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22 Cards in this Set
- Front
- Back
Prokaryotes... A) are bacteria, distinct from archae and eukaryotes B) are very diverse, although none contain a nucleus and many have a cell wall C) live in all sorts of ecological niches, ranging from sewage treatment plants, to hot acid springs, to the human mouth D) B and C E) all of the above |
D) |
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Which of the following statements regarding DNA is NOT correct?
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D) |
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In the absence of lactose and in the presence of glucose... A) CAP binding prevents RNA polymerase activity B) RNA is not transcribed from the LAC operon because of the bacteriophage lambda repressor C) RNA is not transcribed from the LAC operon because the Lac repressor is bound D) RNA is transcribed from the LAC operon because CAP is not bound E) RNA is transcribed from the LAC operon because the Lac repressor is bound |
C) RNA is not transcribed from the LAC operon because the Lac repressor is bound |
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Gene expression is...
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D) Often affected by the extracellular environment, an example being the effect of glucocorticoids on liver cells |
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Which of the following statements about the E. coli tryptophan operon-repressor system is correct? A) Five operators control each of the E. coli tryptophan-synthesizing enzymes B) Low intracellular tryptophan leads to the transcription of genes that encode for tryptophan-synthesizing enzymes C) There is no correct answer D) The presence of tryptophan inhibits the expression of one gene E) When two tryptophan molecules bind the tryptophan repressor, the repressor changes to an inactive conformation |
B) |
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Which of the following is not true regarding eukaryotic gene regulatory proteins?
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A) |
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If you isolated 2 nucleosomes in a test tube, which of the following would you typically have?
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B) |
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Which of the following is not true regarding a gene control region? A) It includes the promoter B) It includes regulatory sequences C) It is always located immediately next to the transcribed region D) It may contain regulatory sequences within the gene's introns E) C and D |
C) |
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The regulatory system that determines the mode of growth in bacteriophage lambda (when infecting E. coli host cells) is a _______. The Cro protein turns on its own synthesis in a ___________, and turns off the repressor protein (cl) in a __________. A) flip-flop device; positive feedback loop; negative feedback loop B) memory device; feed-forward loop; negative feedback loop C) feed-forward loop; positive feedback loop; flip-flop device D) There is no correct answer E) memory device; negative feedback loop; flip-flop device |
A) |
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Histones are...
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D) |
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Why does a cell need to regulate gene expression? |
External environmental conditions
Development cues
In response to hormone signals |
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What is transcriptional regulation? |
Most common type of gene regulation in bacteria
Occurs in the first stage of gene expression before significant amounts of mRNA are synthesized
The lac operon is a well-characterized example of transcription-level gene regulation as well as the ara operon |
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What is the ara operon? |
The ara operon contains genes that encode proteins required to metabolize a sugar like pentose sugar L-arabinose
When arabinose is present in bacterial growth medium, the genes in the ara operon may be transcribed at high levels, but when a more easily metabolized sugar such as glucose is present, there is little transcription of these genes
Proteins produced by lac and ara operons are not easily detectable but a plasmid called pGLO contains a reporter gene that produces an easily visualized gene product that inserts a marker so that the operon was switch on and an easily detectable protein was made |
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What are the parts and functions on this plasmid map of pGLO? |
Arrows indicate open reading frames or genes
araC gene: • Origin of replication for plasmid (ori), a bla gene that codes for a protein that makes the bacterial cell resistant to the antibiotic ampicillin
GFP: • Gene that codes for green fluorescent protein, a fluorescent marker • Made in large amounts in bacterial cells and these cells are exposed to long-wave ultraviolet light that makes GFP bright green fluorescence
PBAD: • The ara operon promoter • Normally regulates the transcription of genes in the ara operon (allow uptake and metabolism of arabinose) but in pGLO these genes have been replaced by the GFP gene • GFP would normally have its own eukaryotic promoter (from jellyfish Aequoria victoria) but instead is controlled by bacterial promoter PBAD
