Study Of The Programmed Translational Frameshifting Genes In Euplotes Octocarinatus

Programmed translational frameshifting is a phenomenon whereby the ribosome is guided toward a triplet nucleotide that is either shifted one nucleotide position downstream(-1 frameshift)or one nucleotide position upstream(+1 frameshift).Programmed ribosomal-1 frameshifting(PRF-1)has been recognized more than 25 years ago as a mode of translational control of specific genes,first in retroviruses and later in bacterial genes,it has also been reported for some eukaryotic genes from yeast to mammals.By far,PRF has been identified in a broad range of organism genes.According to statistics,among all of the organisms the Euplotes genes harbor a highest frequent frameshifting except virus.A recent analysis of gene sequences available in GenBank from ciliates in the genus Euplotes indicates that>5%required one or more +1 translational frameshifts to produce their predicted protein products.Several factors can influent the effect of frameshift,including SD-like sequences,low concentration of release factors,the secondary structures and so on.We recently analyse the transcriptome data of the Euplotes octocarinatus,which predicting that about 11.2%of the genes need one or more +1 frameshifting to produce their protein products,including protein kinase genes in the majority.To investigate these predicted frameshifting genes further,we used the dual luciferase report system in yeast which has been established to measure the frameshift efficiency of genes,then frameshift efficiency was measured in the presence of hybrid peptide chain release factor pBF001.1.Casein kinase ?(CK2)plays a vital role in cell cycle progression,cell proliferation and growth,cell survival and cell death.Analyzing the transcriptome of Euplotes octocarinatus,a ciliated protozoa,revealed seven putative CK2 encoding sequences,including four a subunit and three ?subunit sequences.From macronuclear DNA and cDNA these seven genes were amplified,indicating these genes were expressed in this organism.The genes were named as EoCK2a-1,EoCK2a-2,EoCK2a-3,EoCK2a-4,EoCK2?-1,EoCK2?-2 and EoCK2?-3,respectively.The deduced amino acid sequences showed that four a subunits contained the twelve conserved catalytic subdomains of protein kinases and some near universally-conserved amino acids residues that are characteristic of serine/threonine protein kinases.Sequence analysis suggested that expression of the EoCK2a-1,EoCK2a-2,EoCK2a-3 genes required a + 1 programmed translational frameshift for their translation.2.We used the dual luciferase report system in yeast to measure the frameshift efficiency of EoCK2a-1 after alignment analysis,EoCK2a-1 was inserted into a dual luciferase reporter vector.Meanwhile,plasmid shuffle procedure was used to screen strains YDB447/pDB967.The programmed frameshifting efficiency of EoCK2a-1 was measured in the presence of variant forms of eRF1.As a result,we conclude that EoCK2a-1 was a new frameshifting gene in the Euplotes octocarinatus.3.Recently,a+1 translational frameshift were also showed in MAPK1 genes from E.raikovi and E.nobilii.Then we found the MAPK1 gene of Euplotes octocarinatus needed two +1 frameshifting to express a complete protein.We also used the dual luciferase report system in yeast to measure the frameshift efficiency of MAPK1.As a result,we conclude that EoMAPK1 was a new frameshifting gene in the Euplotes octocarinatus.Therefore,we identified two new frameshifting genes in the Euplotes octocarinatus.These results provide experimental data for further studies of the molecular mechanisms of programmed translational frameshifting in ciliates.