Gene Cloning

The Analysis of the Regulation of Gene Expression 363
Q11.7. You have a limiting amount of protein or cell extract which you use
in an EMSA or footprinting experiment which results in only 10% of the
fragments being bound by the protein. Would you be able to detect the
protein–DNA complex using either the EMSA or footprinting protocol?
A11.7. EMSA would still detect the small percentage of labeled DNA fragments
that have bound protein because you would see the appearance of a
shifted band. You would have difficulty detecting the bound protein using
footprinting protocols because the 90% of unbound fragments in the “+ protein”
lane would give rise to a ladder almost identical to the “– protein” lane.
Q11.8. Are laboratory-based techniques, such as deletion analysis, the only
approach to identify possible binding sites for transcription factors?
A11.8. No, you can also use a bioinformatics approach. It is possible that
your promoter or enhancer contains binding sites for known transcription
factors. Therefore, you can search the DNA sequence for potential target
sequences for known transcription factors. Although this approach may help
you identify potential regulators for your gene, experimental work is still
required to confirm that the transcription factors identified by bioinformatics
really are involved in regulation.
Q11.9. Which approach would you use to clone the genes for a transcription
factor that you suspect contains two different protein subunits and
where neither subunit can bind the DNA target on its own?
A11.9. The only approach that you can use is affinity purification, because
both the one-hybrid assay and an expression library screen a single clone in
each colony or plaque.
Q11.10. In a micro-array constructed with oligonucleotides, should the
oligonucleotide probes be sense or antisense, i.e. the same sequence as the
RNA or the template strand of the DNA?
A11.10. The probes are sense, i.e. have the same sequence as the RNA. This is
because the RNA is purified and then labeled cDNA, that is complementary
to the RNA, prepared. The oligonucleotides need to hybridize to the labeled
cDNA, so like the RNA, are complementary to the cDNA.
Q11.11. Could an oligonucleotide array be used to analyze the labeled RNA
from a nuclear run-on experiment?
A11.11. No, in the nuclear run-on experiment the RNA is directly labeled
and hybridized to the immobilized probe. The immobilized probe therefore
needs to be complementary to the RNA (see answer to Q11.10 for more
detail).
Q11.12. You are interested in the major differences in gene expression
between cells grown in two different conditions. Which approach