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14. What is the general purpose of feedback inhibition? What is the problem if feedback inhibition happens when it isn’t supposed to? 15. How do the effects of insulin resistance compound to decrease cellular responses to insulin? 16. Hypothesize a mechanism by which the GLUT4 transporter’s fusion to the cell membrane could be decreased.

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Trinidad Tremblay
Trinidad TremblayLv2
28 Sep 2019

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Related questions

The overall goal of the research project that we carried out in Experimental Biology is insertion of GFP into target lapA/EF-4 gene.

1. What is the overall goal of the research project that we carried out in Experimental Biology, and how does the experiment aimed at purification of your fusion protein fit in with the overall goal of our research? How does the subcellular localization experiment fit into the overall goal?

2. What method did we use to determine whether your fusion protein was a soluble protein or a membrane bound protein? What positive control did we perform? What result was obtained from the positive control? Was this expected? Explain.

3. Know the methods used for protein purification in general as described in class, and in the file; “protein purification.ppt”

4. What method did we use to attempt to purify your LepA/EF 4 – GFP fusion protein? Why did we use this method, what advantages are there compared to other protein purification methods?

5. What is “Protein A” and how did we use it?

6. What antibody did we use in our purification experiment and why did we use it? Why can’t we use the anti-EF 4/LepA antibody to purify your fusion protein?

DAY 18, NOVEMBER 7: INTRODUCTION TO IN VIVO FUNCTIONAL ASSAYS; ASSAY FOR EXPRESSION TOXICITY AT 30°C VS. 37°C

Purpose: Compare the growth of pPEM109, pIN III, and candidate plasmids in DH5alpha-F’lacIQ -/+ IPTG to compare toxicity of functional LepA/EF-4 (expressed from pPEM109) to empty vector control (pIN III), and EF-4-GFP fusion proteins.

Background: This assay is based on the observation that E. coli is extremely sensitive to elevated levels of LepA/EF-4 expression. In prior work it was demonstrated that LepA/EF-4 expression from pPEM109 is lethal to cells growing in the presence of IPTG [Yaskowiak and March, 1995). Furthermore, pPEM109 can only be maintained in strains that contain the mutated Lac repressor gene, lacIQ, which increases repression of the operon. The wild-type promoter of the lacI gene is rather weak, resulting in ~10 molecules of Lac repressor protein per wild-type cell. The lacIQ gene contains a “promoter-up” mutation that increases transcription and Lac repressor expression 10-fold, resulting in ~100 repressor protein molecules per wild-type cell (Müller-Hill et al., 1998). Strains that only have normal Lac repressor have a slightly elevated level of “leaky” expression from the lac promoter. It was shown by comparing two identical strains (one contained lacIQ and one contained lacI) that pPEM109 could only be maintained in the lacIQ containing strain.

If you consider the structure of LepA/EF-4, and its proposed function, why do you think it would be toxic if expressed at high levels levels? What kind of mutations in the lepA gene could give rise to a mutated protein that is not toxic when expressed? What do you predict for your LepA/EF-4 - GFP fusion protein, will it be toxic when expressed or not?

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DAY 19, NOVEMBER 9: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Lysis and Ultracentrifugation).

Background: LepA/EF-4 was originally found in the cytoplasm, membrane and periplasm when the protein was expressed from a plasmid [March and Inouye (1985) J. Biol. Chem. 260: 7206-7213]. The endogenous protein, expressed from the chromosomal gene in the absence of plasmid expression was located in the membrane [March and Inouye (1985) Proc Natl Acad Sci, USA Vol. 82: 7500-7504]. More recently it has been shown that the cellular location of LepA/EF-4 depends on the Mg2+ stress status of the cell [Pech et al. (2011) Proc Natl Acad Sci, USA Vol. 108: 3199-3203].

Here we will examine the comparative distribution of LepA/EF-4 and LepA/EF-4-GFP in cells by subcellular fractionation of native cellular protein extracts. Do you predict that your EF-4-GFP fusion protein will be localized to the cytoplasm, membrane, or both?

In contrast to our previous denaturing extract preparation, the native preparation maintains the overall shape/folding of proteins so they can localize appropriately prior to analysis. Looking ahead, how will the preparation be different in native vs. denaturing extracts?

