Name:_____________KEY_________________
Student Number: __________________________
BIO315HF—HUMAN CELL BIOLOGY
Midterm Test—October 18, 2010
Professor Danton H. O’Day
Test Length: 1.5h; 100 Marks Total
Answer all questions giving the best or most appropriate answer in each case. No aids allowed.
ANSWER KEY
PART I. TUTORIAL QUESTIONS (30 marks total)
1. Define the following acronyms? (1 mark each; marks deducted for incorrect spelling)
SDS-PAGE
2DE
GFP
FITC
PVDF
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
2 Dimensional Electrophoresis
Green Fluorescent Protein
Fluorescein Isothiocyanate
Polyvinylidene Fluoride
2. List the 4 characteristics that a drug must have in order to be used in the Pharmacological Approach.
(4 marks)
- Drug effect is reversible (1)
- Drug is specific for the target being studied (1)
- Effect of drug is dose-dependent (1)
- Drugs with similar or antagonistic effects give the same or opposite effects (1)
3. List one advantage and one disadvantage of studying a protein that is attached to GFP. (2 marks)
Advantage: (1) It allows researchers to follow the localization and behavior of tagged proteins in
vivo (i.e. in the living cell)
Disadvantage: (1) The presence of GFP may present steric hindrance which may interfere with the
function of the tagged protein or with the interactions that the tagged protein might be involved in.
Also acceptable: usually requires overexpression of the protein of interest. Overexpression may have
negative effects on the cell or in the protein’s normal function.
4. What is the purpose of using SDS? (2 marks)
It denatures proteins by removing their quaternary, tertiary, and secondary structure (1). It also gives
proteins a net negative charge (1). Together, these qualities of SDS allow for proteins to be separated
based on their molecular weights. (0.5 if this was given as an answer by itself)
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Name:_____________KEY_________________
Student Number: __________________________
5a. Using a diagram show how an immunolocalization works. (4 marks)
5b. What additional steps are required to perform a co-immunolocalization? (2 marks)
1. Use a another primary antibody specific for the second protein of interest and follow with a
secondary antibody bound to a fluorescent molecule that gives a different colour (1)
2. View and merge/superimpose the images to determine if the co-localize (1)
6. Having established that two proteins localize to the same site in a cell, describe a technique you could use
to show that these two proteins actually interact with one another. (5 marks)
- Perform a co-immunoprecipitation: (1)
- antibody against protein of interest (1) is bound to a bead/Protein A bead (1)
- antibody-bead complex is mixed with protein sample and complex is pulled down (1)
- pulled down proteins are separated from antibody-bead complex and proteins are analyzed
by western blotting (1)
7. Describe what is being shown in the figure below. You do not need to describe in detail the techniques
that are used. Note: MARCKS and MRP are two different proteins and Scramble is a randomized RNA
sequence. (6 marks)
Shows western blot results of a RNA interference (RNAi) experiment (1)
1. Control lane: Shows the levels of MARCKS, MRP and actin without any RNAi (1)
2. Scramble siRNA lane: Shows that a random piece of RNA does not result in a decrease in MARCKS,
MRP or Actin (1)
3. MARCKS siRNA lane: Shows that MARCKS siRNA specifically targets MARCKS mRNA leading to the
absence of MARCKS protein (1)
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Name:_____________KEY_________________
Student Number: __________________________
4. MRP siRNA lane: Shows that MRP siRNA specifically targets MRP mRNA leading to the absence of
MRP protein (1)
Actin levels were detected in order to show that other proteins were not targeted by RNAi, and it also served
as a loading control (1 mark for either point)
PART II. LECTURE QUESTIONS (70 marks total)
Answer the following questions as specified. Limit your answers to the spaces provided for
each question. (70 marks)
1. List four different ways that proteins associate with the cell membrane. For each one provide one specific
example that we have covered in the course to date. (8 marks)
1 mark for type; 1 mark for example each (note other options possible; check lecture)
single pass—N-CAM, cadherins
multipass—7 Tm receptors (e.g., acetylcholine receptor, etc. etc.)
phospholipid anchor—N-CAM via phosphatidylinositol anchor
Protein protein interactions—7Tm receptors with G protein (various examples possible)
2. Using point form provide evidence with specific examples for the asymmetry of the cell membrane.
Diagrams can be used. (10 marks)





assymmetry of inner and outer leaflet of lipid bilayer (1 mark)
phosphatidylcholine predominate in outer leaflet (1 mark);
phosphatidylserine phosphatidylethanolamine on inner (2 marks)
lipid rafts exist in the cell membrane (1 mark) which are localizations/accumulations of cholesterol,
sphingolipids (2 mark)
carbohydrates of glycolipids (1 mark) and glycoproteins (1 mark) oriented to extracellular space not
cytoplasm; 1 mark for mentioning glycocalyx or ECM (various types)
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Name:_____________KEY_________________
Student Number: __________________________
3. Draw a diagram of an integrin molecule and label it completely. In a short paragraph describe the primary
function of integrins and how a genetic defect for integrin causes a specific human disease. (12 marks).