Whenever environmental conditions of the bacterium would have caused an increase in transcriptional activity from the PBAD promoter, GFP will be produced instead
Only one gene from native ara operon present in the pGLO plasmid is the araC gene and that is has its own promoter |
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Describe the differential regulation of the ara operon in the pGLO system |
AraC is an allosteric activator protein with two binding sites
When arabinose is present in the environment, AraCi (inactive) binds to sugar
This binding causes a conformational change in AraCi to AraCa (active) that allows it to recognize and bind to aral, an activator sequence located just upstream of the PBAD promoter
It is hypothesized that AraCa helps RNA polymerase (RNAP) bind to the PBAD promotor. Therefore, in absence of arabinose: • AraC is not activated (remains as AraCi) and thus cannot bind to aral sequence • RNAP will not bind efficiently to the PBAD sequence • Very little transcription will occur (little GFP will be made)
Second mechanism to regulate transcription involves cyclic adenosine monophosphate (cAMP) and the catabolite activator protein (CAP) |
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How would you get the most efficient binding of RNAP to PBAD? |
In order to get most efficient binding of RNAP to PBAD, not only must two molecules of AraCa bind to the aral sequence, but CAP must bind to the cAMP-CAP binding site (CBS) located just upstream of the aral sequence |
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What are the two forms found of CAP and what happens with CAP in bacterial cells? |
2 forms:
I) When cAMP is bound to CAP, CAP is in its active form and can bind to the CBS sequence
II) But when cAMP is not bound, CAP is inactive and cannot bind to CBS
In bacterial cells:
High concentration of glucose = reduce the synthesis of cAMP = CAP is less likely to be activated and bind to CSB sequence • Consequently even in in presence of arabinose, high concentrations of glucose will prevent the formation of active cAMP-CAP complex and the binding of RNAP to PBAD will not be efficient • Little transcription of GFP will occur
In presence of arabinose and low concentrations of glucose = two molecules of AraCa will bind to aral sequence, activated cAMP-CAP will bind to CBS = binding of RNAP to PBAD will be efficient • Transcription of GFP will be relatively high and it would be expected that bacterial colonies will glow bright green when exposed to long-wave UV light |
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What is the heat shock method? |
Laboratory procedure
Treatment produces bacteria transiently permeable to DNA by introducing brief heat shock
Mechanism Not well understood, believed to involve the neutralization of electrostatic repulsion between DNA and the cell surface induced by interaction with divalent cations and by increased fluidity and permeability of the membrane induced by an increase in temperature |
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How can we differentiate between bacterial cells containing our plasmid of interest and those that do not? |
We cannot differentiate these using a regulate light microscope
Key is in the bla gene found on the pGLO plasmid
This gene codes for beta-lactamase, an enzyme that makes the bacterial cell resistant to the antibiotic ampicillin
This gene is called a "selectable marker" because if we include ampicillin in the E. coli growth medium, only those E. coli cells that have been successfully transformed with the pGLO plasmid and therefore expressed the enzyme beta-lactamase will be able to grown on the medium |
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What is the Aseptic technique? |
Refers to the procedures that are used to establish and maintain a sterile environment or prevent contamination of a pure environment
Procedure goes:
I) Wiping down (decontaminating) your workspace with a bleach or ethanol solution before and after working with bacterial cultures
II) Pipetting bacterial cultures carefully to prevent the formation of aerosols containing bacteria
III) Wearing lab coats, safety glasses and gloves at all times when dealing with bacteria
IV) Minimizing the amount of time that your petri dishes are uncovered
V) Making sure that you do not contaminate materials that may come in contact with your bacterial cultures |
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What is 3C-Seq technology? |
Couples chromosome conformation capture to high throughput sequencing
Measures interaction frequencies between a viewpoint (DNA fragment of choice) and (distal) regulatory elements on a genome wide scale
Recently used for unbiased analysis of spatial organization of the Myb proto-oncogene locus in erythroid cells
Since 3C-based technologies only provide topological information, their functional relevance should be interpreted with caution and needs to be supported by additional experiments |
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What is Myb? |
Myb is a critical hematopoietic regulator required for the proliferation and expansion of all blood progenitors and is dramatically down regulated during terminal differentiation
Failure to silence Myb expression is linked to impaired differentiation and may play a role in leukemogenesis
Myb transcription is regulated by an array of distal intergenic enhancers localizing up to 109 kb upstream of the gene, which are occupied by the essential hematopoietic |