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1 May 2018

9. Your research lab is interested in studying how CREB regulates gene expression in response to cAMP-dependent signaling and cAMP-independent signaling pathways. Your group has discovered that CREB can be phosphorylated by ERK or PKA and that, upon phosphorylation by either of these kinases, CREB is more likely bind to CRE-containing DNA sequences. Your lab groupâs central hypothesis is that (i) CREB is phosphorylated by PKA or ERK in the cytosol, (ii) translocates into the nucleus, and (iii) is then able to bind to CRE-containing DNA sequences to regulate transcription of specific genes. Notably, your group identified c-Fos as an immediate early gene that appears to contain a CRE and increases in expression when CREB is phosphorylated at Ser-133. Propose an experiment that specifically addresses all three of these points using molecular and/or pharmacological manipulations. Be very specific: name and describe the molecular method and/or the specific names/targets of pharmacological manipulations that you propose, and be sure to note specifically which part of the hypothesis you are testing with each manipulation.

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1 May 2018

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As a graduate student, you are working on understanding how a new drug regulates expression of Gene A. When the cells are treated with the new drug, you notice an increase in phosphorylation of CREB and increased expression of Gene A. Your PhD advisor has asked you to design a series of experiments to determine the type of receptor and signaling pathway involved in this response by cells to the drug.Youve started by treating the cells with a membrane permeable analog of cAMP and noted an increase in phosphorylation of CREB and expression of Gene A. Based on this information, you hypothesize that: (1) the new drug binds to a G a S-coupled receptor to increase intracellular cAMP levels; (2) increased intra cellular cAMP levels are directly related to increased phosphorylation of CREB; and (3) phosphorylation of CREB is required for regulation of expression of Gene A. Explain specifically how you would design experiments using pharmacological manipulations (inhibitors or activators of enzymes along this signaling pathway) or genetic manipulations (knockdown, over-expression,specific mutations, or gene editing of enzymes along this pathway or of CREB itself) to prove eachof these three hypotheses. (Hints: in preparing this question, you should have specific drugs and their targets/mechanisms stated or specific mutations in mind that you might make).

QUESTION 1

Which of the following is an example of an extracellular signal molecule?

	a.	IP₃
	b.	cAMP
	c.	Insulin
	d.	Diacylglycerol

2 points

QUESTION 2

Nuclear hormone receptors _____

	a.	Bind to water soluble ligands
	b.	Activate second messengers
	c.	Regulate transcription in response to ligand binding
	d.	Are normally located on the plasma membrane

2 points

QUESTION 3

Which of the following does not occur when a receptor tyrosine kinase is bound by ligand?

	a.	Phosphorylation of the RTK
	b.	Cytoplasmic proteins bind the phosphorylated RTK
	c.	Dimerization of the RTK
	d.	The RTK binds to DNA to regulate transcription

2 points

QUESTION 4

Which of these is a logical signal-transduction pathway?

	a.	An intracellular receptor activates phospholipase C, which cleaves a membrane protein to form IP₃, which then activates the opening of an ER channel protein, which releases cyclic AMP into the cytoplasm, where it binds to an intracellular enzyme that carries out a response.
	b.	A G-protein-linked receptor activates G protein, which activates phospholipase C, which cleaves a membrane lipid to form IP₃, which binds to a calcium channel on the ER, which opens to release calcium ions into the cytoplasm, which bind to an intracellular enzyme that carries out a response.
	c.	An ion-channel receptor opens, allowing a steroid hormone to enter the cell; the steroid hormone then activates protein kinases that convert GTP to GDP, which binds to an intracellular enzyme that carries out a response.
	d.	A tyrosine-kinase receptor activates adenylyl cyclase, which activates phospholipase C, which converts ATP into cyclic AMP, which binds to an intracellular enzyme that carries out a response.

2 points

QUESTION 5

Which of the following is an example of signal amplification?

	a.	activation of an enzyme molecule
	b.	activation of a specific gene by a growth factor
	c.	Breakdown of many cAMP molecules
	d.	activation of 100 molecules by a single signal binding event

2 points

QUESTION 6

Consider a signal transduction pathway that utilizes cAMP as a second messenger to activate PKA (Protein Kinase A). Which of the following situations will result in the cellular response?

		The G protein releases GDP and binds to GTP
		The target protein (adenylyl cyclase) is inactive
		The regulatory subunits of PKA (Protein Kinase A) are bound to the catalytic subunits
		The appropriate G protein is bound to GDP

2 points

QUESTION 7

Which of the following is found in only some signal transduction pathways? [In other words, which is not found in all signal transduction pathways?]

