Exam 3 questions

Question 1

A plasmid that encodes resistance to ampicillin and tetracycline is digested with the restriction enzyme
PstI, which cuts the plasmid at a single site in the ampicillin-resistance gene. The DNA is then
annealed with a PstI digest of human DNA, ligated, and used to transform E. coli cells. (a) What
antibiotic would you put in an agar plate to ensure that the cells of a bacterial colony contain the
plasmid? (b) What antibiotic-resistance phenotypes will be found on the plate? (c) Which phenotype
will indicate the presence of plasmids that contain human DNA fragments?

Answer 1

: (a) tetracycline; (b) tetR ampR and tetR ampS; (c) The tet ampS phenotype indicates that the genefor ampicillin resistance has been interrupted by the insertion of a human DNA fragment.

Question 2

Explain how each of the following is used in cloning in a plasmid: (a) antibiotic resistance genes; (b)
origin of replication; (c) polylinker region.

Answer 2

a) Antibiotic resistance allows a researcher to select for a bacterial cell clone that carries the
plasmid; loss of an antibiotic marker in a strain known to contain the plasmid can be used to infer the
presence of a cloned DNA segment that interrupts the antibiotic resistance gene. (b) An origin of
replication assures that the plasmid will replicate autonomously in the bacterium. (c) Polylinkers have
cut sites for a variety of restriction enzymes, allowing insertion of DNA fragments produced with any of
them.

Question 3

Match each feature of the plasmid pBR322 (at left) with one appropriate description presented (at
right) (see illustration of pBR322 below). Descriptions may be used more than once.
____ ampR sequence
(a) permits selection of bacteria containing the plasmid
____ ori sequence
(b) a sequence required for packaging recombinant plasmids
____ tetR into bacteriophage
____ BamHI sequence
(c) origin of replication
____ PstI sequence
(d) cleavage of the plasmid here does not affect antibiotic sequence resistance genes
(e) insertion of foreign DNA here permits identification of
bacteria containing recombinant plasmids

Answer 3

Ans: a; c; a; e; e

Question 4

Explain briefly the properties of the plasmid pBR322 that make it so convenient as a vector for cloning
fragments of foreign DNA.

Answer 4

pBR322 has two antibiotic resistance markers, so that its presence in a bacterium can be detected,
bacteria that carry it can be selected, and insertion of cloned DNA into one of the resistance markers can
be detected by loss of antibiotic resistance. The plasmid also has an origin of replication, so that it
replicates autonomously in E. coli. Several convenient restriction sites allow easy insertion of restricted
DNA fragments. Its small size facilitates its entry into E. coli by standard transformation protocols

Question 5

What sequences are required in an expression vector (for use with E. coli) that are not essential in a
cloning plasmid?

Answer 5

Regulated expression of the cloned gene requires: (1) a bacterial promoter and (2) its associated
operator; (3) a transcription termination sequence; and (4) a ribosomal binding site. (Fig. 9-10, p.
312.)

Question 6

A scientist wishes to produce a mammalian protein in E. coli. The protein is a glycoprotein with a
molecular weight of 40,000. Approximately 20% of its mass is polysaccharide. The isolated protein is
usually phosphorylated and contains three disulfide bonds. The cloned gene contains no introns. (a)
What sequences or sites will be required in the vector to get this gene regulated, transcribed, and
translated in E. coli? (b) List two problems that might arise in producing a protein identical to that
isolated from mammalian cells and describe each problem in no more than two sentences.

Answer 6

(a) The cloned gene must be preceded by a good E. coli promoter and its associated operator and by a
ribosome-binding (Shine-Dalgarno) sequence. The other end of the gene should have a transcription
terminator sequence. (b) Potential problems are that (1) E. coli enzymes may not glycosylate the protein,
which may affect its folding and activity, and (2) the protein kinases that phosphorylate the protein in
mammalian cells are probably absent in E. coli; therefore, the engineered protein will not be
phosphorylated.

Question 7

Name one enzyme that is always used to make a cDNA library, but is generally not used to make a
genomic DNA library. Describe its function briefly

Answer 7

Reverse transcriptase is used to make first a single-stranded DNA complementary to mRNA, then a
double-stranded DNA.

