Promoter

Question 1

indirect measurement for measuring mRNA levels?

Answer 1

reporter gene system–substitute gene with reporter gene;
endogenous activity of reporter gene should be low or absent in cells to eliminate background; detect expression of reporter genes or enzymes:
- Detection by measuring enzymatic activities of the reporter protein
- Detection with antibodies against reporters by ELISA (enzyme-linked immunosorbent assay) (also called enzyme immunoassay)

Question 2

what is CAT?

Answer 2

a reporter, chloramph acetyltransferase;
is good bc no endogenous activity;
detect with antibody
– Detection of CAT activity with labeled substrate
reaction products separated from substrate by thin-layer chromatography or diffusion into scintillation fluid (use of radioisotope)
– Detection of CAT activity by ELISA
-can also detect via enzymatic activity (acetylates Cam)

Question 3

describe detection of CAT via elisa

Answer 3

anti-CAT-coated micro plate; CAT binds; DIG-labelled anti-CAT antibody binds to bound CAT; anti-DIG POD Fab fragment conjugated to peroxidase; substrate ABTS is added, acted on by peroxidase
can do same thing but 2o Ab is fused to biotin which binds to streptavadin and HRP (substrate TMB)

Question 4

describe luciferase as a reporter

Answer 4

– No endogenous activity
– High specific activity
– Luciferin as a substrate –> light emission – ATP and O2 needed–measure light

Question 5

describe GUS as a reporter

Answer 5

bacterial beta-glucuronidase;
used for plant promoter analysis
quantitative analsysi: tag MUG and convert to MU via GUS, get lfuorescence

Question 6

describe reporter GFP

Answer 6

– No substrates required
– No endogenous activity
– Allows non-invasive means of monitoring gene expression in living cells
– Lack of amplification –> low sensitivity

Question 7

mapping regualtory elements–e.g. delete something, expression of reporter gene decreases, you’ve deleted an enhancer; expression increases, deleted a silencer

Answer 7

ya

Question 8

describe EMSA

Answer 8

DNA incubated with proteins; protein binds to its target site; chop up DNA with DNase I or chemical reagents; incubate the chpped up pieces on denaturing polyacrylamide gel

Question 9

describe DNA footprinting

Answer 9

incubate DNA with protein; degrade wth with cleavage reagents (DNase I or chemical reagents);

Question 10

• DNase I: enzymatic cleavage
– “nicks” dsDNA on each strand independently
– DNA containing A-T tracts may be more resistant to cleavage
– Bulky enzyme: unable to access nucleotides near the edge of the binding site –> larger “footprint”

Answer 10

ya

Question 11

• Chemicalreagents:chemicalcleavage
– Fe(II)-EDTA or 1,10-phenanthroline-Copper(I) + H2O2
– Cleavage is sequence-independent (attacks the deoxyribose moiety)
– Smaller “footprint” defines a tighter core of protein-DNA contacts–cuts very close

Answer 11

ya

Question 12

run a parallel sanger seq nexts to DNA footprinting lane–read sequence that is bound!

Answer 12

ya

Question 13

describe a ChIP assay

Answer 13

• Chromatin immunoprecipitation (ChIP) assay
– Cross-link DNA and proteins in vivo (e.g. with formaldehyde)
– Following cell lysis, chromatin is fragmented by sonication or enzyme digest
– Immunoprecipitate DNA-protein complex with antibody specific for DNA-binding protein
– DNA isolation
– DNA sequences analyzed by RT-PCR, real-time PCR or sequencing

Question 14

how to measure transcriptional activity?

Answer 14

• Direct measurement of mRNA levels – Northern blots
– RT-PCR
– Real-time PCR
• Indirect measurement
– Reporter gene system

Question 15

what are supershifts?

Answer 15

addition of specific antibodies to identify protein components in a complex