		Receptor protein
		Extracellular signal molecule
		Intracellular signaling molecule
		Cellular response
		Effector protein

2 points

QUESTION 8

The enzyme that specifically catalyzes the conversion of ATP to cAMP is:

		Adenylyl phosphatase
		Adenylyl kinase
		Adenylyl dehydrogenase
		Adenylyl cyclase

2 points

QUESTION 9

If an animal cell suddenly lost the ability to produce GTP, what might happen to its signaling system?

		It would use ATP instead of GTP to activate and inactivate the G protein on the cytoplasmic side of the plasma membrane.
		It would not be able to activate and inactivate the G protein on the cytoplasmic side of the plasma membrane.
		It would employ a transduction pathway directly from an external messenger.
		It would be able to carry out reception and transduction but would not be able to respond to a signal.

2 points

QUESTION 10

A hormone signal is sent from the brain to other parts of the body via the bloodstream. Why do only certain cells respond to this signal?

		Hormones do not last long enough in the bloodstream to stimulate a cellular response
		Only cells that have a specific transcription factor gene will be able to respond to the signal
		Only cells in the immediate vicinity of the cell sending the signal will respond
		Only cells that have a receptor for the specific signal will be able to respond to it

cerisehedgehog846

HELP ASAP; Please help me understand my lab results, so be detailed to clearly understand

Then , Please include answers to the question below to include in your response

1. what role does ampicilin (amp) and arabinose (ara) play in the experiment.

2) Because GFP was isolated from jelly fisht, what modification to the GFP DNA had to be made to result in expression in E. coli?

BACKGROUND INFORMATION

Transformation of GFP and the pGLO Plasmid GFP is a commonly used reporter protein in research labs, as the fluorescence creates a marker protein that can be used in many types of cell biology and biochemical studies. In basic research, GFP is often fused to a specific target protein of interest, creating a chimeric reporter protein. GFP has been used as a reporter protein to study blood vessel and tumor progression in mice, brain activity in mice, and malaria eradication in mosquitoes for instance (see website mentioned in notes). In the first week you will use a procedure to genetically transform bacteria with the gene that codes for Green Fluorescent Protein. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. During the second week you will purify the protein away from all of the other proteins produced by the bacteria using chromatography, and during the third week you will evaluate the success of the purification procedure and estimate the size (molecular weight) of GFP by using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fundamental to the process of genetic transformation of bacteria are plasmids. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The occurrence of bacterial resistance to antibiotics is due in large part to the transmission of plasmids which contain antibiotic resistance genes. The pGLO plasmid contains the gene for GFP, and also contains the gene for the enzyme Î²-lactamase (bla), which provides resistance to the antibiotic ampicillin. The Î²-lactamase protein is produced and secreted by bacteria that contain the plasmid. Î²-Lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express Î²-lactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cellsâ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar medium.

The transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly acquired genes. 1) Cells are first treated with a solution of CaCl2, which is thought to neutralize the repulsive negative charges of the phosphate backbone of DNA and the phospholipids of the cell membrane, allowing the DNA to adhere to the cells. This is referred to making the cells âcompetentâ (i.e. the cells are capable, or competent, to take-in exogenous DNA). 2) Cells are then subjected to a heat shock, which is thought to increase the permeability of the cell membrane, allowing the cells to take-in the plasmid DNA. 3) Cells are allowed to grow in a ârecoveryâ phase in the absence of ampicillin. During this time the cells begin to produce the Î²-lactamase protein, which will allow them to survive when they are subsequently placed on agar plates containing ampicillin.

For the first week section of lab

To eac

h of two microfuge tubes, add 250ul of transformation buffer. Keep these tubes on ice.

Using a sterile loop, pick one colony of bacteria from the LB plate. Transfer this colony to one of the microfuge tubes.

Gently vortex the tube until the colony is dispersed. Repeat for the second tube. Add 0.08ug of DNA to one of these tubes (+DNA tube).

Incubate both tubes on ice for 10 minutes. Incubate both tubes at 42Â°C for 50 seconds.

Incubate both tubes on ice for 2 minutes. Add 250ul of LB broth to each tube and incubate at room temperature for 10 minutes. Flick each tube gently. Add 100ul of t

his transformation mix to the appropriate plates.

Results For plates:

+DNA: Has the E. Coli pGlo plasmid and - DNA plates do no have EColi pGLo plasmid .

Amp means ampiclin and Ara means arabinose on agar plates

+ DNA Plate LB/amp/ara--> white 9 colonies, Glow: yes

+DNA LB/amp--> 7 colonies, GLow: no

--DNA LB/AMP- 1 colony so no colony growth and no glovw

-DNA: LB--> many colonies, Glow: no

Please explain significance of result or thand the reason for the results in scientific terms based on the background information provided and your own knowledge to be detailed. Would these results be expected as wll.