Pattern of DNA synthesis in nuclei of feeding and starving Amoeba proteus : A cytofluorometric study

The DNA content in isolated nuclei of Amoeba proteus was determined for each of the three groups of synchronized amoebae over different intervals after division. Several nuclei of each amoeba group were fixed 1 h after division, before the amoebae were fed. About h after division, some amoebae in each group were given food (Tetrahymena pyriformis), while the rest were left starving. Samples of the nuclei of fed and starved amoebae were fixed 24 h and (in different groups) 42–55 h after division. In each group from 22 to 48% of the fed amoebae had divided prior to the last nuclei fixation. Starved amoebae did not undergo division. In all three amoeba groups the nuclear DNA content of fed cells by the end of interphase had increased to 280–300% the value for 1 h amoebae. The nuclear DNA content of starved amoebae of all three groups was also increased, and in two groups it exceeded the initial level more than two-fold. However, in all three groups, it was lower than that of fed amoebae. In all the groups the nuclear DNA content in fed amoebae grew after 24 h, i.e. during the second half of interphase, the increase accounting for from 11 to 48% of the total increase. The hypothesis is put forward that the increase in the nuclear DNA content during the cell cycle of Amoeba proteus is the result of two processes: (1) one-time replication of the DNA of the whole genome; and (2) repeated replication of some part of the DNA. In amoebae the relation of the pattern of nuclear DNA synthesis to the diet is considered.     

Cytoplasmic streaming in Amoeba proteus is inhibited by the actinspecific drug phalloidin

Phalloidin, applied by microinjection into Amoeba proteus inhibits specifically, in a concentration dependent manner, the process of cytoplasmic streaming. Preliminary ultrastructural analysis of phalloidin-injected amoebas shows that extensive arrays of microfilaments are formed. These results are discussed in relation to the specific interaction of phalloidin with actin known to occur in vitro, which involve promotion of actin polymerization and stabilization of F-actin structures.     

Correlation between membrane potential, cell shape, and motile activity in Amoeba proteus

The various motile activities and cell shapes of Amoeba proteus grown in Chalkley's solution are correlated with definitive electrical membrane potentials. The same correlations were found when definitive motile activities and cell shapes were experimentally induced by changing the pH of the culture medium. The highest values of membrane potential (−70 mV) were measured in monopodial amebae during active locomotion. In resting cells, which prevail in acid or basic media, the membrane potential decreases to −5 mV. In those resting cells, which also stop internal cytoplasmic movement at basic pH, the membrane potential turns positive (+9 mV − +30 mV).     

Purification and Characterization of the GroESLx Chaperonins from the Symbiotic X-Bacteria in Amoeba proteus

GroELx and GroESx proteins of symbiotic X-bacteria from Amoeba proteus were overproduced in Escherichia coli transformed with pAJX91 and pUXGPRM, respectively, and their chaperonin functions were assayed. We utilized σ⁷⁰-dependent specific promoters of groEx in the expression vectors and grew recombinant cells at 37°C to minimize coexpression of host groE of E. coli. For purifying the proteins, we applied the principle of heat stability for GroELx and pI difference for GroESx to minimize copurification with the hosts GroEL and GroES, respectively. After ultracentrifugation in a sucrose density gradient, the yield and purity of GroELx were 56 and 89%, respectively. The yield and purity of GroESx after anion-exchange chromatography were 62 and 91%, respectively. Purified GroELx had an ATPase activity of 53.2 nmol Pi released/min/mg protein at 37°C. The GroESx protein inhibited ATPase activity of GroELx to 60% of the control at a ratio of 1 for GroESx-7mer/GroELx-14mer. GroESLx helped refolding of urea-unfolded rhodanese up to 80% of the native activity at 37°C. By chemical cross-linking analysis, oligomeric properties of GroESx and GroELx were confirmed as GroESx₇ and GroELx₁₄ in two stacks of GroELx₇. In this study, we developed a method for the purification of GroESLx and demonstrated that their chaperonin function is homologous to GroESL of E. coli.     

