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Lecture

11-29-12.docx

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Department
Biology
Course Code
BIO-0013
Professor
Mc Vey

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11-29-12
Experiments to show role of enhancers
oRun northern blot with complementary probe for gene X
oRun another northern blot when enhancer is removed
oSee difference in the amount of RNA made
oDecreased RNA on Northern blot when enhancer is removed
Enhancer experiment
oStudied immunoglobulin heave chain/antibody gene found in B cells
oB cells make antibodies
oWanted to see if they could take enhancer elements out and still get RNA
for antibodies
oHuman gene for antibodies
Remove sections of introns, and find one that makes a difference
Use restriction enzyme
Human cell with normal gene—cell 1
Mouse cell without gene inserted—cell 2
Put intact gene in another mouse cell—cell 3
Put modified gene in another mouse cell (deleted intron)—cell4
oWhen northern blot is done
Cell 1 has high lvls of antibody mRNA
Cell 2 not detectable
Cell 3 high
Cell 4 not detectable
To regulate transcription of a gene, specific regulatory proteins need to bind to the
correct DNA sequences (enhancers, silencers, PPE)
How can you test if enhancers are binding sites for regulatory proteins that
activate transcription?
o
oCan a B-cell enhancer work on a RBC gene?
oB-Cell with just Beta-globin gene
oB-Cell with Beta-globin gene and B-cell enhancer
oOn northern blot

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Description
11­29­12 • Experiments to show role of enhancers o Run northern blot with complementary probe for gene X o Run another northern blot when enhancer is removed o See difference in the amount of RNA made o Decreased RNA on Northern blot when enhancer is removed • Enhancer experiment o Studied immunoglobulin heave chain/antibody gene found in B cells o B cells make antibodies o Wanted to see if they could take enhancer elements out and still get RNA  for antibodies o Human gene for antibodies  Remove sections of introns, and find one that makes a difference  Use restriction enzyme  Human cell with normal gene—cell 1  Mouse cell without gene inserted—cell 2  Put intact gene in another mouse cell—cell 3  Put modified gene in another mouse cell (deleted intron)—cell4 o When northern blot is done  Cell 1 has high lvls of antibody mRNA  Cell 2 not detectable  Cell 3 high  Cell 4 not detectable • To regulate transcription of a gene, specific regulatory proteins need to bind to the  correct DNA sequences (enhancers, silencers, PPE) • How can you test if enhancers are binding sites for regulatory proteins that  activate transcription? o o Can a B­cell enhancer work on a RBC gene? o B­Cell with just Beta­globin gene o B­Cell with Beta­globin gene and B­cell enhancer o On northern blot  Beta­globin RNA is only det
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