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

Gene. 2007 Jun 15;395(1-2):108-15. doi: 10.1016/j.gene.2007.02.013. Epub 2007 Feb 28.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Biological Transport, Active
  • Cloning, Molecular
  • Conjugation, Genetic
  • DNA, Protozoan / genetics
  • Genes, Protozoan
  • Genetic Vectors
  • Histones / genetics*
  • Histones / metabolism*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Paramecium caudatum / genetics*
  • Paramecium caudatum / metabolism*
  • Protozoan Proteins / genetics*
  • Protozoan Proteins / metabolism*
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism*
  • RNA, Protozoan / genetics*
  • RNA, Protozoan / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid

Substances

  • Bacterial Proteins
  • DNA, Protozoan
  • Histones
  • Luminescent Proteins
  • Protozoan Proteins
  • RNA, Messenger
  • RNA, Protozoan
  • Recombinant Fusion Proteins
  • yellow fluorescent protein, Bacteria