Protein migration from transplanted nuclei in Amoeba proteus. II. An electrophoretic study

Recently, we showed that protein migration from a nucleus transplanted into a host amoeba involved two classes of nuclear proteins. The chemical character of these proteins has now been investigated using isoelectric focusing and SDS gel electrophoresis. The proteins which migrated into the host cytoplasm from the transplanted nucleus have a range of isoelectric points (pI) between 7.0 and 7.6, and a molecular weight (MW) range of 9 000–110 000 D. This class is likely to be involved with the nucleocytoplasmic transfer of RNA. The second class of migratory proteins had a lower MW and pI range; the majority were between 11 000 and 45 000 D, with pIs between 5.9 and 7.0. This class of migratory proteins exhibited a shuttling character, possibly functioning as cytoplasmic regulators of nuclear activities.     

Quantitative analysis of changes in cell shape of Amoeba proteus during locomotion and upon responses to salt stimuli

A new parameter expressing the complexity of cell shape defined as (periphery)²/(area) in 2D projection was found useful for a quantitative analysis of changes in the cell shape of Amoeba proteus and potentially of any amoeboid cells. During locomotion the complexity and the motive force of the protoplasmic streaming in amoeba varied periodically, and the Fourier analysis of the two showed a similar pattern in the power spectrum, giving a rather broad peak at about 2.5 × 10⁻³ Hz. The complexity increased mainly due to elongation of the cell as external Ca²⁺ increased. This effect was blocked by La³⁺, half the inhibition being attained at 1/200 amount of the coexisting Ca²⁺. On the other hand, the complexity decreased due to rounding up of the cell as the concentration of other cations, such as Sr²⁺, Mg²⁺, Co²⁺, Ni²⁺, Na⁺, K⁺ etc., increased. Irrespective of the opposite effects of Ca²⁺ and other cations on the cell shape, the ATP concentration in amoeba decreased in both cases with increase of all these cations. The irregularity in amoeboid motility is discussed in terms of a dynamic system theory.     

Nuclear actin bundles in Amoeba, dictyostelium and human HeLa cells induced by dimethyl sulfoxide

In a previous study we demonstrated that dimethyl sulfoxide (DMSO) induces the formation of microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium mucoroides [12], in which the microfilaments bound rabbit skeletal muscle heavy meromyosin, forming an ‘arrowhead’ structure, and that this binding could be reversed by Mg²⁺ and ATP. In the present study, we show electron microscopic data demonstrating the occurrence of such microfilament bundles in the nucleus of Amoeba proteus and human HeLa cells, as well as in D. mucoroides. The similarities in the morphology and dimension of the microfilanets, as well as the specific conditions by which they are induced, suggested that these microfilaments are actin. We present evidence that actin is involved in interphase nucleus of a variety of organisms, and that DMSO acts on the molecules to induce microfilament bundles specifically in the nucleus.     

DNA Digestion and Chromatin Condensation during Nuclear Death inTetrahymena

DNA fragmentation and nuclear condensation are key features in the regulated cell death of higher animal cells. Nuclear death also occurs as part of a developmentally programmed process during the sexual life cycle of the unicellular organismTetrahymena.We examined the regulation of nuclear death and the relationship between DNA fragmentation and chromatin condensation in this model system. Nuclear death is accompanied by DNA digestion to low-molecular-weight oligonucleosomal-length fragments, in agree- ment with a previous study [17], indicating an endonuclease-like activity typical of apoptosis in higher organisms. Actinomycin D and cycloheximide block DNA digestion as well as nuclear condensation suggesting that nuclear death is under genetic regulation. DNA digestion is completely blocked by aurin, a general nuclease inhibitor. In addition, when DNA fragmentation is blocked, nuclear condensation also fails to occur. Moreover, a kinetic analysis of DNA breakdown, using agarose gels, shows that some DNA digestion occurs before nuclear condensation has taken place. Thus the initiation of DNA digestion may provide conditions necessary for nuclear condensation. Temporary inhibition of nuclear death aborts the death program since after removal of inhibitors cells revert to a vegetative pathway without having eliminated the old or developed the new macronucleus. Zn²⁺and EGTA, both of which inhibit apoptosis in some cell types, fail to prevent nuclear condensation or DNA digestion inTetrahymena,suggesting a requirement here for an endonuclease which is Ca²⁺-independent and Zn²⁺-insensitive. With the TUNEL assay, DNA breakdown is detected exclusively in the condensed macronucleus (and occasional micronuclei identified as degenerating haploid products of meiosis), but not in precondensed macronuclei. These studies show that apoptotic-like DNA fragmentation occurs after condensation of the degenerating macronucleus. However, early DNA digestion may be critical for nuclear condensation and subsequent degeneration.     