Question 8

A DNA sequence that may be present as only a single copy in a large mammalian genome can be
amplified and cloned using the polymerase chain reaction (PCR). Describe the steps and reaction
components required in a PCR experiment. Illustrate the steps in just one round.

Answer 8

DNA with the desired sequence is heated to convert it to single strands and cooled in the
presence of an excess of oligonucleotide primers that flank the sequence to be amplified. A heatstable DNA polymerase extends the primers, replicating the desired sequence. (See Fig. 9-16.)

Question 9

What are RFLPs and STRs and how are they used in forensic DNA fingerprinting technology?

Answer 9

RFLPs (restriction fragment length polymorphisms) are minor variations among individuals in
DNA base sequence that can be detected by variation in the patterns of fragments that are produced
upon cleavage with restriction endonucleases. When several DNA regions are examined, these patterns
are distinctive for an individual and can be used to determine the identity (or nonidentity) of two
samples of DNA. One of these samples can be from a crime scene, the other from a known individual

Question 10

What is a DNA microarray? How does it resemble and how does it differ from a DNA library?

Answer 10

A DNA microarray is a solid surface upon which are placed DNA fragments from many thousands of
genes. It is in essence a form of DNA library that is arranged physically to allow rapid simultaneous
screening of many thousands of genes

Question 11

Describe how a transgenic mouse is produced. Now describe how a transgender mouse is produced.

Answer 11

Transgenic mouse:
Pronuclear injection into a single cell of the mouse embryo, where it will randomly integrate into the mouse
genome. This method creates a transgenic mouse and is used to insert new genetic information into the
mouse genome or to over-express endogenous genes.
Transgender mouse:
Here you use a female genotype and a male phenotype. Reasonable answers will be given full points.

Question 12

Describe the dependence of the melting point of a fatty acid upon (a) chain length and (b) unsaturation;
(c) explain these dependencies in molecular terms.

Answer 12

All other things being equal, (a) the longer the acyl chain, the higher the melting temperature; and (b)
the more unsaturation, the lower the melting temperature. (c) The melting temperature is a measure of the
thermal energy needed to break the intermolecular interactions that stabilize the
“solid” form of a lipid, which depends upon how well the individual lipid molecules fit into the nearly
crystalline array of lipids. When a shorter acyl chain lies between two longer chains in a nearly
crystalline array of lipid molecules, there is a cavity at the end of the short acyl group that allows
freer motion to the neighboring acyl chains. A cis double bond introduces a “kink” into the acyl chain, so
that it does not pack as easily with its straighter neighbors.

Question 13

What is the most significant chemical difference between triacylglycerols and glycerophospholipids
that leads to their different functions?

Answer 13

Triacylglycerols are nonpolar hydrophobic molecules that can be stored in specialized nonaqueous
cellular compartments. Glycerophospholipids are amphipathic molecules that can serve as structural
components of membranes, which have hydrophilic and hydrophobic regions.

Question 14

Design an experiment to separate the phosphatidyl-ethanolamine, phosphatidyl-choline, phosphatidylserine, and phosphatidyl-threonine.

Answer 14

Phosphatidylethanolamine and phosphatidylcholine differ by one functional group. Choline has a
methyl group and the ethanolamine has an amino group.
You can make the amino group accessible to the carboxy group and create an amidic bond that will easy
to separate them both.
Phosphatidyl serine and threonine are both hydrophilic (polar) – they will be charged because of the
phosphate group (charge is -1) and you can separate the phosphatidyl ethanolamine and choline (charge
0) from serine and threonine using adsorption or thin layer chromatography – here neutral elutes 1st
followed by polar molecules and then – the charged molecules will elute.
Serine and threonine are very similar in structure, except threonine has a methyl group – one methyl
group could possibly make the structure a little hydrophobic – and you could try separating them by
reverse phase chromatography. It will be very hard as it is just one methyl group.

Question 15

Show the basic structure of all glycerophospholipids, sphingolipids and sterols.

