Construction The Yeast Two-Hybrid Library Of Euplotes Octocarinatus And Primarily Screen Proteins Interacting With Polypeptide Release Factors

The process of cellular protein synthesis is an open system which is regulated by interaction between outer elements and endogenous proteins factors to keep all cellular life cycles in order,meanwhile many proteins including some enzymes in cell regulate this process,which form a complicate protein network and a signal transduction pathway.The cDNA library of yeast two-hybrid is an effective way to screen the proteins that interact with the target protein.Using this method,many interacting proteins have been obtained for investigating the protein network and signal transduction pathway.The process of protein synthesis is composed of three steps:initiation, elongation and termination.The last process is regulated by two classes of polypeptide release factors,eRF1 and eRF3 in eukaryotes.The polypeptide release factors are multifunctional proteins,apart from protein synthesis, which are involved in regulation of cell cycle regulation,cytoskeleton organization and the tumorigenesis,especially in nonsense mediated mRNA decay pathway which occurs in a manner coupled to translation termination where normal and abnormal mRNA are metabolized.Euplotes octocarinatus is a kind of unicellular protozoa which has the special usage of codon determined:the codon of UGA encodes cysteine in Euplotes.Comparing another three typical Ciliates Paramecium,Stylonychia, Tetrahymena which deviate from the universal genetic codon by using the stop codons UAA and UGA to encode glutamine,it was found that poly(A)⁺RNA of the Euplotes was readily translated in vitro and yielded a great number of polypeptides with different molecular weights comparable to the data obtained with poly(A)⁺RNA of anther three ciliates.This result provides us the supported idea for constructing the cDNA library of yeast two hybrid.In this study,the total RNA of E.octocarinatus were isolated and the mRNA were reversely transcribed into ds cDNA for constructing the cDNA library of yeast two hybrid Two classes of polypeptide release factors were used as bait protein to screen the constructed library.Isolation of total RNA from E.octocarinatus cells.E.octocarinatus cells fed with Green algae were collected by filtering for getting ride of algae. The total RNA were extracted and analyzed.28s rRNA band in agarose gel and value of OD 260/280 indicated that obtained RNA could be used for next experiment.(1) Reverse transcription of mRNA.The mRNA were transcribed into ds cDNA by using the primers provided by BD Matchmaker^TM Library Contruction & Screening kit.Gel electrophoresis analysis of the products showed,comparing the human placenta cDNA,general size of synthesized cDNA were smaller.Further purification and amplification were conducted for obtaining high quality cDNA.(2) The ds cDNA analysis by PCR using the primers of known genes,i.e. eRF1,eRF3 and so on,indicated that the total cDNA putatively contain most of macronuclus genes of E.octocarinatus.(3) The library vector pGADT7-Rec were digested by restriction enzyme Sma I and cotransformed with confirmed cDNA into AH109 yeast strain. Homologous recombining ligation spontaneously occurs in the cell because both ends of cDNA and the vectors were flanked with same sequences.The titre of the library is 2.437×10⁷ cfu/mL.Accomplished library was analyzed by PCR again to confirme that E.octocarinatus genes have been inserted into the plasmid of pGADT7-Rec in the yeast cell.(4) Screen the library using classâ…¡polypeptide release factor as bait.Firstly, the bait plasmid pGBKT7-eRF3 was constructed and transformed into yeast strain Y187.Mating of AH109/pGADT7-library and Y187/pGBKT7-eRF3 was conducted,the mating percentage was more than 2%.The colonies were selected on nutrition minus plates,such as SD/-Leu-Trp-His for primary screen and SD/-Leu-Trp-His-Ade for restriction screen.200 colonies have been obtained from primary screen.22 of 200 colonies have been obtained on restrict plates.16 of 22 colonies were confirmed byβ-galactosidase filtration assay to be positive colonies.Plasmid were extracted from yeast cell and transformed into E.coli to obtain amplified amount of library plasmid containing target gene.After PCR and restriction enzyme digestion analysis, sequences were conducted.Sequence results demonstrated that #6 and #74 plasmids contain an unknown gene,respectively.The gene in #33 and #105 are homologous to ribosome RNA of Euplotes.The interaction between eRF3 and #6 unknown protein was confirmed again by using yeast two hybrid assay.The primers were designed according to the putative ORF sequence of #6 gene.Gene amplification from macronuclear DNA of Euplotes was done by using the designed primers and telomere primers.So far,a 900bp DNA fragment has been obtained.