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Lecture 16

Lecture 16-November 11th

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Queen's University
BIOL 205
Kenton Ko

Nov 28/2011 – Lecture 16 1. Visualizing Gene expression In situ hybridization - Used to visualize mRNA transcripts - cDNA clone of a gene is transcribed to obtain a ssRNA probe - Embryos are incubated with RNA probe and any unbound probe is washed away - Enzyme conjugated antibodies are added and mRNA expression is visualized under light microscope - Bands of cells will be seen Immunolocalization - CDNA clone of a gene is expressed in bacteria to produce protein which is injected into ivertebrate host - Antibodies to protein are produced (igG) and this is incubated with the embryo Hox gene Expression - Fluorochrome conjugated antibody is added to igG of host species - Visualized under microscope Homeodomain - The final expression of Hox genes in adult depends on control of their expression early in embryogenesis - Hox proteins have a sequence in common, called ahomeodomainwhere these 60 amino acids are found in all homeotic genes and code for DNA binding proteins - This may have arisen by tandem gene duplication of an ancestral gene - This gives a helix-turn-helix motif (a-helices) common in DNA binding proteins, formed by helix 2 and 3 - This protein’s helix 3 contacts with bases in major groove and helix 1 attaches to minor groove - Hox genes regulate the identity of serially repeated structures in vertebrates - In wild type, you have 6 lumbar vertebrae - Hoxa11+/Hoxa11; Hoxd11-/Hoxd11- (double mutant), a sacral vertebrae is convered to lumbar Genetic Screens - Hoxa11-/Hoxa11; Hoxd11-/Hoxd11- has loss of 2 sacral vertebrae and E. Wieschaus, E. Lewis, C. Nusslein- lumbar vertebrae is repeated again. Volhard - In a maternally required gene, if the mom is mutated, she passes her ribosomal toolkit for the egg, which generates all mutant offspring even if they don’t have phenotype m/m (like a mitochondrial disease) - This is a screen to find what comes from mom that affects the embryo - If we look at zygotically required toolkit genes, offspring with m/m will be mutated because they don’t have the zygotic toolkit genes. Segmentation gene mutants 1. Gap mutants - Kruppel and Knirps has a missing gap in segments so that the head region is fused to lower region 2. Pair rule - Even skipped mutants have even segments taken out and odd skipped have odd segments taken out - Paired has even pairs of segments taken out - Runt has odd pairs of segments taken out 3. Segment polarity - Gooseberry reverses the posterior compartment in each segment and leaves Drosophila development the anterior part intact - Patched leaves the posterior part (back) intact and takes out anterior. - Maternal genes code for Bicoid and Nanos development o Bicoid: anterior protein o Nanos: posterior protein - These proteins are at highest expression at the 2 poles and diffuses towards the centre - They also control expression of gap genes and affect gap gene mutants (i.e. Kruppel, Knirps, Giant, Hunchback) which in turn affects  pair-rule genes  segment polarity genes  homeotic genes - On a fluorescent image, small dots represent bags of cells next to each other Gap genes being reprogrammed - The gap genes are activated by specific maternally provided proteins - When bicoid proteins are produced, they bind to hunchback 5’ cis-acting regulatory elements’ binding sites, which activates the reporter gene to express - However, with a mutated bicoid, fewer or none will bind and this can cause diminished or no expression - The bicoid gradient activates gene expression in a concentration dependent fashion, so if the gradient was in the anterior where it has a high [bicoid], then orthodenticle expression is high but if it was in the central embryo, where [bicoid] is low, then the orthodenticle sites (with low affinity) will not turn on the gene. - A combination of maternal-effect and gap proteins control individual pair- rule stripe formation. Hox Proteins and appendage o Even skipped is turned on when bicoid expression falls formation - Maternal effect genes creates a pattern which turns on genes which are transcription factors to make the gaps narrower. Q: What is derepression? Activating the operator again - Expression of gene distal-less (Dll) marks the start of development of appendages - On a fluorescence image, repressed Dll doesn’t give red fluorescence - In wild type, Dll is repressed from A1-A7 - The Ubxhox protein repress Dll but when Ubx is mutated, Dll becomes expressed in A1 - UbxAb-A dble mutated, Dll is expressed from A1-A7 - Other mutations include: 1. Slp (sloppy paired mutation) - Sloppy paired is a segment polarity gene that defines the anterior of each stripe - Thus in A1 to A7, the anterior would be expressed 2. En mutation (Engrailed) Q: Slp = activates anterior and En = - A mutation on the Cis-Regulatory element, defines posterior of each stripe activates posterior, why is it that the - Thus, A1-A7 will have the posterior expressing posterior is not expressed A: It’s a mistake it should be all 3. Slp, En mutations expressed - Causes complete expression with both anterior and posterior being expressed. Nov/ 30/2011 – Lecture 17 1. miRNAs - Mature microRNAs (miRNAs) are a class of naturally occurring, small non- coding RNA molecules, about 21–25 nucleotides in length. MicroRNAs are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression in a variety of manners, including translational repression, mRNA cleavage, and deadenylation - Thus, you can have few genes but many proteins How miRNAs are produced Q: what is the purpose of that bulge? - miRNA genes are transcribed by RNA pol 2 and binds to a promoter near DNA sequence that encodes the hairpin loop of pre-miRNA - miRNA encode no proteins, it folds up and creates a dsRNA in itself - This is capped, tailed and s
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