experimental approaches for gene control regions

Question 1

which technique do we use to identify important regions within gene control regions

Answer 1

linker scanning mutagenesis

Question 2

what is the purpose of linker scanning mutagenesis

Answer 2

to identify important regions within gene control regions

Question 3

explain the process of linker scanning mutagenesis

Answer 3

make a reporter gene construct containing a region upstream of the gene of interest fused to a reporter gene

transfect the construct into cultured cells

make a series of overlapping mutations, then observe reporter-gene expression

Question 4

in linker scanning mutagenesis, what result will show that a region is important within the control region

Answer 4

low level of expression will tell us that that region is important, because those are the parts being interfered with

Question 5

what important regions of control regions were identified via linker scanning mutagenesis

Answer 5

the TATA box and two other additional elements

Question 6

how do we determine whether a regulator is an activator or a repressor

Answer 6

by using in vivo transfection assay

Question 7

what is the purpose of in vivo transfection assay

Answer 7

to determine whether a regulator is an activator or a repressor

Question 8

describe the procedure for in vivo transfection assay

Answer 8

construct two plasmids: one has the gene, the other has the regulator binding site + a reporter gene

transfect the reporter-construct on its own to determine level of reporter gene expression

transfect both plasmids into cells and determine reporter-gene expression

Question 9

based on the results from in vivo transfection assay, how do we determine if a regulator is an activator or repressor

Answer 9

after putting both plasmids into a cell, if reporter-gene expression increases, it’s an activator

decrease=repressor