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BMP5748: Molecular Biology of parasites Structure of the course.

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1 BMP5748: Molecular Biology of parasites Structure of the course

2 23.9. General intro: DNA replication (Chapter Genes 10) Classic paper: Replication of kinetoplast DNA maxicircles. Hajduk, Klein, Englund, Cell Volume 36, Issue 2, p483–492, February 1984 / Identification of ORC1/CDC6-interacting factors in Trypanosoma brucei reveals critical features of origin recognition complex architecture. PLoS One. 2012;7(3):e32674. doi: 10.1371/journal.pone.0032674. Epub 2012 Mar 8. 25.9. Introducing mutations during replication: DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families. Nucleic Acids Res. 2014 Feb;42(4):2270-81. doi: 10.1093/nar/gkt1174. Epub 2013 Nov 18.Nucleic Acids Res. How to find startpoints of replication in the apicoplast: Multiple replication origins within the inverted repeat region of the Plasmodium falciparum apicoplast genome are differentially activated. Mol Biochem Parasitol. 2005 Jan;139(1):99-106.Mol Biochem Parasitol. 30.9. What to do with chromosome ends: Telomerases background Intro, Articles: The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus. Nucleic Acids Res. 2005 Feb 18;33(3):1111-22. Telomere length affects the frequency and mechanism of antigenic variation in Trypanosoma brucei. PLoS Pathog. 2012;8(8): e1002900. doi: 10.1371/journal.ppat.1002900.Nucleic Acids Res.PLoS Pathog. 2.10. Genomics: Working with genome databases: TriTryp e PlasmoDB. Chararcterizing genetic traits without whole genome sequencing: Optimized Multilocus Sequence Typing (MLST) Scheme for Trypanosoma cruzi. PLoS Negl Trop Dis. 2014 Aug 28;8(8):e3117PLoS Negl Trop Dis.

3 7.10. Transcription/splicing: General intro (Genes 10) The basics: Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing. Cell. 1986 Nov 21;47(4):517-25 RNA decay: Transcriptome-wide analysis of trypanosome mRNA decay reveals complex degradation kinetics and suggests a role for co-transcriptional degradation in determining mRNA levels. Mol Microbiol. 2014 Aug 22. doi: 10.1111/mmi.12764.Cell.Mol Microbiol. 9.10. RNA editing: The classics: Major transcript of the frameshifted cox2 gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. Cell 46, 819-826 (1986) RNA interference analyses suggest a transcript- specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei. J Biol Chem. 2005 Jan 28;280(4):2429-38.J Biol Chem. 14.10. Control is essential: variant gene expression in protozoans Review for Trypanosoma brucei :Antigenic variation in African trypanosomes. Mol Biochem Parasitol. 2014 Jul;195(2):123-129. Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature. 2008 Dec 11;456(7223):750-4. Strand- specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts in Plasmodium falciparum. BMC Genomics. 2014 Feb 22;15:150.Nature.

4 16.10. Novel tools for parasite manipulation: Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol. 2014 Aug;32(8):819-21. Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One. 2014 Jun 27;9(6):e100450. Inducible knockdown of Plasmodium gene expression using the glmS ribozyme. PLoS One. 2013 Aug 30;8(8):e73783. 21.10. final exam During the course: Small cloning tasks as self training in basic procedures “homework”

5 Structure of the presentations: -You work in teams of two, presenting groups are randomly chosen at each data* - Introductions (given in red letters) are presented by volunteers (who don´t have to prepare the paper seminars) - The group that presented one paper will not be presenting another on the same day - Imagine you did the study: “You sell the fish” - Tell us why the study was done – what´s the goal of the study - Go into details of the methods (watch your colleagues are there many “??“ in their faces?) - Get the background (especially new papers) if the message cannot be understood from the paper itself - Use the pics form the paper but add other figures from other sources if this helps to explain the effects/results * Unprepared presentations/absence discounts one point from max 10 of the final grade


7 - discover the mechanism of allelic exclusion in in var gene transcription in P. falciparum - This is done by measuring var gene transcription events in subnuclear level Aim of the study:

8 Metodos Plasmideos de transfecção Real time PCR FISH RNA-FISH Cy5 núcleo Microscopia confocal


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