urea-SDS gel electrophoresis and about twenty-one proteins as evidenced by acid-urea gel electrophoresis. It appears that the nucleoid is attached to the inner membrane since it does contain cytochromes.     

RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.     

Cost-effective and rapid (3 h) method for combined extraction of DNA,RNA and protein from a single tea leaf sample for genomic and proteomic analysis

This is the first report about the simultaneous extraction of nucleic acids and proteins from tea leaf tissue. Using the present protocol, the DNA, RNA and protein were simultaneously isolated from a single tea leaf sample. The method also maintained the quality and quantity of the isolated biomolecules. The method is cost-effective and takes only 3 h to isolate the starting molecules (DNA, RNA and protein) of central dogma of biology. It was also demonstrated that the isolated DNA, RNA and protein could be successfully used for genomics and proteomic analysis in tea plant which was verified by performing marker study, gene cloning, cDNA preparation, gene expression study and 2-DE.     

Chromosome fractionation. II. Features of the composition and behavior of biphasic polymer systems of metaphase chromosomes obtained from HeLa cells at acidic and neutral pH values

Metaphase chromosomes (MCh) isolated at pH 2.1 and 6.5 slightly differ in their composition and contain in average: 17% of DNA, 9% of RNA and 74% of protein. The three-fold increase of protein content in MCh was not accompanied by the change in nonhistone protein composition. The behaviour of chromosomes isolated at pH 2.1 (acid) and 6.5 (neutral) in two-phase polymer system is shown to be very similar. The function of certain chromosome components in the determination of MCh behaviour in two-phase systems and possible usefulness of acid and neutral MCh for fractionation of total MCh are discussed.     

Rapid and Reliable Method of Extracting DNA and RNA from Sweetpotato, Ipomoea batatas (L). Lam

A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg/g leaf tissue). The extracted DNA was completely digested by restriction endonucleases indicating the absence of common contaminating compounds. The absorbancy ratios of A260/A230 and A260/A280 of isolated RNA were approx. 2 and the yield was about 0.2 mg/g fresh wt. CIPK and tublin genes were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total DNA and RNA isolated by this method was of sufficient quality for subsequent molecular analysis.     

Perturbation of mammalian cell division. II. Studies on the isolation and characterization of human mini segregant cells.

A method is described for the isolation, according to size, of mini segregants produced by the abnormal cleavage of reversibly arrested mitotic HeLa cells. Many of these mini segregants contain small amounts of DNA, as judged by Feulgen staining and chromosome analysis. After fusion with mitotic HeLa cells, the interphase chromosomes of the mini segregants are seen as either monovalent or bivalent prematurely condensed chromosomes (PCC), some of which are damaged. A proportion of isolated mini segregants synthesize DNA, RNA and protein. Fusion of mini segregants with interphase HeLa cells gives rise to cells with 'hybrid' karyotypes.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein

Analysis of E.coli chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli deoxyribonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17,000 and 9,000 (proteins 1 and 2, respectively). 2) Proteins 1 and 2 occur in approximately equal molar amounts in the isolated E.coli chromosome, and their relative content corresponds to one molecule of protein 1 plus one molecule of protein 2 per 150-200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deoxyribonucleoprotein suggesting a fairly uniform distribution of the proteins 1 and 2 along DNA. 4) The protein 2 is apparently identical to the DNA-binding protein HU which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes with staphylococcal nuclease proceeds through discrete deoxyribonucleoprotein intermediates (in particular, at approximately 120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite conclusions about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.Images     

Selective cleaning of the cell debris in human chromosome preparations studied by scanning force microscopy

The chromosome structure is one of most challenging biological structures to be discovered. Most evidence about the structure comes from optical microscopy. Scanning force microscopy (SFM) can achieve molecular resolution and allows imaging in liquids. However, little information about the chromosome structure has been revealed by SFM. In this work, a mild enzymatic treatment is applied to the chromosomes to remove selectively the RNA and proteins coming from the cell. The resulting SFM images indicate that a protein film with embedded RNA molecules covers chromosomes in standard cytogenetic preparations. The thickness of the protein layer is 15-35 nm and the RNA adheres preferentially to the chromosome surface. The cell material film results in a quite smooth chromosome surface without evidence of any structural detail. After treatment, the chromosome was cleaned from cell residues and individual chromatin fibers at the surface were resolved. Furthermore, insights about the higher order structure of the chromosome can be inferred.     

仿刺参体壁总RNA提取方法的建立

目的:通过对TRIzol一步法进行改进,建立一种从富含胶原蛋白、多糖及色素的仿刺参体壁提取总RNA的有效方法。方法:样品在液氮中研磨并用TRIzol匀浆后再进行抽提;对TRIzol一步法提取的总RNA进行DNaseⅠ消化和酚氯仿抽提,用2.5mol/L的醋酸钾沉淀,并加入适量糖原(10mg/mL)与RNA共沉淀。结果:琼脂糖凝胶电泳和紫外分光光度法以及RT-PCR检测结果表明,改进的方法能够有效去除基因组DNA、蛋白、多糖及色素的污染,RNA的产率提高。结论:制备的总RNA纯度高,完整性好,能够满足mRNA差异显示RT-PCR等分子生物学研究的要求,是一种提取仿刺参体壁及其他富含黏多糖、胶原蛋白和色素的动物组织总RNA的有效方法。     

