Direct observation of histone H2B-YFP fusion proteins and transport of their mRNA between conjugating Paramecia

Abstract

Cytoplasmic exchange between conjugating cells of Paramecium caudatum has been implicated by mating experiments using wild-type and behavioral mutant cells. To observe macromolecular transport between mating cells, we cloned and expressed the P. caudatum histone H2B gene as a fusion protein attached to an enhanced yellow fluorescent protein (YFP) named PcVenus. Significant fluorescent signals derived from histone H2B–PcVenus were detected throughout the macro- and micronuclei of transformant cells after microinjection of the expression vector. The normal growth and high mating reactivity of the transformants indicated that H2B–PcVenus functioned normally. Seven hours after a transformant cell expressing histone H2B–PcVenus was mated with an untransformed complementary mating-type cell, fluorescence derived from histone H2B–PcVenus was emitted from the macronuclei of the untransformed cell. About 48 h later, the fluorescent signal was detected not only in the macro- and micronuclei of untransformed cells but also in the macronuclear anlagen of both mating cells. This suggests that conjugant cells share parental histones during meiosis and subsequent DNA rearrangement. Single-cell RT-PCR analysis demonstrated the presence of H2B–PcVenus mRNA in untransformed cells 15 and 24 h after conjugation. We concluded that at least the mRNA of histone H2B–PcVenus was transferred from the transformed, to the untransformed cell during conjugation.