Development of a reporter gene construct that can be used for the localization of RNA pyrophosphohydrolase (rppH) in cells of Chlamydomonas reinhardtii cells.

Abstract

Abstract

The concept that messenger RNA (mRNA) degradation in E. coli begins with endonucleolytic cleavage has been challenged by the recent discovery that the conversion of the 5’ terminus from a triphosphate to a monophosphate is required prior to endonucleolytic activity. RNA pyrophosphohydrolase (RppH) initiates the degradation of transcripts by removing pyrophosphate from the 5'-end of mRNAs which allows binding of RNase E/ RNase J in bacteria. A putative RNA pyrophosphorylase is present in cells of the unicellular green alga Chlamydomonas reinhardtii and several lines of evidence suggest that the protein is involved in mRNA degradation in the chloroplast. In order to determine the location of the protein in Chlamydomonas cells, the 5’ region of the rppH gene was tagged to a codon optimized green fluorescent protein and introduced into Chlamydomonas cells. This study focuses on developing a reporter vector construct that can be used to transform the Chlamydomonas reinhardtii nuclear genome. GFP (Zsgreen 1) was codon optimized by using a Codon Usage Database. The reading frame of the optimized synthetic GFP was adjusted and cloned into the pBluescript-5’rppH SK+ vector. The 5’rppH-GFP gene fragment was then sequentially cloned into the pE coli-Cterm 6HN protein expression vector, intermediate vector and finally in the pChlami transformation vector. All constructs were verified by restriction cutting and sequencing. Transformants were screened for the presence of the chimeric GFP gene by PCR. The screening experiments have led us to choose one transformant that could be used for further work. In addition although fluorescence was not checked, the optimized synthetic GFP was expressed in E. coli (BL21DE3) cells confirming the functionality of the synthetic GFP construct in vivo.