The effect of growth and starvation on the lysis of the ruminal cellulolytic bacterium Fibrobacter succinogenes.

Growing cultures of Fibrobacter succinogenes assimilated more ammonia than could be accounted for by cellular protein, RNA, or DNA and released large amounts of nonammonia nitrogen. The difference between net and true growth was most dramatic at low dilution rates, but mathematical derivations indicated that the lysis rate was a growth rate-independent function. The lysis rate was sevenfold greater than the true maintenance rate (0.07 h-1 versus 0.01 h-1). Because slowly growing cells had as much proton motive force and ATP as fast-growing cells, lysis was not a starvation response per se. Stationary-phase cells had a lysis rate that was 10-fold less than that of growing cells. Rapidly growing cells were not susceptible to phenylmethylsulfonyl fluoride, but phenylmethylsulfonyl fluoride increased the lysis rate of the cultures when they reached the stationary phase. This latter result indicated that autolysins of stationary-phase cells were being inactivated by a serine proteinase. When growing cells were treated with the glycolytic inhibitor iodoacetate, the proteinase-dependent transition to the stationary phase was circumvented, and the rate of lysis could be increased by as much as 50-fold.     

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in <Emphasis Type="Italic">Alstroemeria aurea</Emphasis>

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry at different developmental stages of pollen tubes cultured in vitro revealed changes in the nuclear phase at 9 and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cells showed a tendency to increase. This association was suggested as the male germ unit (MGU). When sperm cells, vegetative nuclei, and partial MGUs were collected separately from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial MGUs was counted as 2C. Therefore, the second change in the nuclear phase, which results in an increase in 2C nuclei, is possibly related to the formation of MGUs.     

Lia1p, a novel protein required during nuclear differentiation for genome-wide DNA rearrangements in Tetrahymena thermophila

Extensive genome-wide rearrangements occur during somatic macronuclear development in Tetrahymena thermophila. These events are guided by RNA interference-directed chromatin modification including histone H3 lysine 9 methylation, which marks specific germ line-limited internal eliminated sequences (IESs) for excision. Several genes putatively involved in these developmental genome rearrangements were identified based on their proteins' localization to differentiating somatic nuclei, and here we demonstrate that one, LIA1, encodes a novel protein that is an essential component of the genome rearrangement machinery. A green fluorescent protein-Lia1 fusion protein exhibited dynamic nuclear localization during development that has striking similarity to that of the dual chromodomain-containing DNA rearrangement protein, Pdd1p. Coimmunoprecipitation experiments showed that Lia1p associates with Pdd1p and IES chromatin during macronuclear development. Cell lines in which we disrupted both the germ line and somatic copies of LIA1 (DeltaLIA1) grew normally but were unable to generate viable progeny, arresting late in development just prior to returning to vegetative growth. These mutant lines failed to properly form Pdd1p-containing nuclear structures and eliminate IESs despite showing normal levels of H3K9 methylation. These data indicate that Lia1p is required late in conjugation for the reorganization of the Tetrahymena genome.     

Isolation and characterization of nuclei from Neurospora crassa.

A procedure was developed for isolating nuclei from either the conidial or germinated conidial growth phase of Neurospora crassa. A frozen conidial suspension was lysed by passage through a French pressure cell, and the nuclei were freed from the broken cells by repeated homogenization in an Omni-Mixer. Pure nuclei were obtained from the crude nuclear fraction by density banding in a Ludox gradient. The final nuclear yield was 20 to 30%. The nuclei had a deoxyribonucleic acid (DNA):ribonucleic acid (RNA):protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6-diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 micron in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70 to 75% yield by dissociation with 2 M sodium chloride and 5 M urea had a DNA:RNA:protein ratio of 1:1.05:1.7. Chromatin reconstituted from this preparation exhibited a level of RNA polymerase template activity lower than that of pure Neurospora DNA, but the maximum level of reconstitution obtained was only 10%. Fractionation of Neurospora chromatin on hydroxylapatite separated the histones from the chromatin acidic proteins. The normal complement of histone proteins was present in both the reconstituted and dissociated chromatin preparations. The acidic protein fraction exhibited a variety of bands on sodium dodecyl sulfate gel electrophoresis ranging in molecular weight from 15,000 to 70,000. The gel pattern was much more complex for total dissociated chromatin than for reconstituted chromatin.     

Analysis of Nuclear DNA content in plant cells by Flow cytometry

Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro. Cell nuclei were isolated either mechanically (chopping, syringing) or by a hypotonic lysis of isolated protoplasts. Although both methods gave similar results, a slight shift to lower ploidy levels was observed after protoplast isolation from intact tissues and calli. No differences were observed if the two methods were compared using cell suspension cultures. The results showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissues and variousin vitro cultures.     

