Analysis Of Internal Eliminated Sequences Developmentally Excised From Icronucleus In Ciliated Protozoan

Ciliates contain two types of nuclei:a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes.The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei,and a new macronucleus develops from a new diploid micronucleus.The old macronucleus is destroyed.This conversion consists of amplification,elimination, fragmentation,and splicing of internal eliminated sequences(IES) on a massive scale.In order to understand {he molecular basis of this nuclear differentiation process,we analyse a number of IESs which have been found from Paramecium tetraurelia,Tetrahymena thermophila and Euplotes octocorinatus.The results provide more evidence for origin and evolution of IESs.We contrast 43 IESs from GenBank database.Results suggest that IESs may be the products of a great deal of point mutation in chromosome.The mutation induced a lot of A/T and excision of IESs.In order to find some new IESs,we cloned 4 genes,including Centrin,β-Tubulin,eRF1and eRF3,from the micronuclei of Euplotes octocorinatus in accordance with the 4 genes macronuclei sequences.But the results presented that there is no IES in their micronuclei genes. We also cloned a new Rab gene from the micronuclei and macronuclei. The rab gene from macronucleic DNA was 827 bp in length,including non-coding regions and telomeric sequences at both ends.The open reading frame was 633 bp in length.The rab gene had four in-frame TGAs encoding cysteine in E.octocarinatus.The rab gene use TAG as stop codon.The result of BLAST in NCBI indicated that the rab shares a homology of 45%-54%at the amino acid level with rab8 proteins from a number of other eukaryotes, suggesting the rab gene was a rab8 homolog.The rab gene was therefore designated Eorab8.But the contrasted results suggest that the Eorab8 has no IES in its micronucleic DNA.