Answer 15

All glycerophospholipids have two fatty acids in ester linkage with C-1 and C-2 of glycerol; often the fatty
acid at C-1 is saturated, and that at C-2 is unsaturated. C-3 of glycerol is joined to an alcohol-containing
head group through a phosphodiester linkage, which is negatively charged at neutral pH. (See Fig. 10-10, p.
351.)
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Question 16

Show the structure of sphingosine and indicate the relationship between sphingosine and ceramide.

Answer 16

The structure of sphingosine is shown in Fig. 10-13, p. 353, which also shows that the
attachment of a fatty acyl group to sphingosine in amide linkage converts it to ceramide.
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Question 17

. Describe the differences between the glycosphingolipids corresponding to the A, B, and O human
blood group antigens.

Answer 17

The type O structure is found in all three glycosphingolipids. In both type A and type B, there is
an added sugar; this sugar differs between type A and B.

Question 18

Show the structure of isoprene; explain what is meant by isoprenoid compounds and give an example.

Answer 18

The structure is shown on p. 359. Isoprenoid compounds contain chains that consist of multiple
isoprene units. (See Fig. 10-22, p. 362, for examples.)
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Question 19

What do all these compounds have in common: vitamin A, vitamin K, ubiquinone, and dolichol?

Answer 19

They are all lipids with potent biological activities derived from isoprenoid precursor

Question 20

Build a MS/MS spectrum acquired in positive ionization mode that corresponds to peptide
PANCAKES. Indicate the y ions and y-H2O ions, as well as b2, a2, b3, and a3 ions. Now
build an ESI-MS spectrum for a 14,000 Da protein (positive ionization mode) and a 9,000
protein (negative ionization mode).

Answer 20

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Question 21

If beeswax, cholesterol, and phosphatidylglycerol were dissolved in chloroform, then subjected to thinlayer chromatography on silica gel using a mixture of chloroform/methanol/water as the developing
solvent, which would move fastest?

Answer 21

In this chromatography, the least polar compound (beeswax) moves fastest and the most polar
(phosphatidylglycerol, which has a negative charge on its head group) moves the slowest. (See Fig.
10-24, p. 364.)

Question 22

What are the principal features of the fluid mosaic model of membranes?

Answer 22

: The principal features of the fluid mosaic model of membranes include: (1) a lipid bilayer in
which individual lipids are free to move laterally but not across the bilayer; (2) integral membrane
proteins, which penetrate or span the bilayer, associating with lipid acyl chains by hydrophobic
interactions and exhibiting lateral mobility; (3) peripheral membrane proteins, which associate
noncovalently with the lipid head groups and protruding domains of integral membrane proteins, and
which are sometimes tethered to the membrane by a covalent lipid anchor.

Question 23

Draw the structure of a biological membrane as proposed by the fluid mosaic model. Indicate the
positions and orientations of phospholipids, cholesterol, integral and peripheral membrane proteins,
and the carbohydrate moieties of glycoproteins and glycolipids.

Answer 23

Phospholipids and sterols are found in both faces of the lipid bilayer. Integral membrane proteins
penetrate or span the lipid bilayer, but peripheral membrane proteins associate at the membrane
surface with lipid head groups or integral membrane proteins. The carbohydrate moieties of
glycolipids and glycoproteins are invariably on the outside face of the plasma membrane. (See Fig.
11-3, p. 373.)

Question 24

. Draw a hydropathy plot for a hypothetical integral membrane protein with 3 transmembrane segments
and containing 322 amino acids. Be sure to label the x- and y-axes appropriately, including numerical
values.

Answer 24

The plot should have a y-axis that is labeled “hydropathy index” and an x-axis that is
labeled “residue number” or “position in the protein.” The y-axis should have values from
+3 to –3; the x-axis should have an integral number that has a range of zero to several
hundred. The plot should show three distinct peaks corresponding to the 3 transmembrane
segments.
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Question 25

The bacterium E. coli can grow at 20°C or at 40°C. At which growth temperature would you expect
the membrane phospholipids to have a higher ratio of saturated to unsaturated fatty acids, and why?

Answer 25

At 40°C, the membranes of E. coli will contain more saturated fatty acids than at 20°C. The cell
regulates fatty acid composition to achieve the same fluidity in its membranes, regardless of growth
temperature. Saturated fatty acids counterbalance the fluidizing effect of high temperature.