Segregation following interspecific transfer of isolated nuclei between Phytophthora parasitica and P. capsici

Nuclei isolated from metalaxyl-resistant (MR) protoplasts of Phytophthora parasitica were transferred into chloroneb-resistant (CnR) protoplasts of Phytophthora capsici and vice versa, with an average success rate of 2.6 x 10(-4) (protoplasts with donor nuclei/regenerated protoplasts), using a selective medium containing only the fungicide tolerated by the nuclear donor. No colonies appeared when self-fusion products of donor nuclei or recipient protoplasts were exposed to the selective medium. Colonies produced by the nuclear transfer formed sectors commonly, and differed from the parental types in appearance. All the zoospores produced by the nuclear hybrids were of normal size, and one-fifth of them contained both MR and CnR genes. Since zoospores are mostly uninucleate, these results indicated the occurrence of chromosome re-assortment or mitotic crossing-over following the production of transitory tetraploids, followed by diploidization during zoosporogenesis, thus suggesting the completion of events leading to a parasexual cycle. Hyphal fragment cultures from a nuclear hybrid tested showed considerable variation in growth rate, mycelial morphology, and level of resistance to metalaxyl, indicating uneven distribution and continuous segregation of different types of nuclei in mycelia during vegetative growth.     

Cadmium induced apoptotic changes in chromatin structure and subphases of nuclear growth during the cell cycle in CHO cells

CHO cells were grown in the presence of 1 M CdCl₂ and subjected to ATP-dependent replicative DNA synthesis after permeabilization. By decreasing the density of the cell culture replicative DNA synthesis was diminishing. At higher than 2 × 10⁶ cell/ml concentration Cd had virtually no effect on the rate of DNA replication. Growth at higher cell concentrations could be supressed by increasing Cd concentration. After Cd treatment cells were synchronized by counterflow centrifugal elutriation. Cadmium toxicity on cell growth in early and mid S phase led to the accumulation of enlarged cells in late S phase. Flow cytometry showed increased cellular and nuclear sizes after Cd treatment. As the cells progressed through the S phase, 11 subpopulations of nuclear sizes were distinguished. Apoptotic chromatin changes were visualized by fluorescent microscopy in a cell cycle dependent manner. In the control untreated cells the main transitory forms of chromatin corresponded to those we have published earlier (veil-like, supercoiled chromatin, fibrous, ribboned structures, chromatin strings, elongated prechromosomes, precondensed chromosomes). Cadmium treatment caused: (a) the absence of decondensed veil-like structures and premature chromatin condensation in the form of apoptotic bodies in early S phase (2.2–2.4 average C-value), (b) the absence of fibrous structures, the lack of supercoiled chromatin, the appearance of uncoiled ribboned chromatin and perichromatin semicircles, in early mid S phase (2.5–2.9 C), (c) the presence of perichromatin fibrils and chromatin bodies in mid S phase (2.9–3.2 C), (d) early intra-nuclear inclusions, elongated forms of premature chromosomes, the extrusion and rupture of nuclear membrane later in mid S phase (3.3–3.4 C), (e) the exclusion of chromatin bodies and the formation of clusters of large-sized perichromatin granules in late S phase (3.5–3.8 C) and (f) large extensive disruptions and holes in the nuclear membrane and the clumping of incompletely folded chromosomes (3.8–4. C).     

水稻OsMS2基因在花药发育中的功能分析

拟南芥MS2(MALE STERILITY 2)是一个调控花药花粉发育的关键基因。水稻OsMS2(Os03g07140)基因与拟南芥MS2的序列具有高度同源性。利用RNA干扰技术研究OsMS2基因在水稻花药发育过程中的功能。与野生型水稻相比, 转基因 植株营养生长阶段正常, 但雄性育性降低。转基因植株雄性育性降低与RNA干扰引起的OsMS2基因表达水平降低有关。进一步对转基因植株花药进行细胞学观察, 结果表明OsMS2基因表达水平的降低导致绒毡层细胞退化延迟, 小孢子壁的形成出现异常。扫描电镜观察结果显示, 小孢子壁光滑, 不能形成正常的外壁。以上结果表明OsMS2基因在水稻花药发育过程中起重要作用。     

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus.

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

Reactivation of nuclear RNA polymerase activity in growth stimulated epithelial cells

The activation of endogenous RNA polymerases has been studied in mouse kidney epithelial cells which have been induced to grow in culture. Slide cultures were assayed for RNA polymerase, in situ, after brief fixation. Enzyme activity was detected by autoradiography. Polymerases I (nucleolar) and II (nucleoplasmic) were distinguished by localization and by the activities in the presence of different ions as well as sensitivity to α-amanitin.Kidney epithelial cells resuming their growth activity when inoculated in vitro show an early and rapid increase in nuclear size (nuclear protein content) which precedes cell multiplication. Both nucleolar and nucleoplasmic polymerase activities appear at an early phase of growth activation and increase during the whole period of growth activation in proportion to increase in nuclear size. RNA polymerases may also be activated in cells which have not undergone an increase in nuclear size if assayed in the presence of a high salt concentration (0.4 M ammonium sulphate). The role of modification of chromatin structure for genetic reactivation is discussed.     

