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Pgm like genes in Paramecium

Simran Bhullar
Eric Meyer - ENS Paris, FR
Mariusz Nowacki - University of Bern, CH
Thursday, February 20, 2014 to Friday, February 28, 2014

I was working as a Post-doctoral Fellow in Prof. Mariusz Nowacki’s laboratory at University of Bern, before joining Prof. Eric Meyer’s laboratory in Paris.

During my stay in Mariusz’s lab, I was working on a project wherein we had identified nine genes similar to the domesticated piggybac transposase, PiggyMac (Pgm). All nine PiggyMac-like genes (named Pgml1 till Pgml9) show conserved transposase domain and are significantly up-regulated during new MAC development. To investigate their role in the genome rearrangement, RNAi-mediated gene silencing was carried out for the single genes as well as for their ohnolog pairs. Three of these gave lethal phenotype and also showed impairment in IES excision as reflected by the retention of the most IESs tested by PCR. All but one of the remaining six showed variable degree of retention of the different IES tested. We hypothesize that these transposases might work either in conjunction with Pgm or on their own depending on their catalytic abilities. Deep sequencing of the zygotic MAC DNA obtained from the cells depleted in those transposases that give lethal phenotype, showed retention of almost all the IESs (Pgml2), while significant number of IESs was retained in other two sequenced genomes.

This project had been carried out extensively in Bern, but some of the planned experimental part was left. For the overall picture of the effect of these genes, we are keen to see if the genome defense smallRNAs (scnRNAs, and the recently discovered iesRNAs) produced in the cells depleted in Pgml1, Pgml2 and Pgml8 proteins are different from those of the control cells.

The purpose of the STSM in Mariusz’s lab was to complete this part.

The technical procedure in the detection of the small RNAs, involved the  5'-end-labelling of the total RNA, with [γ- 32P] by the exchange reaction of T4 polynucleotide kinase, denaturation and running on the 15% polyacrylamide-urea gel. The RNA was collected from vegetative samples and from four different time points of autogamy from the cultures submitted to dsRNA-induced silencing of the Pgml1Pgml2Pgml8 gene. I did the gel twice with different RNA concentration, but I can not draw any conclusion. To address this question, I will have to repeat the gels.

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