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Study of genome evolution analyzing the genetic code of Condylostoma magnum (Ciliophora)

Name: 
Valentina Serra
Team: 
Petroni - University of Pisa, Italy
Host: 
Nowacki - University of Bern, Switzerland
Date: 
Wednesday, February 4, 2015 to Friday, April 17, 2015
Report: 

Recently, one new and interesting genetic code, not only differing from the universal one but also from those previously discovered in ciliates, was found in Condylostoma magnum (Swart et al., in preparation), a ciliate belonging to Class Heterotrichea. In the genetic code of C. magnum UAA and UAG are translated in glutamine, UGA can be encoded as tryptophan, or infrequently as a selenocysteine, and only if a SECIS element is present, while both UAG and UGA are used as stop codons. It is thus inferred that C. magnum UAG and UGA codons might either be coding or non-coding, and thus the problem of how C. magnum's translation system can discriminate between sense and nonsense codons arises.

During my study period in Bern University I had the possibility to investigate this topic, learning some new molecular tools and giving my contribution to this research. Summaries of carried out activities are listed below.

1) MASS SPEC ANALYSIS

C. magnum proteins were extracted from >30.000 cells (5X protein loading buffer), then loaded on a 10% SDS page gel, stained and sent for Mass Spec analysis to the Mass Spectrometry and Proteomics Laboratory at the Children’s University Hospital in Bern. Data output were used to verify the incorporation of tryptophan at UGA codons, and to determine the rate of translational readthrough of stop codons.

2) tRNATrp VERIFICATION AND ANALYSIS

In order to find a possible modification from noncognate to cognate anticodons I used RT-PCR approaches to obtain sequences from two tRNATrp isoforms in C. magnum. cDNA was obtained using Superscript III Reverse Transcriptase Kit, (Invitrogen) from single cell or either from C. magnum RNA extraction. I also used specific adaptors ligated to the 3' and 5' ends of a different divergent candidate tRNATrp (T4 RNA Ligase 1, NEB ) to attempt to reverse transcribe and amplify this molecule from total RNA and to delimit its ends. All the products were cloned (pGEM-T Easy Vector, Promega; DH5-apha E. coli chemocompetent cells) and sequenced (Microsynth company). More than 80 clones were sequenced but only one showed anticodon modification (ACU) compatible with UGA codon. We decided that we should continue with high throughput sequencing to investigate these tRNAs further.

3) ACTIN-GFP CONSTRUCT FOR MICROINJECTION AND GENE EXPRESSION IN C. MAGNUM
I obtained a sequence of the 5' UTR and upstream region of the most highly expressed C. magnum actin paralog, by single cell PCR. This sequence was then ligated to a truncated GFP sequence that has previously been codon optimized for Paramecium tetraurelia (T4 DNA Ligase, Promega), to which I had added a 3' UTR plus poly(A) cleavage/addition site extension. This construct was selected by PCR and cloning, and will be tested for expression in the near future.

4) RNA EXTRACTION FOR tRNA SEQUENCING

We had some difficulty with RNA degradation initially, but finally were able to obtain RNA for tRNA sequencing. About 1000 cells of C. magnum were used for RNA extraction with miRNeasy Mini Kit (Quiagen). Extracted RNA has been sent for sequencing to Fasteris company and we are waiting for data output.

 

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