Question 26

You have a mixture of gulose, chitobiose and heparin. Suggest a procedure for separation of
these three molecules from each other (i.e. in 3 different vials). Also, describe a test that will
allow you to determine the purity of the three fractions.

Answer 26

The physical and chemical property of gulose is very similar to glucose. Gulose is a monomer and
chitobiose is a dimer (monomer=chitin) it has beta 1,4 linkage.
Heparin is alpha 1,4 linkage and a huge polymer with approximately 30 kDa.
1
st you would use gel filtration to separate heparin from gulose and chitobiose.
Since gulose is a monomer and chitobiose is a dimer, you would again do a gel filtration and separate the
two.

Question 27

Describe three factors that contribute to the high degree of sensitivity of signal transduction systems.

Answer 27

The sensitivity of signal transduction results from (a) the high affinity of receptors for signal
molecules; (b) cooperative binding of signal molecules to receptors; (c) signal amplification by enzyme
cascades

Question 28

Explain how amplification of a hormonal signal takes place; illustrate with a specific example.

Answer 28

Amplification occurs when one molecule of signal (epinephrine, for example) elicits the
formation of many molecules of some enzyme (for example, protein kinase A). This occurs when a
single hormone molecule binds to its specific receptor in the plasma membrane and causes the
activation of several molecules of Gs
, each of which activates an enzyme (adenylate cyclase) that,
acting catalytically, produces many molecules of cAMP for every active molecule of enzyme. Each
of these many molecules of cAMP can activate protein kinase A that, acting catalytically, phosphorylates many molecules of target protein (for example, glycogen synthase). (See Fig. 12-7, p. 429.)

Question 29

Identify the relationship between these three MS/MS spectra (similarities, differences, etc).

Answer 29

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Question 30

Draw Alpha-L-2N-acetyl-4S-glucosamine-(1-6)-beta-L-4S mannosamine (1-2) beta-D-fructofuranose

Answer 30

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Question 31

Draw Adenine N7 – x non-glycosidic-L-ribofuranose alpha (1-1) beta-D-3N-acetylglucosamine-4-
threonine

Answer 31

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Question 32

Compare and contrast the modes of action of epinephrine, acting through the -adrenergic receptor,
and of insulin, acting through the insulin receptor

Answer 32

[The mechanisms of epinephrine and insulin action are summarized in Figs. 12-4, p. 424 and
12-15, p. 441]. The adrenergic receptor indirectly activates a catalyst (adenylate cyclase), which
produces a second messenger (cAMP). The insulin receptor is itself a catalyst when occupied with
insulin; its tyrosine kinase activity phosphorylates and activates another protein kinase, which
initiates a cascade of phosphorylations of other proteins. (See Fig. 12-14, p. 440.)

Question 33

Determine the number of proteins and their masses in the spectra shown below

Answer 33

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Question 34

List five guidelines that are used for protein purification. Now describe the principles of protein
identification and protein characterization.

Answer 34

Define objectives, define properties of the target protein and critical impurities, develop analytical
assays, minimize sample handling at every stage, minimize use of additives, remove damaging
contaminants early, use different technique at each step, minimize number of steps.
Protein identification – amino acid composition and sequence of protein as well as its mass identified by
mass spectrometry
Protein characterization – characterization of purified protein, 3D structure (NMR, X-Ray), its activity.
Determination of protein characteristics such as domains, oligomeric state, post-translational
modifications and protein-protein and protein-ligand interactions.

Question 35

Discuss the health benefits of ALA, EPA & DHA

Answer 35

ALA: Alpha-Linolenic acid is an Omega-3 fatty acid. It is one of the two essential fatty acids that are necessary for
health and cannot be produced within the human body. ALA consumption has preventative effect against cardiovascular
diseases.
EPA: Eicosapentaenoic acid (EPA) is an omega -3 fatty acid. It is a polyunsaturated fatty acid (PUFA) that acts as
precursor for prostaglandin-3
DHA: Docohexaenoic acid (DHA) is an omega-3 fatty acid that is a primary structural component of human brain, skin,
sperm and retina