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Enhancer Elements

-control transcription in time and space
-each tissue expression pattern driven by enhancer and enhancer can sit anywhere near gene
-more complex organisms have more enhancers
-generally individual enhancer controls expression of gene in single tissue
-tissue expressed in multiple tissues has several enhancers



-if deleted completely lose expression of that gene in tissue of interest



-boost expression of primary
-deleted, slight reduction in expression of gene of interest
-slight abnormal development of tissue



-influenced by environmental factors and stress


Locating Enhancers

-used to characterize expression pattern of gene
-need to generate set of transcriptional reporters that consist of individual genomic fragments, a minimal core promoter, and a reporter like bacterial lacZ, jellyfish GFP or firefly luciferase
-these constructs must be inserted into genome and reporter must be visualized in tissue of interest
-review slide


Using Fireflies to Measure Transcription Levels from Enhancer

-fireflies glow because of addition of oxygen to luciferin
-catalyzed by luciferase
-gene that encodes luciferase can be used in determining transcription levels of individual enhancers
-luciferase gene fused to minimal core promoter and enhancer element
-construct inserted into genome of living organism or transfected into immortalized cell line
-endogenously expressed transcriptional activators will bind to enhancer while TFIID will recruit RNA Pol II to Inr element
-activators release RNA Pol II from promoter thereby allowing it to transcribe luciferase gene


Strength of Activator

-strength of activator determines how many mRNA copies will be produced and this directly affects how much luciferase protein will be found in cell line/tissue


After RNA Pol II transcribes luciferase

-cells and/or tissue homogenized and luciferin substrate added to lysate
-reaction will glow and level of bioluminescence correlates directly to strength of transcriptional activators that are bound to enhancer element
-mutations that remove transcriptional activaotrs or alter the binding sites within the enhancers will eliminate luminescence


UAS/GAL4 Expression System

-GAL4 in yeast controls expression of number of genes
-self-contained transcription factor as it contains both DNA binding domain and activation domain
-binds to DNA and aids in phosphorylation of C-terminal tail of RNA Pol II
-binds to UAS


Manipulation of UAS/GAL4 System

-manipulated to direct expression of target genes to specific domains in many systems
-in order for system to work a transgenic fly is engineered to contain GAL4 gene fused to minimal core promoter and genomic enhancer
-in fly GAL4 gene transcribed in cells under control of enhancer element
-second transgenic fly engineered to contain target gene fused to minimal promoter and cluster of UAS sites
-fily will never express target gene since genome does not contain GAL4


Transgenic Fly Cross

-in order to direct expression of target gene to particular tissue or population the two transgenic flies must be crossed together
-progeny will contain both elements
-genomic enhancer will direct expression of GAL4 to specific domain
-within the domain, GAL4 binds to UAS sites and stimulates expression of target gene
-target gene can be transcriptional reporter or another developmental gene


Compound Eye

-development is dependent upon activity of 14 nuclear factors that form intricate regulatory network
-expression of any of these genes in non-retinal tissue is sufficient to induce formation of ectopic eyes


Ectopic Eye Expression

-used UAS/GAL4 system to forcibly express eyeless gene in developing wings and halteres
-eyeless is member of Pax6 class
-human Pax6 gene can induce eye development in flies and fly Eyeless gene can induce eye formation in a mouse
-tells a lot about evolutionary connections between organisms