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DAY 20, NOVEMBER 14: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (SDS PAGE and Transfer)

Procedure:

SDS PAGE Analysis

1. Each section should have the following control samples to analyze from DAY 19:

Extract Fraction Treatment

pPEM109 Input +IPTG

pPEM109 Sol +IPTG

pPEM109 Mem +IPTG

In addition, each student should have 3 experimental samples for their candidate:

Extract Fraction Treatment

Candidate Input +IPTG

Candidate Sol +IPTG

Candidate Mem +IPTG

-------------------------------------------------------------------------------------------------------------------

DAY 21, NOVEMBER 16: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Western Blot detection)

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DAY 23, NOVEMBER 28: PURIFICATION OF NATIVE LEPA/EF 4 – GFP FUSION PROTEINS BY AFFINITY CHROMATOGRAPHY.

Purpose: In order to examine whether LepA/EF 4 – GFP fusion proteins retain function our prior experiments examined whether fusion proteins retained expression toxicity, and whether fusion proteins localized to the membrane. In order to examine other aspects of function, such as GTP – binding and ribosome - binding, purified protein is required. In this experiment we examine the feasibility of purifying LepA/EF 4 – GFP fusion proteins using affinity purification.

Background: Scientists are often interested in recovering proteins that have been produced in bacteria for research or therapeutic purposes. The process of protein purification from bacteria is medically relevant. It can be used as a method to mass-produce proteins of interest. For instance, the human insulin gene can be cloned into a plasmid and transformed into E. coli. The protein can then be purified and used to treat patients with diabetes.

The process of purifying a protein means separating this protein from other cellular components (proteins, DNA, ions, etc.). Protein purification is described in all biochemistry books, and in Chapter 5, Section A, of the textbook used in the Emmanuel College Biochemistry class (Fundamentals of Biochemistry, Voet, Voet and Pratt). This book is on reserve in the Emmanuel College Library. Here we will test the feasibility of using affinity chromatography, and magnetic beads to purify LepA/EF 4 fusion proteins in one step.

Preparation of magnetic beads for affinity purification of native LepA/EF 4. This procedure will be done the day before by the Emmanuel College Lab staff.

Background. The primary antibody that we employed to detect LepA/EF 4 – GFP in our Western blotting experiments can also be used to purify native, functional LepA/EF 4 – GFP fusion protein. Commercially available Protein A magnetic beads will be used for this purpose. Protein A is a very stable bacterial protein that has a high affinity for IgG produced from any animal species. Protein A magnetic beads contain tiny magnetized beads with Protein A covalently attached to their surface. Remember that the primary antibody that was employed in our prior experiments was affinity-purified rabbit IgG that was specific for GFP epitopes. The application of GFP primary antibody to Protein A magnetic beads will result in a tight, non-covalent attachment of GFP – specific IgG onto the magnetic beads. These beads would specifically recognize and bind to GFP that was present in a crude cell lysate. The application of a magnetic field could then be employed to collect the beads and the bound LepA/EF 4. By this method the collection and wash steps would be very rapid and very efficient. After washing away unbound proteins, purified LepA/EF 4 – GFP could be eluted from the magnetic beads. HOWEVER, the GFP IgG was non-covalently attached to the Protein A, and therefore some will leak out in the elution step. This would contaminate the purified LepA/EF 4 – GFP fusion protein with IgG. To avoid this problem, GFP IgG will be chemically cross-linked onto the Protein A magnetic beads by the procedure described in Sisson and Castor [Journal of Immunological Methods (1990), Vol. 127, 215-220].

10.Give the mechanism of action and the result for each of the following therapeutic agents used to treat type I hypersensitivity reactions.

a.antihistamines

b.cromolyn sodium

c.theophylline epinephrine

d.cortisone (10 marks)

11.Outline the events that occur in an asthma attack. How do steroids prevent asthma attacks? (5 marks)

12.How are transfusion reactions type II responses? What happens in a transfusion reaction? (5 marks)

13.Mycobacterium tuberculosis is an intracellular bacterial pathogen. An individual’s exposure to this pathogen can be tested by injecting it into the skin. What type of reaction would you expect in an individual who had been exposed to M. tuberculosis? What is the principle behind this test? (5 marks)

