HAU16008-04-2 - Gene Discovery and Gene Regulation Lecture...

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1 Gene Discovery and Gene Regulation Lecture 4, Thursday, October 30, 2008 Regulation of Transgene Expression Regulation of Transgene Expression Core Concepts : Position Effects DNA Methylation Transcriptional Silencing (TS) Post Transcriptional Gene Silencing (PTGS) RNAi (RNA interference) Co-suppression Epigenetic Regulation
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2 Section of an apple leaf disc infected with Agrobacterium Infecting Agrobacterium cells Leaf Disc Cut Edge Immature Anthers Embryogenic Callus Kanamycin Selection Germination GREEN HOUSE TESTING PCR Validation Transformation of Grapevine Aguero , Uratsu and Meredith T-DNA FATE Single Copies Multiple Copies Cis Mediated Variation Trans Mediated Variation -Al elic -Paramutation Unlinked Copies -Ectopic -Cocuppression Linked Copies Homology Dependent Gene Silencing Post-Transcriptional Gene Silencing Position Effects Position Effects DNA METHYLATION DNA METHYLATION T-DNA fate: Epigenetic mechanisms responsible for the variation and/or inactivation of transgene expression
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3 Position Effects: Variation in gene expression due to insertion site of T-DNA -Hetro vs Euchromatin -Proximity to other chromosomal structural regions, centromere, ARS (autonomous replication regions) etc -Interference due to transcriptional activity of other proximal genes -DNA methylation -Addition of buffer DNA does not work -MARS (matrix attachment regions) or SARS (scaffold attachment regions) can normalize transgene expression Analysis of DNA Methylation Methylated bases in DNA -5mC Methylation sensitive restriction endonucleases Inheritance of DNA Methylation Methylation and its propagation during replication Methylation imprinting – dominant and recessive traits/genes Genome restructuring Methylation and the regulation of transposable elements
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4 Homology Dependent Transgene Inactivation Gene-allele interactions -allelic/homologous/regional/paramutation -atopic/cosupression Epigenetic mechanisms -regulation of transcription -post transcriptional regulation Gene Silencing -expression of antisense -expression of sense or cosupression -self complimentary/double stranded RNA Hannon 2002 Double Stranded RNA DIRECT SDE1 – RNA dependent RNA Polymerase si-RNA Systemic Graft- Transmissible RNA interference HC-pro p25 Aberrant RNA INDIRECT Cmv2b VIGS: Virus Induced Gene Silencing TS: Transcriptional Silencing - DNA methylation RRF1 dsRNA Amplification mi-RNA Translation Arrest Antisense Sense + Antisense Self Compli-mentary Sense DNA Dicer Nuclease mRNA RNAi: RNA interference mRNA degradation R NA I nduced S ilencing C omplex RISC ? ? Mechanism that Regulate Gene Silencing in Plants AAAA protein mRNA DNA RNAi TS DICER and RISC (RNA-induced siencing complex): Hannon 2002 2 molecules of dicer bind and cleave RNAaseIII cleavage product 21 nucleotide product – siRNA Single strand of siRNA is incorporated into RISC RISC is a multicomponent nuclease Unwound siRNA guides mRNA selection Hairpin precursor RNA also cleaved by dicer Single stranded temporal RNA (stRNA) stRNA target translation
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This note was uploaded on 12/03/2010 for the course ECONOMY Eco 100 taught by Professor Taidat during the Spring '10 term at Hanoi University of Technology.

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HAU16008-04-2 - Gene Discovery and Gene Regulation Lecture...

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