Structure (8 marks): Need to indicate the following on diagram
Cell and matrix binding domains (1 mark)
Calcium binidng domain (1 mark)
Extracellular domain (1 mark)
Transmembrane domain (1 mark)
Intracellular domain (1 mark)
Disulphide linkage (1 mark)
Cysteine-rich domain (1 mark)
Talin and alpha-actinin binding (1 mark)
Integrins & Disease (4 marks; must be in essay format; if not -2 marks)
 Humans: Leukocyte adhesion deficiency (1 mark)
 Can't make Beta2 Subunit (1 mark)
 WBCs can't stick to endothelium; an initial step in fighting infections and inflammation (1 mark)
 Leads to persistent bacterial infections (1 mark)
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Name:_____________KEY_________________
Student Number: __________________________
4. Using point form, short sentences and diagrams, outline the diverse ways that signaling events are shut
down. (10 marks).
1.
2.
3.
4.
Stop production of ligand (1 mark)
Modify receptor to prevent ligand binding (1 mark)
Remove receptor and ligand by RME (1 mark)
Remove second messenger (1 mark). Examples: Pump calcium back into ER (1 mark). PDE
hydrolysis of cAMP (1 mark).
5. Remove phosphate groups from target proteins (1 mark). Examples: Calcineurin dephosphorylates
MLC (1.5 marks). Protein phosphatase dephosphorylates phosphorylase kianse (1.5 marks).
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Name:_____________KEY_________________
Student Number: __________________________
5. Using point form, discuss the functions of ZO1, showing its cellular localization in normal and diseased or
infected cells. Diagrams can be used. (15 marks)
-ZO1 is localized in tight junctions (1 mark)
-ZO1 is a structural tight junction protein (1 mark); mouse KOs verified this (1 mark)
-ZO1 is a NACO—must define term: protein that moves between nucleus and Adhesion Complexes (2
marks; can also use term NACOs)
-NACOs can respond to infections or cellular disruption by leaving the adhesion junction and moving into
the nucleus to regulate gene activity (2 marks)
-Example: Infection with Helicobacter pylori (1 mark) (which causes gastrointestinal ulcers, cancer; 1 mark)
leads disruption of intestinal/stomach lining (1 mark); this disruption leads to the release of ZO1 from the
tight junction which moves into the cytoplasm and nucleus to regulate gene activity (1 mark)
-Experimental evidence; human gastric epithelial cells (1 mark; needs to show understanding of cell if not
exact cell type) infected in vitro with H. pylori show by immunolocalization (1 mark) movement from cell
membrane to cytoplasmic vesicles (1 mark)
-1 mark for clarity or some original insight
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Name:_____________KEY_________________
Student Number: __________________________
6. Using point form explain the structure and role of gap junctions in human cells. In a concluding, wellorganized paragraph, describe how the study of mammary gland development in mice has helped our
understanding of connexin protein localization and function. Diagrams can be used. (15 marks)
Structure/Role of Gap Junctions (8 marks based on minimum 8 points from the following information)
 Gap junctions are made up of clusters of closely packed connexons
 Connexons consist of pairs of transmembrane channels
 The connexon hemichannel in one cell membrane docks with a connexon hemichannel in an adjacent cell
 The connexons are hexameric: they consist of arrays of 6 connexin protein subunits
 About 20 connexin subunit isoforms exist (give examples e.g., Cx43, etc)
 A connexon may be made of the same (homohexameric) or different (heterohexameric) subunits
-Connexons regulate the flow of small molecules (less than 5000MW; e.g. Ca2+, cAMP) between connected
cells
-The flow of materials though connexons is regulated by phosphorylation (mention experiments)
-Connexons form spontaneously
Mammary Gland Gap Junctions (Paragraph format; 7 marks based on content below; -4 if not in
essay format)
Different types of connexin proteins are present in different regions of the developing breast indicating
variations in the types of gap junctions that function in those regions. Focusing in on human breast duct,
Cx26 is expressed primarily in the duct luminal cells while Cx43 is localized in gap junctions in the
contractile myoepithelial cells that regulate the release of the milk from the alveolus. Both of these gap
junction proteins undergo developmental changes. If Cx26 gene expression is knocked down in mice at
puberty alveolar development is impaired and lactation is prevented. If the Cx43 gene is knocked out the
mice die at birth. A conditional knockout for Cx43 has not been generated. There is also evidence that in
normal breast tissue both Cx26 and Cx43 have tumour suppressive roles.
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Name:_____________KEY_________________
Student Number: __________________________
Worksheet—Use this page for rough work—it will not be marked
This page will not be
marked
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