14.Leukocyte adhesion deficiency type 1 occurs in individuals with a mutation in a protein required for neutrophil extravasation. Patients with this condition usually do not live past childhood. Explain what they are susceptible to and the reasons behind the shortened lifespan. What could be done to treat people with this condition? (7 marks)

15.What is chronic inflammation and what are the causes? What cytokines when released can promote the development of chronic inflammation? (3 marks)

16.Discuss the links between Type 2 diabetes, obesity, and inflammation. (6 marks)

1.When something appears blue, it is absorbing all colors except__________.

yellow

blue

red

green

orange

2.During heavy exercise, lactic acid can build up in ourmuscles. Why? How does this resemble the metabolism of bacteria andyeast?

lactic acid is results from insufficient oxygen during sugarmetabolism; it is fermentation

lactic acid accumulates when we are dehydrated; bacteria andyeast use the same pathway

lactic acid is built up from carbon dioxide; bacteria and yeaststore energy in this way

3.Why must a cell keep a similar concentration of dissolvedsubstances with the fluid surrounding them?

to avoid dilution of potential energy

to avoid osmotic imbalance and rapid gain or loss of water

to maintain the correct amount of dehydration and hydrolysis

4.What is entropy?

the amount of energy in a system

the amount of disorganization

the amount of organization

5.Burning a log is the reverse reaction found in photosynthesis.What products would be formed in burning a log?

carbon dioxide and water

glucose and oxygen

oxygen and carbon dioxide

oxygen and water

6.The main reason that cellular respiration needs to occur stepby step instead of a single, big reaction is

cells don't store enough oxygen

too much energy would be released for the cell to harness

cells don't have many mitochondria.

cells produce the enzymes needed for cellular respiration veryslowly

7.Enzymes are catalysts because they operate to:

raise activation energy

lower activation energy

supply activation energy

supply the reactants

8.When ATP powers a reaction it becomes ADP. The ADP

can reform ATP by an exergonic reaction

is a waste product for lysosomal breakdown

can reform ATP by an endergonic reaction

can reform ATP by an oxidation reaction

can reform ATP by an equilibrium reaction

9.The energy source that drives photosynthesis is

water

glucose

carbon dioxide

oxygen

sunlight

10. What is the process by which enzymes regulate their ownactivity?

osmosis

self-propulsion

feedback inhibition

11.Coupled reactions are reactions in which a

endergonic reaction drives a spontaneous reaction

exergonic reaction drives a spontaneous reaction

endergonic reaction drives an exergonic reaction

exergonic reaction drives an endergonic reaction

12.How is water used in photosynthesis?

as a substrate for ATP synthase

as an electron donor

to combine with ADP forming ATP

as an electron acceptor

13.Why are the “Dark Reactions” of photosynthesis referred to as“Dark”?

they do not require light

they can only occur at night

they generate excess sugar and the leaves turn brown

14.What is the difference between potential and kineticenergy?

potential is stored, kinetic is being used

potential is being used, kinetic is ready to be used

potential is negative, kinetic is positive

15.The products of cellular respiration are

carbon dioxide, water, and ATP

glucose, water, and ATP

glucose, carbon dioxide, and ATP

oxygen, ATP, and water

carbon dioxide, glucose, and water

16.All of the following are correct about energy except:

The biological world gets its energy from the sun.

Chemical bonds store potential energy.

A boulder perched on a hill has kinetic energy.

Energy is defined as the ability to do work.

All forms of energy can be converted to heat.

17.What is needed for the end product of glycolysis, pyruvate,to enter the citric acid cycle?

More glucose for energy

ATP

Oxygen

Carbon dioxide

18.The enzyme that forms a transmembrane channel in mitochondriaand phosphorylates ADP

a carrier protein

ATP synthase

acetyl CoA

19.Fermentation is most common in

plants

microorganisms

animals

20.During adsorption of sunlight by photosystems, H+ ions aregenerated. Where do they come from? What are they used for?

from the breakdown of sugar; they help form water

water; they help form sugar

from carbon dioxide; they help dissolve NaCl

21.Glycolysis

does not use oxygen

uses oxygen

uses carbon dioxide

22.Photosynthesis is

not dependent on chlorophyll

a process that produces glucose and oxygen

a process that produces water and carbon dioxide

23.Breakdown of glucose generates many ATP molecules, each ofwhich can be used independently. Why is ATP ideally suited to powerbiological reactions?

