Analysis of alternatively spliced CrXrn1-GFP reporter gene mRNAs in the green alga Chlamydomonas reinhardtii
Mentor 1
David Higgs
Location
Union Wisconsin Room
Start Date
24-4-2015 2:30 PM
End Date
24-4-2015 3:45 PM
Description
All organisms depend on effective regulation of gene expression to coordinate growth and development. In photosynthetic eukaryotes, such as plants and algae, the regulation of chloroplast gene expression is highly controlled. Nucleus-encoded proteins help to regulate photosynthesis in the chloroplast in response to developmental cues and the environment. In the single-celled green alga Chlamydomonas reinhardtii, a 5’ to 3’exoribonucleolytic activity has been shown to be part of a normal mechanism for the degradation of some chloroplast mRNAs and may help regulate photosynthesis. All eukaryotic organisms appear to have 5’ to 3’ exoribonucleases (Xrns) that are involved in regulating gene expression through the degradation of mRNAs. Using sequence analysis of the nuclear genome of C. reinhardtii we identified at least three predicted Xrn genes, CrXrn1, CrXrn2, and CrXrn3. The CrXrn1 is of particular interest as RT-PCR and sequence analysis indicate that this gene produces two mRNA splice variants: CrXrn1a and CrXrn1b, and the predicted protein sequence of CrXrn1b contains a putative N-terminal chloroplast targeting sequence. We are testing both CrXrn1a and CrXrn1b (CrXrn1a and CrXrn1b proteins, respectively) to determine the sub-cellular location of these proteins. For this, we developed a series of transgenic C. reinhardtii strains with different CrXrn1-GFP (green fluorescence protein) reporter genes. We have used confocal microscopy to determine the sub-cellular targeting of these different GFP fusion proteins, and these data suggest that CrXrn1b is targeted to the chloroplast, as predicted. The current focus of this study centers on analyses of the CrXrn1-GFP mRNAs in the transgenic strains. Using Northern Blot, RT-PCR and sequencing, we are investigating the size, sequence and relative abundance of the CrXrn1-GFP mRNAs to assess the alternative splicing and comparing the expression levels of the variants. Data and conclusions from these experiments will be presented and discussed.
Analysis of alternatively spliced CrXrn1-GFP reporter gene mRNAs in the green alga Chlamydomonas reinhardtii
Union Wisconsin Room
All organisms depend on effective regulation of gene expression to coordinate growth and development. In photosynthetic eukaryotes, such as plants and algae, the regulation of chloroplast gene expression is highly controlled. Nucleus-encoded proteins help to regulate photosynthesis in the chloroplast in response to developmental cues and the environment. In the single-celled green alga Chlamydomonas reinhardtii, a 5’ to 3’exoribonucleolytic activity has been shown to be part of a normal mechanism for the degradation of some chloroplast mRNAs and may help regulate photosynthesis. All eukaryotic organisms appear to have 5’ to 3’ exoribonucleases (Xrns) that are involved in regulating gene expression through the degradation of mRNAs. Using sequence analysis of the nuclear genome of C. reinhardtii we identified at least three predicted Xrn genes, CrXrn1, CrXrn2, and CrXrn3. The CrXrn1 is of particular interest as RT-PCR and sequence analysis indicate that this gene produces two mRNA splice variants: CrXrn1a and CrXrn1b, and the predicted protein sequence of CrXrn1b contains a putative N-terminal chloroplast targeting sequence. We are testing both CrXrn1a and CrXrn1b (CrXrn1a and CrXrn1b proteins, respectively) to determine the sub-cellular location of these proteins. For this, we developed a series of transgenic C. reinhardtii strains with different CrXrn1-GFP (green fluorescence protein) reporter genes. We have used confocal microscopy to determine the sub-cellular targeting of these different GFP fusion proteins, and these data suggest that CrXrn1b is targeted to the chloroplast, as predicted. The current focus of this study centers on analyses of the CrXrn1-GFP mRNAs in the transgenic strains. Using Northern Blot, RT-PCR and sequencing, we are investigating the size, sequence and relative abundance of the CrXrn1-GFP mRNAs to assess the alternative splicing and comparing the expression levels of the variants. Data and conclusions from these experiments will be presented and discussed.
