Transcript Replication of kinetoplast DNA maxicircles.

BMP5748: Molecular Biology of parasites
Structure of the course
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.
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.
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.
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):e3117
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.
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 transcriptspecific 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.
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. Strandspecific RNA-Seq reveals widespread and developmentally regulated transcription
of natural antisense transcripts in Plasmodium falciparum. BMC Genomics. 2014
Feb 22;15:150.
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”
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
Aim of the study:
- 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
Metodos
Plasmideos de transfecção
Real time PCR
FISH
RNA-FISH
Cy5
núcleo
Microscopia
confocal