ATP has many hydrogen bonds

ATP has enough energy to power one biological reaction

ATP readily dissolves in water

24.Objects that are not actively moving but have the capacity todo so are said to possess:

kinetic energy

entropy

potential energy

living energy

25.The first stage of cellular respiration, called ___________,takes place in the cytoplasm of the cell and needs no oxygen.

photorespiration

citric acid cycle

glycolysis

oxidation

26.Diffusion

requires energy

utilizes proteins to move molecules across a membrane

moves molecules against a concentration gradient

does not require energy

cannot occur without a membrane present

27.What is the name of the molecule on which an enzymeworks?

substrate

monomer

polymer

28.ATP contains

phenylalanine

two phosphate groups

three nitrate groups

three phosphate groups

29.How does chlorophyll function in photosynthesis?

by absorbing carbon dioxide

by absorbing the sun's energy

by absorbing water

30.Objects that are moving are said to possess:

living energy

potential energy

entropy

kinetic energy

31.The respiration pathway that evolved first is probably

glycolysis

aerobic respiration

citric acid cycle

32.The energy source for the process of photosynthesis is

oxygen

sunlight

carbon dioxide

chlorophyll

glucose

33.Burning wood generates heat and light. What type of reactionis this?

exergonic

kinetic energy

endergonic

potential energy

equilibrium

34.What is meant by a “coupled reaction” in terms of energy?

many poymers are built simultaneously

energy released by one reactions powers another reaction

some reactions occur in the same organelle

35.The First Law of Thermodynamics states:

energy is constantly being created by entropy events

energy can be changed from one form to another, but cannot becreated or destroyed

energy can be used and then destroyed because of entropy'sactions on it

energy can be destroyed while it is producing entropy

36.How do we dispose of the carbon derived from the glucose thatis metabolized during respiration?

by breathing out

via our urine

it is broken down in lysosomes

37.Both plant and animal cells have mitochondria because theyboth

carry on respiration

carry on photosynthesis.

require water

38.What is Activation Energy?

the energy needed to initiate a reaction

the energy that transports water into cells

potential energy

39.If you put a plant in a chamber and shine a light on it

oxygen will decrease and carbon dioxide will increase

oxygen will increase and carbon dioxide will decrease

oxygen and carbon dioxide will increase

oxygen and carbon dioxide will decrease

40.What are the inputs and the output molecules for the overallphotosynthetic process?

input sugar and oxygen; output carbon dioxide and water

input oxygen and water; output sugar and carbon dioxide

input carbon dioxide and water;output sugar and oxygen

41.What kind of reaction is photosynthesis?

endergonic

kinetic energy

exergonic

potential energy

equilibrium

42.In the process of glycolysis, glucose is cleaved into a pairof three-carbon molecules called

lactate

pyruvate

acetyl CoA

ATP

alcohol

43.Entropy is

order

disorder

complexity

Both order and disorder are correct

Both complexity and disorder are correct

44.Glycolysis takes place in the _____________ and the citricacid cycle and electron transport chain take place in the___________.

cytoplasm; endoplasmic reticulum

mitochondria, chloroplast

mitochondria; cytoplasm

cytoplasm; mitochondria

45.The organelle that carries out photosynthesis in plants isthe

mitochondria

chloroplast

ribosome

chlorophylllysosome

46.The energy to power the Calvin cycle comes from

cellular respiration

oxygen

the light reactions of photosynthesis

47.Which of the following can be broken down into intermediateproducts that enter cellular respiration?

Proteins

Lipids

Carbohydrates

All of these.

48.When cellular product levels get too high, the responsibleenzyme is inhibited. This is an example of

promotion

denaturation

equilibrium

a coenzyme

feedback inhibition

49.How is respiration the opposite of photosynthesis?

respiration breaks sugar down to carbon dioxide; photosynthesisbuilds sugar from carbon dioxide

respiration builds sugar from carbon dioxide; photosynthesisbreaks sugar down to carbon dioxide

respiration requires energy input; photosynthesis gives offexcess energy

50.The molecule that absorbs sunlight for photosynthesis is

oxygen

carbon dioxide

chlorophyll

glucose

sunlight

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