MCB 317 FINAL EXAM 2025/2026
QUESTIONS AND ANSWERS 100% PASS
Discovering eukaryotic promoter - ANS a series of deletional analysis required
What two requirements for mutational analysis of transcription control regions (promoter) do
we need? - ANS a systemic way to make mutants
a quantitative way to assay for transcription
deletion analysis - ANS systemic way to used to define borders of transcriptional control.
sequence between last functional txn and no txn is the border.
How did we make delusion mutants? - ANS PCR with two restriction enzyme sites
Make two primers specific to genes, on one end add a sequence that corresponds to one
restriction enzyme(HIND), and the other a different restriction enzyme (BAM) to the other end
and amplify
linker scanner mutagenisis - ANS a systemic way to determine 10bp necessary for txn
Boxes indicate a place where we've lost transcription, so we need that DNA.
if we lost transcription with 3 substitutions, our promoter needs those 3 elements
1 @COPYRIGHT 2025/2026 ALLRIGHTS RESERVED
,construction of linker scanner mutant - ANS confusing image of take a 3' deletion and a 5'
deletion mutant and clone them together which will change the 6-8 base pairs that make up
the resctriction site. we alter sequence by substitution using restriction enzyme
in linker scanner, what codes for the 6-8 base pair substitution? - ANS the substitution is a
restriction enzyme site of Zho, so when using PCR, we use 2 different restriction enzyme like
bam and hind
site directed mutagenesis allows us to.... - ANS make single base substitution in order to see
if particular nucleotides are required for txn
steps of sire directed mutagenesis: - ANS 1. isolate a plasmid DNA from bacteria with your
gene that is methylated and denature strands
2.make primers with one base pair mismatch
3. add mutagenic oligonucleotide primers with one mismatch each and angel, now the 2
plasmids are hemimethylated
4.digest the plasmids with an "oddball" restriction enzyme that cleaves only methylated DNA
5. transform into bacteria...we now have clones with one mismatch
why does bacteria methylate DNA? - ANS to recognize viral DNA and cleave it up if it is not
methylated.
do most restriction enzymes cut hemimethylated DNA? - ANS no because they keep to
protect DNA when they replicate
summary of finding promoters: - ANS deletion analysis: border
linker scanner: subregions
site directed mutagenisis: individual nucleotides
mutational/genetic analysis of DNA can be used to study: - ANS any DNA sequence-
dependent process
2 @COPYRIGHT 2025/2026 ALLRIGHTS RESERVED
, promoters, enhancers, origins of rep ORF, CEN, TEL, a
how did we quantify transcription? - ANS in vitro assays: cell extract with no other DNA,
radioactive nucleotides, and cloned DNA, run gel, quantify radioactive RNA and determined txn
efficient by radioactive RNA product...
the problem: DNA polymerase will run until it falls off, so we can't determine where promoter is
from the size of RNA
how did we determine where the promoter STARTS/ make bands of defined sites. -
ANS using linear DNA to get an RNA of defined length. so if RNA was 30 nucleotides, then we
can figure out 30 nucleotides back where promoter began.
what did the initial result of systemic (mutants) and quantification (northern blot) of RNA tell
us? - ANS promoters are sufficient for transcription. (which is false, but that's what we
believed at time)
most control regions: - ANS most control regions are not ESSENTIAL/ABSOLUTE to
transcription, only for its "basal" level of activity
response element: - ANS a DNA sequence that binds a transcription factor or protein
virtually every DNA sequence regulating any process are binding sites for proteins - ANS DNA
has no function other then that.
do all genes have TATA box? - ANS no. some lack certain elements, different promoters can
be made of different combinations of elements. no element is essential in the sense that they
are found at all promoters
is any element essential? - ANS no, there are promoters that lack certain elements. no
element found at all promoters
3 @COPYRIGHT 2025/2026 ALLRIGHTS RESERVED
QUESTIONS AND ANSWERS 100% PASS
Discovering eukaryotic promoter - ANS a series of deletional analysis required
What two requirements for mutational analysis of transcription control regions (promoter) do
we need? - ANS a systemic way to make mutants
a quantitative way to assay for transcription
deletion analysis - ANS systemic way to used to define borders of transcriptional control.
sequence between last functional txn and no txn is the border.
How did we make delusion mutants? - ANS PCR with two restriction enzyme sites
Make two primers specific to genes, on one end add a sequence that corresponds to one
restriction enzyme(HIND), and the other a different restriction enzyme (BAM) to the other end
and amplify
linker scanner mutagenisis - ANS a systemic way to determine 10bp necessary for txn
Boxes indicate a place where we've lost transcription, so we need that DNA.
if we lost transcription with 3 substitutions, our promoter needs those 3 elements
1 @COPYRIGHT 2025/2026 ALLRIGHTS RESERVED
,construction of linker scanner mutant - ANS confusing image of take a 3' deletion and a 5'
deletion mutant and clone them together which will change the 6-8 base pairs that make up
the resctriction site. we alter sequence by substitution using restriction enzyme
in linker scanner, what codes for the 6-8 base pair substitution? - ANS the substitution is a
restriction enzyme site of Zho, so when using PCR, we use 2 different restriction enzyme like
bam and hind
site directed mutagenesis allows us to.... - ANS make single base substitution in order to see
if particular nucleotides are required for txn
steps of sire directed mutagenesis: - ANS 1. isolate a plasmid DNA from bacteria with your
gene that is methylated and denature strands
2.make primers with one base pair mismatch
3. add mutagenic oligonucleotide primers with one mismatch each and angel, now the 2
plasmids are hemimethylated
4.digest the plasmids with an "oddball" restriction enzyme that cleaves only methylated DNA
5. transform into bacteria...we now have clones with one mismatch
why does bacteria methylate DNA? - ANS to recognize viral DNA and cleave it up if it is not
methylated.
do most restriction enzymes cut hemimethylated DNA? - ANS no because they keep to
protect DNA when they replicate
summary of finding promoters: - ANS deletion analysis: border
linker scanner: subregions
site directed mutagenisis: individual nucleotides
mutational/genetic analysis of DNA can be used to study: - ANS any DNA sequence-
dependent process
2 @COPYRIGHT 2025/2026 ALLRIGHTS RESERVED
, promoters, enhancers, origins of rep ORF, CEN, TEL, a
how did we quantify transcription? - ANS in vitro assays: cell extract with no other DNA,
radioactive nucleotides, and cloned DNA, run gel, quantify radioactive RNA and determined txn
efficient by radioactive RNA product...
the problem: DNA polymerase will run until it falls off, so we can't determine where promoter is
from the size of RNA
how did we determine where the promoter STARTS/ make bands of defined sites. -
ANS using linear DNA to get an RNA of defined length. so if RNA was 30 nucleotides, then we
can figure out 30 nucleotides back where promoter began.
what did the initial result of systemic (mutants) and quantification (northern blot) of RNA tell
us? - ANS promoters are sufficient for transcription. (which is false, but that's what we
believed at time)
most control regions: - ANS most control regions are not ESSENTIAL/ABSOLUTE to
transcription, only for its "basal" level of activity
response element: - ANS a DNA sequence that binds a transcription factor or protein
virtually every DNA sequence regulating any process are binding sites for proteins - ANS DNA
has no function other then that.
do all genes have TATA box? - ANS no. some lack certain elements, different promoters can
be made of different combinations of elements. no element is essential in the sense that they
are found at all promoters
is any element essential? - ANS no, there are promoters that lack certain elements. no
element found at all promoters
3 @COPYRIGHT 2025/2026 ALLRIGHTS RESERVED
