Regulating Gene Expression Flashcards
In regards to the cDNA library problem, if one wants to express a eukaryotic protein in E. coli most eukaryotic genes will not work. Why?
The genes contain introns.
note: the way around this problem is to make cDNA using reverse trasncriptase
The first step in creating the cDNA library is isolating the total ___ from a cell
mRNAs
In the second step of the cDNA library, the mRNAs are converted to cDNA by reverse transcriptase and DNA polymerase I. Isolated mRNA serves as a ___ for copying the RNA into DNA
primer
This nicks the RNA, leaving segments as primer for DNA polymerase I
RNase H
This fills in the blanks, removes primers, and fills again
DNA pol I
This makes the DNA blunt ended
T4 poly
Adding linkers prepares the cDNA for ___ into a vector
cloning
How could one isolate a gene for a particular protein with an unknown nucleotide sequence?
synthesize an oligodeoxynucelotide probe which would be complimentary to one of the strands of the cloned gene
Which oligonucleotides should be used when making the probe?
sequences that contain AAs with the least degenerate codons (such Trp and Met; Asn and Lys have minimum wobble)
The Radioactive-antibody test. Describe process
- a plastic disk coated with anti-insulin antibody is touched to the surface of colonies on a petri dish
- insulin molecules from an insulin-producing colony bind to antibodies
- radioactivity adheres to the disk at the positions of insulin-producing colonies
PCR permits as high as ___ fold increase in the concentration of a single DNA molecule
10^10
This PCR technique produces a fingerprint of an organism that would allow identification of disease causing organisms. - With the set of short, random primers, different organisms will produce unique PCR products visualized by gel electrophoresis
RAPD analysis (random amplified polymorphic DNA)
In regards to use of PCR in genetic testing, ___ can be designed to detect specific ___ in affected patients
primers; mutations
PCR is so sensitive that only one or few copies of target DNA are required. True or false?
true
The DNA polymerase enzyme is isolated from ___ bacteria so that it is able to resist denaturation even in boiling water
thermophilic (heat-loving)
These are proteins which are found in relatively constant amount in cells. Their rates of synthesis and degradation are relatively constant in a given cell type regardless of the metabolic state of the cell
constitutive proteins
These are proteins which are usually found in low concentrations in a given cell type but whose concentration can be increased markedly in response to the proper stimulus
inducible proteins
True or false? eukaryotic cells contain much less DNA than is found in prokaryotic cells
false; contain much more
___ ____ are most important for regulation in eukaryotes
transcriptional activators
____ is an inhibitor of dihydrofolate reductase, an enzyme indirectly important to the synthesis of thymidine and thus DNA (anticancer drug)
Methotrexate
Cancer cells can develop resistance to methotrexate by increasing the number of genes for DHFR to 40 to 400 copies per cell. These extra copies recombine to somehow form ____ ____ chromosomes
double minute
note: these extrachromosomal elements are inherited in an unstable fashion
These are called “jumping genes”
transposons
True or false? Although humans have many transposons in their genomes, there is little to fear because many are inactive and they jump infrequently
true
note: however, transposons have been found to cause mutation
Transposons in bacteria sometimes contain ____ ____ genes and can “jump” onto plasmids which can be taken up by sensitive strains as one mechanism for resistance transfer
antibiotic resistance
In regards to transcriptional activators, the activator sequences, known as ____ are further activated by tissue-specific proteins/hormones
enhancers
Enhancers are ___ acting DNA sequences. they activate transcription only in the ____ where they reside
cis; chromosome (DNA molecule)
enhancers are often effective in either orientation. true or false?
true
Enhancers only affect transcription nearby where they are located. true or false?
false; can affect transcription thousands of bps away
enhancers affect the transcription of any gene in their vicinity. true or false?
true
How do enhancers work?
transcriptional activators bind to the enhancer and the enhancer stimulates RNA polymerase transcription of specific genes
This is a 72 bp sequence which can enhance the transcription of many genes. An experiment with this enhancer shows that permissive enhancers can stimulate any promoter in the vicinity
SV40
This enhancer is stimulated by zinc and increases the transcription of the gene for the metal-binding protein. An experiment with this enhancer shows that certain enhancers are not specific for a given promoter
Metallothionein
Cloning this enhancer with the polymer large T antigen caused pancreatic tumors in transgenic mice. Showed that enhancers can be tissue specific.
pancreatic B-cell tissue enhancer
Albumin has an enhancer which binds a liver specific transcription factor. This explains why albumin is synthesized exclusively by the ___
liver
Small regions of proteins make direct contact with the DNA and bind by ___ bonds and ___ ___ ___ forces
hydrogen; van der walls
In regards to types of binding, these proteins consist of at least 2 connected coils of an alpha-helix with one of the coils lying flush in a major groove and the other turned away about 90 degrees
helix-turn-helix
What type of binding domain to Lac Repressor proteins contain?
helix-turn-helix
In regards to binding domains, the estrogen receptor contains several ___ ___
zinc fingers
Zinc finger domains bind to the ___ groove of DNA
major
In regards to binding domains, this is a modified or smaller region of a much larger DNA binding protein. A leucine at every 7th position in the “zipper” alpha helix.
leucine zipper
The leucine zipper holds the DNA binding ___-___ domains in the correct position for interaction with DNA
alpha-helical
The transcription factor ___-___ __ binds to the major groove of DNA through multiple interactions which bonds or loops in the protein structure. Shows similarities to Ig light chain antigen binding domains
NF-Kappa Beta
Jak-STAT is a signaling pathway vital for the function of ____
cytokines
JAKs are ___ kinases which phosphorylate specific tyrosines on STATs. STATs are a family of transcription factors which migrate to the ___ after being phosphorylated to stimulate transcription of genes which mediate cytokine function
tyrosine; nucleus
Loss of gene for STAT 1 causes loss of response to what?
viral or bacterial infection
Loss of STAT 4 causes what?
no TH1 cell function
Loss of STAT 5 causes what?
no breast development or lactation
Post-trancriptional controls appear to play a role in regulating which mRNAs actually reach the ____
cytoplasm
Regulation of this could be a control point in mRNA synthesis
splicing
Certain messages are unstable because the message contains an extra ___-___ rich sequence near the 3’ end
A-U
What is masked RNA?
mRNA that is stored in the cell and not translated - repressor proteins bind to it
What do Micro-RNAs do?
small RNA transcripts which regulate the levels of translation of messages.
note: they form double-stranded segments which signal nuclease degradation or block translation
True or false? Micro-RNAs degrade the message
true
