Molecular and cellular studies on endonuclear symbionts

1.草履虫消化液泡内pH的酸化是由液泡膜上的一个质子颗粒(V-ATPase)引起的，这种酸化是全孢子菌逃离液泡所不可缺少的现象。2.钝顶冬孢子菌的繁殖形态不仅在低温(4ﾟC)下促进寄主细胞的分裂，而且在25ﾟC中也促进宿主细胞的分裂。3.当繁殖形态分化为具有感染性的长体时，大量的细胞壁抗原(IR-4-1抗原)被释放出来，或感染体不断地脱落抗原，脱落的抗原附着在大核包膜上，使包膜无法通过大核膜内的免疫球蛋白等大分子。这可能是宿主死亡的一个原因，当大核充满了传染性的长形式。无IR-4-1抗原的突变株即使在宿主的大核中充满了感染性形式也不能诱导宿主死亡。4.克隆了钝顶螺旋体中的应激蛋白基因groes和groEL同源物。5.纯化了钝顶螺旋体感染型特异的4.5K蛋白，并对其氨基酸序列进行了分析，并克隆了该基因。免疫电子显微镜和多克隆抗体显示抗原定位于周质。6.间接免疫荧光显微镜和抗草履虫尾部肌动蛋白抗体免疫印迹分析表明，钝顶螺旋藻含有一种带有抗体表位的蛋白质，采用脉冲场凝胶电泳法(PFGE)，波纹草履虫、钝顶螺旋藻和直肠草履虫的基因组大小分别为1800、1740和1800kBP。

1.Acidification of inside pH of Paramecium digestive vacuole is induced by a proton pomp (V-ATPase) of the vacuole membrane, and the acidification is indispensble phenomenon for Holospora's escape from the vacuole.2.Reproductive forms of Holospora obtusa promotes the host cell division not only in low temperature (4ﾟC) but also in 25ﾟC.3.When the reproductive forms differentiate to the infectious long forms, large amounts of cell-wall antigen (IR-4-1 antigen) are released, or the infectious forms are continuously shedding the antigens, and the shedded antigens stick to the macronuclear envelope and make the envelope unable to pass large molecules such as immunoglobulins inside the macronuclear envelope. This may be a cause for the host death when the macronucleus is filled with the infectious long forms. A mutant strain without the IR-4-1 antigen could not induce the host death even if the host macronucleus was filled with the infectious forms.4.Genes of stress proteins, groES and groEL homolog, were cloned in H.obtusa.5.Infectious form-specific 4.5 K protein of H.obtusa was purified and aminoacid sequences was clarified in the total length, and also the gene was cloned. Immunoelectron microscopy with a polyclonal antibody showed that the antigen was localized in the periplasm. Northern blot analysis using the cDNA showed that transcriptio was carried out in intermediate form of the bacterium which appeared in differentiation from the reproductive to the infectious form.6.Indirect immunofluorescence microscopy and immunoblot with antibodies against Paramecium caudatum actin showed that H.obtusa had a protein with an epitope for the antibody.Using pulse field gel electrophoresis (PFGE), is was found that genome sizes of H.undulata, H.obtusa and H.recta were 1800,1740 and 1800 kbp respectively.