Cell cycle and DNA content of mitotic cells in brain ganglia ofdrosophila larvae

The programmes of replication of hetero- and euchromatin regions, mitotic cell cycle and the DNA content in metaphases in brain ganglia from late third instar larvae ofDrosophila melanogaster (wild type and a tumour bearing mutant, 1(2)gl, strain) and ofDrosophila nasuta were examined by autoradiography of [³H]thymidine labelled (continuous or pulse) cells and by cytophotometry, respectively. Brain ganglia labelled continuously with [³H]thymidine for 24 hin vitro showed a significantly high proportion of cells with incorporation of radioactivity restricted to heterochromatin only. Pulse labelling of brain ganglia from larvae ofDrosophila melanogaster andDrosophila nasuta followed by chase for different time intervals showed that (i) the frequency of labelled metaphases was more than 50% within 15 to 30 min of chase and remained higher than 50% in nearly all the chase samples till 24 h, (ii) euchromatin labelled metaphases appeared with a low frequency within 1 to 4 h chase period but the heterochromatin labelled metaphases continued to be more common in the later chase samples also, (iii) single chromatid labelled second cycle metaphases were seen within 1 to 4 h after the pulse, but their frequency did not increase in the later samples. Cytophotometry of feulgen-DNA and Hoechst 33258 stained metaphases in late third instar larval brain ganglia revealed a greater variation in the DNA content of individual metaphases, although the means were close to the expected 4 C content. It appears that in relation to the known asymmetric cell divisions of neuroblast and other neural cells, the mitotically active cells in brain ganglia comprise a heterogenous population with widely varying lengths of the different phases of cell cycle; some of them may not cycle regularly and may possibly have a discontinuous S-phase.     

Isolation of the Lembadion-factor,A morphogenetically active signal,that induces Euplotes cells to change from their ovoid form into a larger lateral winged morph

A morphogenetically active substance released by the predatory ciliate Lem-badion bullinum is recognized by ciliates of the genus Euplotes, which are potential prey organisms of Lembadion. The substance (L-factor) induces cells of the genus Euplotes to become less compact, which reduces their likelihood of becoming engulfed. Under the influence of this Lembadion- derived signal, E. octocarinatus develops extended wings and dorsal and ventral ridges and transforms within a few hours from its typical ovoid morph into an enlarged circular morph. This takes place without cell division. We have isolated the L-factor and report that it is a protein with a mass of 31,500 Da. The factor has been purified to chromatographic and electrophoretic homogeneity and was found to be active at concentrations as low as 10^?12 mol/L. © 1992 Wiley-Liss, Inc.     

Nucleolar apparatus in the macronucleus of Didinium nasutum (Ciliata): EM and 3D reconstruction

The three-dimensional (3D) organization of nucleoli in the somatic nuclei (macronuclei) of recently fed and starved Didinium nasutum was reconstructed on the basis of serial ultra-thin sections. It was shown that nucleoli, looking on the single sections like individual separate structures, appeared to be parts of the large complicated branchy nucleolar networks. A 30 h starvation did not lead to disintegration of this network, but stimulated formation of numerous vacuoles in the granular component of nucleoli, which becomes more condensed. Unlike starved D. nasutum, in fed ciliates numerous holes appeared in the fibrillar component located at the periphery of nucleoli. These holes may presumably serve as channels for transporting newly synthesized rRNA. To our knowledge, this is the first report of a 3D reconstruction of the nucleolar apparatus in ciliates.     

过表达EoRPL11下调HEK293T细胞中蛋白质的翻译活性(英文)

核糖体蛋白L11(RPL11)是真核生物核糖体的重要组成部分.RPL11参与核糖体的生物发生及其它的一些细胞调控过程.本研究在人细胞中研究了游仆虫RPL11(EoRPL11)的亚细胞定位及对蛋白质合成的调控功能.通过激光共聚焦显微镜观察发现,融合绿色荧光蛋白的EoRPL11分布于细胞核中,并集中于核仁上;将EoRPL11和海肾荧光素酶报告基因共转染HEK293T细胞后发现,细胞内海肾荧光素酶的酶活性明显下降,并呈现一种剂量依赖性关系;实时定量PCR分析则表明,海肾荧光素酶的mRNA水平并没有明显改变;同时,细胞的增殖也受到了一定的抑制.以上结果表明,EoRPL11是核蛋白,并且其过表达可能在翻译水平上抑制细胞内总蛋白质的合成.     