从少量培养细胞中同时提取微量蛋白和RNA的方法探讨

为建立一项从少量培养细胞中同时提取RNA和蛋白质的技术 ,向 2～ 3× 10 5细胞中加入 1ml自制RNA提取试剂 ,RNA抽提后剩下的中下两相 ,用异丙醇、盐酸胍和无水乙醇抽提蛋白质 .同时用进口Tripure试剂、经典的异硫氰酸胍 苯酚 氯仿一步抽提RNA法和分子克隆实验手册裂解液制备蛋白质的方法 ,作为对照 .自制试剂提取的总RNA ,18S、2 8S清晰可见 ,2 8S比 18S带亮度强 2～ 3倍 ,带与带之间无拖尾现象 ,5S隐约可见 ,而且成功地进行了Northern印迹、RT PCR分析 ,与经典方法差异不大 ;用此法所提蛋白质 ,经SDS PAGE检测 ,蛋白分离效果很好 ,无杂质 ,且Western印迹检测Giα蛋白 ,可见一条清晰的特异带 ,与常规提取蛋白质 ,结果相似 .从微量细胞中同时提取的RNA和蛋白质 ,得率高、纯度好 ,具有化学完整性和生物学性质     

Increased degradation rates of protein in aging human fibroblasts and in cells treated with an amino acid analog.

Isolated polytene nuclei from Drosophila hydei salivary glands were subjected to various in vitro conditions, and structural and functional alterations were observed. The conditions required to best maintain the structure were different from those needed for maximal RNA synthesis. However, both the structure and function were adequately maintained at a moderate ionic strength. Functionally the RNA synthetic activity of giant chromosomes under these conditions shows a close resemblance to the in vivo situation according to several criteria. Morphologically chromosomes from isolated nuclei show an intact and distinctive banding pattern in squash preparations, but they are much more condensed than chromosomes derived directly from cells. Decreasing the ionic strength reduces the degree of condensation but also diminishes the RNA synthetic activity of the isolated nuclei. Increasing the ionic strength results in maximal endogenous RNA polymerase activity, but considerably alters the chromosomal morphology. The chromosomes from isolated nuclei did not exhibit any template activity with exogenously added RNA polymerase B from Drosophila hydei. The possible implications of these findings are discussed.     

Rapid ephemeral cell sensitization as the mechanism of histone-induced and protamine-induced enhancement of transfection by poliovirus RNA

Summary The mechanism of transfection enhancement by protamine and histone was investigated, using poliovirus RNA and sheet cultures of the CLI line of chimpanzee liver cells. The transfection obtained using inocula prepared by premixing the viral RNA with a large excess of basic protein could be quantitatively accounted for by direct sensitization of the cells by the excess free basic protein postinoculation. Such direct sensitization to transfection was very rapid; at 25 °C with protamine chloride or histone at 1 mg/ml peak sensitivity was reached in about 16 and 25 seconds, respectively. Sensitivity then rapidly decreased, but could be partially restored later by a second pretreatment with fresh basic protein. The original sensitization, desensitization, and resensitization phases of the cell sensitivity curves are transfectionspecific, since ordinary infection with intact poliovirions was not influenced by the pretreatments with basic protein, except that prolonged pretreatment with histone decreased sensitivity to intact virus. The viral RNA transfectivity in inocula premixed with protamine chloride at 1 mg/ml and held at 0 °C was labile, with an initial half-life of only 5 minutes; the transfectivity of RNA similarly held in buffer alone or with histone was completely stable at 0 °C for at least 1.5 to 2 hours. With the best basic protein transfection method (cell sensitization by protamine), the specific transfectivity of RNA isolated from purified poliovirions was 130,000 plaque-forming units/g RNA.     

Architecture of metaphase chromosomes and chromosome scaffolds

We have developed procedures for depositing intact mitotic chromosomes and isolated residual scaffolds on electron microscope grids at controlled and reproducible levels of compaction. The chromosomes were isolated using a recently developed aqueous method. Our study has addressed two different aspects of chromosome structure. First, we present a method for improved visualization of radial chromatin loops in undisrupted mitotic chromosomes. Second, we have visualized a nonhistone protein residual scaffold isolated from nuclease-digested chromosomes under conditions of low salt protein extraction. These scaffolds, which have an extremely simple protein composition, are the size of chromosomes, are fibrous in nature, and are found to retain differentiated regions that appear to derive from the kinetochores and the chromatid axis. When our standard preparation conditions were used, the scaffold appearance was found to be very reproducible. If the ionic conditions were varied, however, the scaffold appearance underwent dramatic changes. In the presence of millimolar concentrations of Mg++ or high concentrations of NaCl, the fibrous scaffold protein network was observed to undergo a lateral aggregation or assembly into a coarse meshlike structure. The alteration of scaffold structure was apparently reversible. This observation is consistent with a model in which the scaffolding network plays a dynamic role in chromosome condensation at mitosis.     

Simultaneous banding of rat embryo DNA, RNA, and protein in cesium trifluoroacetate gradients

A procedure for the simultaneous banding of cellular DNA, RNA, and protein by centrifugation in cesium trifluoroacetate (CsTFA) gradients is described. Starting with homogenates of Day 11 rat embryos, this procedure was used to separate total DNA, RNA, and protein. Under the conditions used DNA banded at a peak density of 1.63 g/ml, RNA at a peak density of 1.83 g/ml, and protein at a peak density of 1.40 g/ml. Nucleic acids isolated from CsTFA gradients were judged to be protein free. RNA isolated by this method is apparently free of DNA contamination; however, DNA isolated by this method does contain some RNA (less than 5% contamination).