Nuclear DNA content and phytohormone requirements of normal, crown gall and habituated tissues of Nicotiana tabacum var. White Burley

Determination of the nuclear DNA content of leaves and normal, habituated and Crown gall callus tissues of Nicotiana tabacum var. White Burley were performed using cytophotometry on Feulgen stained preparations. Several aspects concerning the reliability of the Feulgen technique for DNA determinations were investigated.Crown gall callus tissue used in this study had both a higher nuclear DNA content and chromosome number than normal callus (3.2C versus 2.5C). Both have a higher DNA content than the diploid tobacco leaf cells (2C).The normal callus tissue failed to grow on medium without indole acetic acid and kinetin when cultured in tubes. From this normal callus two habituated lines growing without both phytohormones were selected by culturing the normal callus first in the absence of either indole acetic acid or kinetin. Changing the culture conditions of the normal callus by using culture flasks instead of tubes resulted in a remarkably faster growth rate of the tissue. This was accompanied by an acquisition of the habituation characteristics since it was possible now to grow this tissue also directly on medium lacking both phytohormones. All habituated tissues showed a higher nuclear DNA content compared to the normal callus tissue from which they were derived. Interestingly, one of the tissues acquired a nuclear DNA content not different from that of Crown gall tissue. By changing the culture conditions of Crown gall callus tissue no concomitant change in nuclear DNA content occurred.The results suggest a correlation between the acquisition of a special chromosome complement and the loss of phytohormone requirement resulting in autonomous growth.     

Nuclear DNA endonuclease activities on partially apurinic/apyrimidinic DNA in normal human and xeroderma pigmentosum lymphoblastoid and mouse melanoma cells

DNA endonuclease activities from nuclear proteins of normal human and xeroderma pigmentosum (XP), complementation group A, lymphoblastoid and Cloudman mouse melanoma cells were examined against partially apurinic/apyrimidinic (AP) DNA. Non-histone chromatin-associated and nucleoplasmic proteins, obtained from isolated nuclei, were subfractionated by isoelectric focusing and assayed for DNA endonuclease activity against linear, calf thymus DNA. All of the nine chromatin-associated and three of the nucleoplasmic fractions, which lacked DNA exonuclease activity, were tested for DNA endonuclease activity against both native and partially AP, circular, duplex, supercoiled PM2 DNA. In all three cell lines, four chromatin-associated, but none of the nucleoplasmic fractions, showed increased activity against DNA rendered AP by either heat/acid treatment or by alkylation with methyl methanesulfonate (MMS) followed by heat. One chromatin-associated activity, with pI 9.8, which was not active on native DNA, showed the greatest activity on AP DNA. AP activity was moderately decreased in XP cells and slightly decreased in mouse melanoma cells, as compared with normal cells, in the fraction at pI 9.8. Little or no increased activity was observed in any of the endonucleases from any of the cell lines on MMS alkylated DNA.     

Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae)

Checker mallows (Sidalcea, Malvaceae) constitute a western North American genus of annuals and perennials that have been regarded as taxonomically difficult because of complex patterns of morphological variation putatively stemming from hybridization and polyploidy. In recent molecular phylogenetic investigations extensive polymorphism was observed in the internal and external transcribed spacers (ITS and ETS) of 18S–26S nuclear ribosomal DNA for some Sidalcea samples. To resolve the evolutionary basis for this polymorphism and to readdress the evolutionary impact of hybridization in Sidalcea we cloned and sequenced the polymorphic DNAs and included the clones in phylogenetic analyses together with direct sequences of non-polymorphic samples. The positions of cloned spacer sequences in the phylogenetic trees suggest that S. reptans and two subspecies of S. malviflora may have been influenced by past hybridization with lineages of the “glaucescens” clade. Polymorphic sequence patterns in other taxa may be a result of extensive interbreeding within young clades, in keeping with the minimal sequence divergence, largely overlapping geographic distributions and morphology, and ploidy variation in these groups. Other possible explanations for polymorphic sequences in members of Sidalcea include slow concerted evolution relative to mutation rates, incomplete lineage sorting, and recent pseudogene formation.     

Nuclear plasmids in the simple eukaryotes Saccharomyces cerevisiae and Dictyostelium discoideum

High copy number nuclear plasmids are becoming recognized as common genetic components of simple eukaryotes. Like bacterial plasmids, eukaryotic plasmids ensure their persistence in dividing cells by having a partitioning system and a regulated means of amplifying copy number to correct inherent fluctuations in partitioning. By virtue of their small size and autonomy from the chromosomes, eukaryotic plasmids are useful for studying not only features of eukaryotic replicons but many aspects of gene regulation and DNA organization in nucleated cells.     

Serpin genes AtSRP2 and AtSRP3 are required for normal growth sensitivity to a DNA alkylating agent in Arabidopsis

Background  

The complex responses of plants to DNA damage are incompletely understood and the role of members of the serpin protein family has not been investigated. Serpins are functionally diverse but structurally conserved proteins found in all three domains of life. In animals, most serpins have regulatory functions through potent, irreversible inhibition of specific serine or cysteine proteinases via a unique suicide-substrate mechanism. Plant serpins are also potent proteinase inhibitors, but their physiological roles are largely unknown.     

石刁柏雄性偏向核质体DNA的克隆与分析

该研究以雌雄异株植物石刁柏为材料,利用基因组消减杂交技术对石刁柏雌雄核基因组中的性别差异核质体DNA（nuclear plastid DNA,NUPTs）进行了分离和分析。结果表明：（1）通过构建消减杂交文库共获得了52个雄性偏向序列,序列长度分布在63~297 bp之间,其中有19个差异序列属于叶绿体来源序列（命名为Ao1~Ao19）,且这些序列与石刁柏叶绿体基因组的相似性均大于84%,Ao19与石刁柏叶绿体基因组相似性为100%。（2）利用基因组半定量PCR对19个NUPTs序列的性别差异分析表明,有4条序列为稳定的雄性偏向NUPTs序列,分别为Ao1、Ao3、Ao10和Ao18。（3）序列比对表明,转移到核基因组的NUPTs主要来源于叶绿体基因组的反向重复区（包含IRa和IRb区）,说明石刁柏叶绿体基因组重复区序列更容易向核基因组进行转移形成雄性偏向的NUPTs序列。     

Nuclear reconstitution of demembranatedOrychophragmus violaceus sperm inXenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon- densation and pronuclear formation from demembranated plant (Orychophragmus violaceus)sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.     

Detection of Ehrlichia and Anaplasma DNA in Ixodes (Exopalpiger) trianguliceps Bir. ticks in Tyumen Province

This paper presents data of study (PCR with fluorescent hybridization probes) of Ixodes (Exopalpiger) trianguliceps Bir. collected from small mammals in the southern taiga forests of Tyumen Province. DNA of Ehrlichia and Anaplasma was detected in ticks of this species for the first time. Cases of mixed infection were also observed.