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

水稻OsMS2基因在花药发育中的功能分析

拟南芥MS2（MALE STERILITY2）是一个调控花药花粉发育的关键基因。水稻OsMS2（Os03g07140）基因与拟南芥MS2的序列具有高度同源性。利用RNA干扰技术研究OsMS2基因在水稻花药发育过程中的功能。与野生型水稻相比,转基因植株营养生长阶段正常,但雄性育性降低。转基因植株雄性育性降低与RNA干扰引起的OsMS2基因表达水平降低有关。进一步对转基因植株花药进行细胞学观察,结果表明OsMS2基因表达水平的降低导致绒毡层细胞退化延迟,小孢子壁的形成出现异常。扫描电镜观察结果显示,小孢子壁光滑,不能形成正常的外壁。以上结果表明OsMS2基因在水稻花药发育过程中起重要作用。     

Nick-forming sequences may be involved in the organization of eukaryotic chromatin into ∼50 kbp loops

Phenomena involving the disassembly of chromosomes to ∼50 kbp double-stranded fragments upon protein denaturing treatments of normal and apoptotic mammalian nuclei as well as yeast protoplasts may be an indication of special, hypersensitive regions positioned regularly at loop-size intervals in the eukaryotic chromatin. Here we show evidence in yeast cell systems that loop-size fragmentation can occur in any phase of the cell cycle and that the plating efficiency of these cells is ∼100%. The possibility of sequence specificity was investigated within the breakpoint cluster region (bcr) of the human MLL gene, frequently rearranged in certain leukemias. Our data suggest that DNA isolated from yeast cultures or mammalian cell lines carry nicks or secondary structures predisposing DNA for a specific nicking activity, at non-random positions. Furthermore, exposure of MLL bcr-carrying plasmid DNA to S1 nuclease or nuclear extracts or purified topoisomerase II elicited cleavages at the nucleotide positions of nick formation on human genomic DNA. These data support the possibility that certain sequence elements are preferentially involved in the cleavage processes responsible for the en masse disassembly of chromatin to loop-size fragments upon isolation of DNA from live eukaryotic cells.     

通过不对称体细胞杂交向小麦转移青苗碱谷DNA

普通小麦"济南177"(Triticum aestivum L.cv.Jinan177)经继代培养和选择,形成了性质不同的愈伤组织,一种生长迅速,易于形成悬浮系,游离的原生质具有旺盛的分裂能力,但不能分化,称为Cha9;另一种具有一定能力,但游离原生质体分裂能力低,称为176.二者之一来源的原生质体与紫外线照射的青苗碱谷原生质体融合均不能获得再生植株.而将源于Cha9和176的两种原生质体混合,与经紫外线照射的青苗碱谷原生质体在PEG诱导下融合,融合产物再生了大量植株.再生的愈伤组织及植株经表现型、细胞学、有工酶、RAPD分析,证明了其杂种性质,用不麦叶绿体特异的简单得复序列(SSR)引物分析了再生杂种的叶绿体遗传组成情况.在不同的杂种克隆中,同时带有Cha9和176的遗传物质并含有供体核及胞质基因组的克隆4具有较高的分化能力,再生了大量生长旺盛的完整植株.     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

通过不对称体细胞杂交向小麦转移青苗碱谷DNA(英文)

普通小麦“济南177”(Triticum aestzvum L.cv.Jinan 177)经继代培养和选择,形成了两种性质不同的愈伤组织,一种生长迅速,易于形成悬浮系,游离的原生质体具有旺盛的分裂能力,但不能分化,称为Cha 9;另一种具有一定分化能力,但游离的原生质体分裂能力低,称为176。二者之一来源的原生质体与紫外线照射的青苗碱谷原生质体融合均不能获得再生植株。而将来源于Cha 9和176的两种原生质体混合,与经紫外线照射的青苗碱谷原生质体在PEG诱导下融合,融合产物再生了大量植株。再生的愈伤组织及植株经表型、细胞学、同工酶、RAPD分析,证明了其杂种性质,用小麦叶绿体特异的简单重复序列(SSR)引物分析了再生杂种的叶绿体遗传组成情况。在不同的杂种克隆中,同时带有Cha 9和176的遗传物质并含有供体核及胞质基因组的克隆4具有较高的分化能力,再生了大量生长旺盛的完整植株。     

Affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized. Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix. After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500).     

Temperature-sensitive multicellular mutants of Wangiella dermatitidis.

Three temperature-sensitive morphological mutants of Wangiella dermatitidis were isolated and characterized. The mutants grew in the yeastlike morphology at the permissive temperature (25 degrees C) but expressed a multicellular (Mc) phenotype at the restrictive temperature (37 degrees C). Cultures of Mc 2 and 3 incubated at the restrictive temperature showed rapid reductions in the percentage of budded cells in the population. In contrast, budding continued for several generations in cultures of Mc 1. Incubation of cultures of Mc 2 and 3 at the restrictive temperature for 48 h resulted in nearly total conversion of yeastlike cells to the multicellular form; about 50% of the cells of Mc 1 had converted to multicellular forms after 48 h at the restrictive temperature. Studies using radiolabeled compounds documented that DNA, RNA, and protein synthesis continued at the restrictive temperature. The results suggest that multicellularity is the result of inhibition of bud emergence and cell separation without inhibition of growth nuclear division, and cytokinesis.