Molecular and cellular studies on endonuclear symbionts

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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.