Exam 4: Lecture 6 Flashcards Preview

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Cone Cells in Drosophila Retina

-each unit eye contains four lens secreting cells called cones
-different than cone photoreceptors
-secrete overlying lens


Lens of Drosophila

-made of roughly 30 protein layers
-main protein constituent of each layer is drosophila crystallin (dcy)
-homologous to crystallin proteins that make up human lens
-lens has different refractive index than surrounding air
-lens channels photons of light onto photoreceptors


Development of Insect Retina

-photoreceptor cells instruct four undifferentiated cells to adopt a cone cell fate
-process of cone cell recruitment is thought to occur via EGF Receptor Pathway and Notch Pathway
-these pathways activate D-Pax2 gene in cells destined to become cone cells


D-Pax2 gene

-key target of EGF Receptor Pathway and Notch Pathway
-required for formation of cone cells


Photoreceptor cells

-produce ligands that bind to membrane bound receptors on presumptive cone cell surface


Paired Box (Pax)

-founding member of Pax family of TF's is Drosophila Paired protein
-contains special binding domain called PAIRED domain
-over years number of similar proteins have been identified in all organisms
-different Pax proteins grouped into distinct classes based on protein structure


Pax1 and Pax9

-contain paired DNA binding domain and an eight amino acid octapeptide used in transcriptional repression


Pax 2, Pax5, and Pax8

-contain paired DNA binding domain, octapeptide and first helix of homeo DNA binding domain
-lacks second and third helices so these proteins do not interact with DNA through this motif
-Drosophila D-Pax2 belongs in this class


Pax4 and Pax6

-have intact paired and homeodomains
-can bind to DNA using both domains


D-Pax2 in Cone Cells

-in wild-type retina is expressed in just the four cone cells
-loss of D-Pax2 expression leads to complete loss of all cone cells and roughening of external surface of compound eye


Isolation of Rough Eye

-convenient way to screen for genes that affect eye development


Lab Induced Mutation

-some use x-rays which will cause deletions within genome
-others use chemicals like EMS to induce single base changes
-some use transposable elements to jump around genome and inserting themselves into genes
-mutations that eliminate expression of D-Pax2 within cone cells are located in eye-specific enhancer
-D-Pax2 also expressed in other tissues-but these flies only eye enhancer has been disrupted


D-Pax2 Eye Specific Enhancer

-approximately 500bp in length
-sufficient to drive expression of reporter construct just within cone cells
-contains binding sites for at least 4 proteins
-Pointed and Yan TF's are downstream members of EGF REceptor signaling cascade
-Su(H) DNA BP is most downstream member of Notch Pathway
-Lozenge is TF that is expressed in all cells of developing retina (not developmentally regulated)
-binding sites make up only small fraction of DNA found within this enhancer
-some sequences conserved in other Drosophila species and may represent binding sites for additional proteins


Sequences in D-Pax2

-some found solely in Drosophila melanogaster
-may simply be necessary for proper spacing between binding sites
-binding sites nearly always separated from each other since proteins that bind are large and bulky
-sites too close, DNA BP's will not have enough room to bind onto enhancer



-bound by DNA BP's.
-some contain single recognition sites for multiple TF's
-some contain multiple recognition sites for several different BP's
-transcriptional output dependent upon number of TF binding sites on enhancer



-review EGF Receptor Pathway
-review Notch Pathway


Activation of D-Pax2 Enhancer

-mutations that alter ability of Pointed (Pnt), Suppressor of Hairless (Su[H]) or Lozenge (Lz) to bind to D-Pax 2 eye enhancer results in complete loss of D-Pax2 expression within presumptive cone cells
-results in failure of cone cells to develop and structure of eye is disrupted (since lens is not secreted)



-combinatorial code of TF's required to activate expression of target genes
-Lz DNA BP as well as EGF Receptor and Notch Pathways are required to activate expression of D-Pax2 gene and specify cone cell fates
-loss of any of the factors will inactivate D-Pax2 and presumptive cone cells will either remain undifferentiated or will be transformed into photoreceptor cell


Complexity of D-Pax2 Enhancer

-within lies five sites for Su(H) binding, three sites for Lz binding and four sites for Pnt/Yan binding
-transcriptional reporter that preserves native spacing of these sites and nature of intervening sequences drives expression in all four cone cells
-construct in which the native spacing has been preserved, but sequence has been mutated fails to drive expression of reporter
-likewise, construct that eliminates intervening sequences also fails to drive expression of reporter
-results imply that DNA bases that lie between Lz, Pnt/Yan, and Su(H) sites are important for D-Pax2 expression in eye


Order of Site

-also important
-scrambling the order while preserving the correct spacing is insufficient to maintain proper D-Pax2 enhancer expression


Intervening Sequences

-important or just a subset?
-deleting or changing individual regions can be used to test requirements for each intervening segment


Dissecting D-Pax2 Enhancer

-individual intervening segments either deleted or altered
-resulting modified enhancers are assayed for ability to drive expression of reporter construct in cone cells
-wild type construct used as control for comparison
-most intervening sequences are required for proper expression
-exceptions: deletions of 3rd intervening sequence (no effect)
-deletion of 5th intervening sequence leads to up-regulation of D-Pax2 expression
-suggests that during normal development this region is bound by a transcriptional repressor-in its absence the enhancer has increased activity


Pax 3 and 7

-have all 3 motifs
-paired and homeodomains and octapeptide