Lecture 26 Outline of Last Lecture I. RNA InterferenceA. The Mechanism and Function of RNA interferenceB. CRISPR SequencesC. Comparison and Application of RNAiII. TransposonsIII. TGS, miRNA, and lncRNA as RegulatorsIV. Vocabulary and Sample QuestionsOutline of Current Lecture I. Transposable ElementsII. McClintock Tranposon DiscoveryA. The Ac-Ds SystemB. Examples of transposable elementsIII. Genetics of DevelopmentA. 5 classes of Mutations affecting Drosophila Embryonic DevelopmentB. Maternal Effect and Hox GenesIV. Genetic Model Organisms for DevelopmentV. Genetic ScreensVI. Key Vocabulary and Sample QuestionsCurrent LectureI. Transposable ElementsTransposable Elements (TEs) are called jumping genes because they are genetic elements with the capacity to move from one chromosomal location to another (50bp – 10kb in size)Number of copies in a genome greatly varies: 1 – several hundred thousand copiesThey can cause mutationsTEs can insert into genes, changing sequence and phenotypeThey can result in abnormal recombinationThere are two classes of TEsTransposons: move via a DNA intermediateRetrotransposons: move via an RNA intermediateDNA Transposons can move using two mechanisms of transposition:1) ReplicativeTransposase enzyme copy + pastes the TE into a new locusBIOLOGY 305 1st Edition2) ConservativeTransposase cut + pastes the TE into a new locusRetrotransposons are always replicative (copy + paste)The original location is transcribed (RNA is produced)This RNA is translated, producing a protein called Reverse TranscriptaseThis protein catalyzes a copy of DNA using reverse transcriptionThis DNA is inserted in a new siteTypes of TEs:Bacteria – Tn transposonsYeast – Ty elementsDrosophila – P-elements, copia elementsHumans: LINEs and SINEs (long and short interspersed elements)II. McClintock Tranposon DiscoveryBarbara McClintock – found that genes would give rise to mottled phenotypes (corn that have spots)By crossing corn, she proposed that there are genes that can jump around in the genomeC gene encodes for color: The C+ gene is purple while the C- gene is whitec-/c- : all whiteC+/_: all purplecu/cu: white with purple spots (her notation: u means ‘unstable’ gene element)A. The Ac-Ds SystemA transposable element can insert itself into a gene, disrupt its function and cause a mutation. Mutation by movement of TEs are entirely different mutations mentioned beforeThe tranposase gene recognizes inverted repeats (IR) autonomous = functional transposase with IR presentNon-autonomous = transposase genes are mutated but IR is presentAc - activator: transposonDs – dissociation element: is an Ac element with non-functional transposaseTransposase are trans acting, IR are cis—actingTheir mechanism of transposition is conservative: Cut + Paste:1) AC: element on its own = transposon is able to move2) Ds: on its own = not able to move3) Ac + Ds = allows both able to moveTwo phenotypes caused by Ac/Ds elements1) Chromosome breakage near the centromere of chromosome 92) Inactivation of the C genecu alleles : u for unstable, C inactivated by Ac or DsReversion of cu to C+ : excision of Ac or DsDevelopment of a corn kernel with genotypes:cAc/cAccDs/cDs, AcThe zygote has unstable c on both zygotes, when it develops the transposon jumps out of the gene, and they all become wild-type = excision of Ds/Ac = purple phenotype:All of those that are white will have cu/cuWe know that white phenotype can become purple, what about when they are purple with white spots?It is technically possible: low probability of the transposon to jump out of the C locusc- is stable so there are no transposons (we don’t need to know how its mutated)Summary with wild background/Ac background:B. Examples of transposable elementsThey are often genomic parasites but this is not the whole story: they can affect how the genome evolves Transposons can take an enhancer and make it moveableThey can affect splicing, transcription termination, gene expression, produce RNA in two directions, and the chromatin regulation (often with RNAi):Ex: Transposons are responsible for red and white wineCabernet grapes have red color, which is disrupted by transposons being inserted in the promoterHas a ruby intermediate: terminal repeats are retainedOranges – gene important for pigment is turned off for orange grapesAn insertion of a retrotransposon turns on the geneCTR recombination occurs:III. Genetics of DevelopmentThe study of the role of genes in the formation of an organism: How can a mouse gene sequence for an eye inserted into a fly allow the fly to grow a regular compound eye?Drosophila are a model organism for development:Fertilized Egg  multinucleate  blastoderm  differentiaties  grastrulates segmentation in larvaeLewis/Volhard/Wieschaus: Discovered specific genes involved in drosophila development – conducted genetic screens for maternal and zygotic mutations affecting early embryonic developmentA. The 5 classes of Mutations affecting Drosophila Embryonic DevelopmentMutations: some are maternally affected, some are gap genes, some are pair-rule where they are missing ‘every other segment’, some polarity genes where only fragments of segments are missing, and homeotic gene mutations develops as if they are located elsewhere…(In order of precedence)Each class of genes regulates the expression of the next class: all of these are TFs or signaling moleculesMaternal effect genes: for recessive mutationsEmbryonic development begins earlier than zygotic transcription. Maternally supplied mRNA is the process behind maternal effect genes, this mRNA directs initial formation of the body planWhere is a gene expressed? What is the phenotype of the mutant?Mutations in a gene will affect parts of the body where a gene product is needed. localization of protein to body part = affected by mutantThus, we use genetic screens to identify a number of genes as important players in a given developmental process:B. Maternal Effect Genes and Homeotic GenesEx: Example of the Maternal Effect Gene in bcd Gradient of bicoid protein can be seen across organism’s AP axisBicoids (bcd) – a protein produced in mRNA that comes from the mother that are not spread across the whole embryo, while front part of the embryo carries the majority of it1) RNA in situ hybridization – detected a high concentration of mRNA in anterior end2) Immunolocalization: Using antibodies for the protein to detect