Exam 4: Lecture 7 Flashcards
1
Q
Enhancer Elements
A
- 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
2
Q
Primary
A
-if deleted completely lose expression of that gene in tissue of interest
3
Q
Secondary
A
- boost expression of primary
- deleted, slight reduction in expression of gene of interest
- slight abnormal development of tissue
4
Q
Shadow
A
- rare
- influenced by environmental factors and stress
5
Q
Locating Enhancers
A
- 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
6
Q
Using Fireflies to Measure Transcription Levels from Enhancer
A
- 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
7
Q
Strength of Activator
A
-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
8
Q
After RNA Pol II transcribes luciferase
A
- 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
9
Q
UAS/GAL4 Expression System
A
- 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
10
Q
Manipulation of UAS/GAL4 System
A
- 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
11
Q
Transgenic Fly Cross
A
- 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
12
Q
Compound Eye
A
- 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
13
Q
Ectopic Eye Expression
A
- 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