Teratogenic development in chicken embryos associated with a major deletion in the rRNA gene cluster

A new strain of chickens (mPNU) that segregates a severely deleted rDNA cluster was studied. Individuals heterozygous (+/p²) and homozygous (p²/p²) for the deletion were found to have 56 and 27%, respectively, of the normal complement of rRNA genes (290 copies/cell). Morphogenesis, cellular rRNA levels, and nucleolar sizes, were investigated and compared in normal +/+, +/p², and p²/p² embryos. Cellular rRNA contents were similar among the three genotypes at stage X, but subsequently during gastrulation, p²/p² levels were reduced to 56% and eventually to 43% of +/+. Viability and morphogenesis were normal in p²/p² embryos until the initial primitive streak stage of gastrulation. However, further development was abnormal and characterized by disrupted axis formation. In +/+ and +/p² embryos, rRNA levels and nucleolar sizes increased during early development; however, the profile of these increases differed temporally and quantitatively between the genotypes. The +/p² embryos, at the full streak stage of gastrulation, exhibited reduced rRNA levels and nucleolar sizes (80% of +/+), yet the +/p² embryos developed normally. These studies establish a minimum copy number requirement lower than previously demonstrated, that is, a rDNA genotype with only 56% of the normal gene complement (～160 genes) is compatible with early embryonic viability. Also, a rRNA threshold was detected: rRNA levels that were 56% of +/+ failed to support normal gastrulation; however, even under the circumstance of reduced rRNA levels (43% of control), some aspects of gastrulation apparently continue (cell migration and invagination). The teratogenic development of p²/p² embryos is a biological consequence unique from that found in other metazoan models of rDNA-deficiency, and will be useful as a model to investigate mechanisms of vertebrate gastrulation and axis formation.     

一种游仆虫皮层纤维结构的扫描电镜研究

应用扫描电镜研究了一种游仆虫皮层纤维结构。结果显示,口围带托架由横向组排在一起的小膜托架单元组成,每个小膜托架是由40-50 nm直径的纤维编织成的长方形薄片;波动膜托架是横向平行排列和纵向平行排列两部分共60多股纤维交错编织成的网状结构;额腹横棘毛区表膜下含有纵纤维层、球形纤维层和深部纤维层三层纤维,其纵纤维层在相应于游仆虫皮层脊（或脊和唇）之间的表膜下方被纵沟分成几部分;背面表膜下含有纵纤维,这层纤维下似乎也有球形纤维层。此外,作者也推测了这些皮层纤维结构在游仆虫皮层形态的保持、支持纤毛器和纤毛器运动及形态发生等方面的作用。     

Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.     

Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a parasite infecting the hypotrichous ciliate Euplotes woodruffi, with observations on microsporidian infections in ciliophora

A new microsporidian species, Euplotespora binucleata n. gen., n. sp., from the brackish-water ciliate Euplotes woodruffi is described and defined on the basis of life history characteristics, light and electron microscopic features, and small subunit (SSU) ribosomal DNA (rDNA) sequencing. The life cycle of E. binucleata n. sp. probably has rather short merogonic and relatively long sporogonic phases. Some uninuclear meronts and sporonts, along with diplokaryotic sporoblasts and spores, were found in experimentally infected host cells. Such a peculiar life cycle has been induced experimentally in Euplotes eurystomus and constitutively microsporidian-free stocks of E. woodruffi. Spores of E. binucleata n. sp. are monomorphic, ovoid-cylindrical in shape, 3.44+/-0.17 x 1.65+/-0.22 microm in size, and characterized by a diplokaryotic condition and a large posterior vacuole. The polar tube is isofilar, 4.5-5.5 microm in length when ejected, and lacking a distinctive coiled region (half-coiled). The polaroplast is divided into two regions: the anterior part has a few lamellae close to the anchoring disc; and the posterior part is a rounded body (sack), about one-quarter of the spore length. Spores do not appear to cluster together as a group. Each spore is surrounded by a sporophorous membrane closely adjacent to the exospore layer. A phylogenetic analysis of SSU rDNA sequences by different methods placed E. binucleata n. sp. in a clade with representatives of the microsporidian genera Cystosporogenes and Vittaforma. Observations of microsporidia in several other ciliates are discussed in view of the microsporidian infection frequency in the phylum Ciliophora.     

Expression and localization of VCX／Y proteins and their possible in-volvement in regulation of ribosome assembly during spermatogenesis

Variable Charge X/Y (VCX/Y) is a human testis-specific gene family that localized on X and Y chromo-somes. In this study, VCY protein was expressed in E. coli in the form of glutathione-S-transferase (GST)fusion protein. With the purified fusion protein as antigen, the anti-GST-VCY antibody was generated andthe localization of VCY protein in human testis was determined by immunohistochemistry. In the testisseminiferous epithelium, VCY proteins were highly expressed in nuclei of germ cells. Using propidium io-dide staining and green fluorescent protein (GFP) tag technologies, VCY and VCX-8r proteins were mainlylocalized in the nucleoli of COS7 cells. In addition, the colocalization for VCY and VCX-8r in COS7 cellswas also observed. With VCY cDNA as bait, a cDNA fragment of acidic ribosomal protein PO was obtainedusing yeast two-hybrid system. All the information above indicates that VCX/Y protein family might beinvolved in the regulation of ribosome assembly during spermatogenesis.