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
