Direct observation of histone H2B-YFP fusion proteins and transport of their mRNA between conjugating Paramecia.
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.
Takenaka
Yasuhiro
Y
Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
Yanagi
Akira
A
Masuda
Hiromi
H
Mitsui
Youji
Y
Mizuno
Hiroshi
H
Haga
Nobuyuki
N
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D016054:DNA, Protozoan / Q000235:genetics; D017125:Genes, Protozoan; D005822:Genetic Vectors; D006657:Histones / Q000235:genetics* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D048488:Paramecium caudatum / Q000235:genetics* / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D016054:DNA, Protozoan; D006657:Histones; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria