Biochemistry Genetics Sem 1 Part A

Course Handouts 1-13
Biochemistry and Genetics I

copyright DeVry Inc. 2005
September 2005
Semester 1
Drs. Grogan, James, Larsen, LaVille, Meisenberg, Smolanoff and Mrs. Lambert
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Handout Contents
Handout Page Title
Number Numbers
1 1-16 Introduction to Biomolecules
2 17-33 Enzymatic Reactions
3 34-46 DNA and Gene Expression
4 47-55 The Human Genome and Mutations
5 56-67 Chromosome Aberrations
6 68-84 Methods in Molecular Medicine
7 85-91 Mendelian Inheritance
8 92-100 Plasma Proteins
9 101-108 Biochemistry and Genetics of Blood
10 109-115 Hormone Biochemistry
11 116-127 Glycolysis, TCA and Ox-Phos
12 128-138 Single-Gene Disorders and Traits
13 139-143 Coagulation
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Welcome Message,
The faculty of the Biochemistry Department at Ross University

welcome you to the first semester biochemistry course. The following
handouts are provided to supplement lectures and reading material in
your text. Please understand that this handout set is in no way a sure
indication of what you will be required to know for exams. This
material can be viewed as the minimum information students are
expected to know. Additional reading material for this course can be
found in the library. Reserve materials, texts and journals in this
subject area are also available. We encourage you to use these
materials in addition to your assigned materials throughout the
semester.
Enhancing and updating your knowledge of biochemistry
requires you to take the long view, and develop habits which foster
lifetime learning. Discussion of medical biochemistry topics with
faculty and other students is helpful for integrating this core
knowledge with your other subjects.
The following web-sites are a small sampling of additional
material which can be accessed online. We encourage your use of
internet resources to enhance your integration of course material.
General Biochemistry Courses:

http://www.kumc.edu/biochemistry/bioc800/biocindx.htm
http://web.indstate.edu/thcme/mwking/
http://tutor.lcsf.ucsb.edu/instdev/sears/biochemistry/
Metabolism: http://www.gwu.edu/~mpb/
Clinical Biochemistry:
http://www.qub.ac.uk/cm/cb/text/studgide/index.htm
Database searching for journal articles, and biochemical
information:
http://www3.ncbi.nlm.nih.gov/Entrez/index.html
Enjoy your learning experience in this course! And remember, the

faculty are here to help you succeed.
Sincerely yours,
The Biochemistry Faculty
3
ROSS UNIVERSITY SCHOOL OF MEDICINE
BIOCHEMISTRY and GENETICS I
Handout 1
INTRODUCTION TO BIOMOLECULES
I. ELEMENTS AND MOLECULES
Biomolecules contain only a few of the 92 elements that exist in nature:

carbon hydrogen, oxygen (all), nitrogen (proteins, nucleic acids), sulfur (proteins),
phosphate (nucleic acids, some proteins, many metabolic intermediates). Some
elements occur in charged form: sodium, potassium, calcium, magnesium,
chloride. Important atomic weights (in Dalton units) are:
Carbon (C): 12 Nitrogen (N): 14

Hydrogen (H): 1 Sulfur (S): 32
Oxygen (O): 16 Phosphorus (P): 31
If you know the composition of a molecule, you know its molecular weight.
Example: Glucose (C6H1206) has a molecular weight (MW) of 180. One mol is
the molecular weight in grams (g). One mol of glucose is 180 g. Concentrations
of dissolved molecules are often given in mol per liter (mol/L, or M), millimol per
liter (mmol/L), or milligrams per 100 ml (90mg/dl). Can you calculate its molar
concentration?
Composition of the human body :
Class of Molecule Content (%) MW (Daltons)

Water 60.0 18
Inorganic salt, soluble 0.7 = atomic weight
Inorganic salt, insoluble 5.5 = atomic weight
Protein 16.0 5 x 103 to 1 x 106
Triglyceride (fat) 13.0 ≈ 800
Membrane lipids 2.5 400 to 1000
Carbohydrates 1.5 >1000
(polysaccharides)
Nucleic acids 1.2 2 x 104 to 2 x 108
II. COVALENT BONDS AND NONCOVALENT INTERACTIONS
1. Covalent Bonds.
The covalent bonds that connect the atoms in molecules are formed by
binding electron pairs which orbit both atoms. Formation and breakage of
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covalent bonds requires chemical reactions which, in the body, are catalyzed by
enzymes.
Electronegativity is the tendency of an atom to attract electrons. The order
of electronegativities of atoms in biomolecules is:
O>N>S>C≈H
2. Noncovalent interactions.
The binding electron pair of a covalent bond is displaced towards the more
electronegative atom. This creates a dipole, with partial positive and negative
charges at opposite ends. Opposite charges attract each other, therefore
dipole-dipole interactions can form between different polarized bonds.
Hydrogen bonds are a type of dipole-dipole interaction involving a hydrogen
bound to an electronegative atom (O or N). The water molecule is a dipole with a
partial negative charge on the oxygen and partial positive charge on the
hydrogens:
δ+ δ+
H H
O
δ-
The unusual physical properties of water (for example its high boiling point)
are caused by hydrogen bonds between water molecules.
Ions carry either a positive charge (cations) or a negative charge (anions).

They form ion-dipole interactions with water. That is why many salts are water
soluble: the interactions with water can overcome the electrostatic interaction
(“salt bond”) in the salt crystal. Organic molecules that contain positive and
negative charges interact strongly with water, and most of them are water
soluble. Also molecules that form hydrogen bonds with water are water soluble.
Molecules that have mostly nonpolar bonds, (especially the hydrocarbons which
consist of carbon and hydrogen) are insoluble in water.
Example:
H
O O O HC CH
3 3
H2N NH2 H2N CH3 H3C CH3 H
> > >
Urea Acetamide Acetone Propane
Higher solubility Lower solubility
Polar interactions (ion-ion, ion-dipole, dipole-dipole) are also important for

interactions within and between biomolecules. Hydrophobic interactions are
formed between nonpolar groups (usually hydrocarbon) in the molecules.
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Nonpolar groups occupy space between the water molecules preventing their
interactions. Therefore, hydrophobic groups aggregate to minimize their
interface with water. Van-der-Waals interactions are weak noncovalent forces
between neighboring molecules. All noncovalent bonds are weak. They form
and break spontaneously, therefore all noncovalent bonding is reversible.
III. BONDS IN BIOMOLECULES.
Functional groups determine chemical reactivity and physical properties

(solubility, melting point, boiling point) of the molecule. Most important functional
groups (R= “residue”, the remainder of the molecule):
Alkyl groups: R-CH3 (methyl), R-CH2-CH3 (ethyl) etc. Nonpolar
(hydrophobic) groups. Most common in lipids.
Hydroxy group: R-OH
Non-ionizable, forms dipole-dipole interactions. Occurs in carbohydrates,
ethanol, some amino acids.
O
O
Carboxy group: R
This is the most important acidic group in biomolecules. In fatty acids, amino
acids, and many metabolic intermediates. The carboxy group carries a negative
charge at pH = 7.
R O R O
Carbonyl group: H (adehyde), R (ketone)

Non-ionizable, forms dipole-dipole interactions. Occurs in monosaccharides.
Amino group: R N H (primary amine)
H CH3
R N
R N
CH3 CH3
(secondary amine) (tertiary amine)
CH3
+
R N CH3
CH3
(quaternary ammonium salt)
These are the most important basic groups in biomolecules. In amino acids
and biogenic amines. The nitrogen of aliphatic amines carries a positive charge
at pH = 7.
Sulphydryl group: R-SH Weakly acidic. In cysteine, and coenzyme A.
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OH
R H
Hemiacetal: OR'
Non-ionizable, in the ring forms of monosaccharides. Most biomolecules are
large molecules (macromolecules) which are formed when functional groups in
their building blocks react with the formation of water. These reactions are called
condensation reactions. The reversal of a condensation reaction is called
hydrolysis. Condensation reactions are endergonic (energy-requiring), while
hydrolysis reactions are exergonic (energy-releasing):
Carboxylic ester: From carboxy group and hydroxy group.

O O
R' OH + HO R'' R' O R'' + H O H
Example: Triglycerides (fat)
Phosphate ester: From hydroxy group and phosphate.

O O
O P OH + HO R'' O P O R'' + H O H
O O
Example: Many intermediates of carbohydrate metabolism. All esters
can be cleaved by acid and, especially, alkaline hydrolysis.
Phosphodiester: From hydroxy group and phosphate ester.

O O
R' O P OH + HO R'' R' O P O R'' + H O H
O O
Examples: Nucleic acids, phospholipids.
Mixed anhydride: From two different acids (e.g., carboxylic acid and
inorganic phosphate).
O O O O
O P OH + R' OH R' O P O + H O H
O O
In some metabolic intermediates.
Phosphoanhydride: From phosphate and phosphate (-ester).

O O O O
R' O P OH +O P OH R' O P O P O + H O H
O O O O
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Example: Energy-rich bonds in ATP.
Ether: From two hydroxy groups.

R' OH + R'' OH R' O R'' + H O H
In some O-methylated and hydroxy group.
Acetal: From hemiacetal and hydroxy group.
R' O R' O
R'' OH + HO R''' R'' O R''' + H O H
H H
In Carbohydrate, where they are known as glycosidic bonds.
Thioesters: From sulfhydryl group and carboxy group.

O O
R' OH + HS R'' R' S R'' + H O H
In coenzyme A thioesters (acetyl-CoA, fatty acyl-CoA).
Amides: From (carboxylic) acid and ammonia or amine.

O O
R' OH + NH3 R' NH2 + H O H
O O
R' OH + H2N R'' R' N R'' + H O H
H
Example: Peptide bonds in proteins.
The formation of anhydride bonds and thioester bonds requires more energy
than the formation of the other bonds. These bonds are called energy-rich
bonds.
IV. ISOMERS
Isomers are chemically different molecules of identical composition. There

are three different types:
Positional isomers differ in the positions of atoms or functional groups within
the molecule. Example:
H
H O
H OH
H OH O
H OH H OH
H H
Glyceraldehyde Dihydroxyacetone
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Geometric isomers differ in the relative geometric position of different parts
of the molecule, for example cis-trans isomers at double bonds. Substituents at
double bonds are planar, and they don’t rotate:
R' H R' H
R'' H H R''
Cis-isomer Trans-isomer
Optical isomers arise when a carbon has four different substituents making it
an asymmetric carbon. If the molecule has only one asymmetric carbon, the
isomers are mirror images or enantiomers. Also disastereomers differ only in
the position of some groups in space, but they are not mirror images. Unlike
positional isomers and disastereomers, enantiomers have identical
physicochemical properties. But they rotate the plane of polarized light in
opposite directions. Optical isomerism is also called chirality. Example:
COOH COOH
H NH3+ NH3+ H
R R
D-amino acid L-amino acid
Alternative positional, geometric and optical isomers are not equivalent

biologically. Enzymes can distinguish between the isomers.
V. ACIDS AND BASES
A proton can be transferred from one water molecule to another in a

spontaneous, reversible reaction:
2 H2O H3O+ + OH-
In distilled water, [H3O+] = [OH¯] = 10¯4 M, and
[H3O+] x [OH¯] = 10¯14 M2
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This means that any rise in [H3O+] must be compensated by a decline in [OH¯]
and vice versa. The concentration of [H3O+] is usually written as the “proton
concentration” [H+], although free protons are uncommon in water.
The proton concentration is expressed as the pH value. The pH value is the
negative logarithm of [H+].
pH = 7 neutral ([H+] = 10¯7)

pH < 7 acidic ([H+] > 10¯7)
pH > 7 alkaline ([H+] < 10¯7)
The pH of cells and body fluids has to be kept constant. Normal blood pH:
7.4.
Acids are substances which donate protons to water. They increase [H+] and
decrease the pH. Bases are substances which accept protons from wate: They
decrease [H+] and increase the pH.
Many biomolecules contain acidic and basic groups which undergo reversible
protonation/deprotonation reactions. The protonation state of such groups
depends on the solvent pH. This can be understood in terms of mass action: H+
is a reactant, therefore its concentration drives protonation and deprotonation
reactions.
The major acidic group in biomolecules is the carboxy group, which
undergoes the following ionization reaction:
OH O
R' + H2O R' + H3O+
O O
This is a nonenzymatic, instantaneous, freely reversible equilibrium reaction.

Note that the carboxylate anion (R-COO¯) is itself a base, ready to accept a
proton and reform the carboxylic acid in the reverse reaction. The carboxylate
anion is the conjugate base of the carboxylic acid. Acids are uncharged in the
protonated form and negatively charged (anionic) in the deprotonated form.
The major basic group in biomolecules is the primary amino group:

R' NH2 + H3O+ R' NH + 3
The ammonium salt (R-NH3+) is itself an acid, ready to release a proton and
reform the primary amino group in the reverse reaction. The ammonium salt is
the conjugate acid of the amine. Bases are positively charged (cationic) in the
protonated form and uncharged in the deprotonated form.
The protonation/deprotonation reaction.
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OH O
R' + H2O R' + H3O+
O O
Can be described by its dissociation constant KD:
KD = [R-COO¯] x [H+] or [H+] = KD x [R-COOH]

[R-COOH] [R-COO¯]
We can put this whole equation into the negative logarithm:
pH = pK - Log [R-COOH]
[R-COO¯]
= pK + Log [R-COO¯]
[R-COOH]
The pK value is an intrinsic property of the ionizable group. If the pH equals
the pK, the group is half-protonated; if pH > pK it is mostly deprotonated;if pH <
pK, it is mostly protonated.
Important ionizable groups in biomolecules:

Acidic groups, deprotonated at pH 7: Carboxy group, phosphate ester,
phosphodiester.
Acidic groups, protonated at pH 7: Sulfhydryl group, phenolic hydroxy
group.
Basic groups, protonated at pH 7: Aliphatic amino groups.
Basic groups, deprotonated at pH 7: Aromatic amines.
VI. FATS AND CARBOHYDRATES
1. Triglycerides (“fat”) are esters formed from glycerol and fatty acids.
H2C OH
HO H
C OH
H2
Glycerol:
H2 H2 H2 H2 H2 H2 H2
C C C C C C C COOH
H3C C C C C C C C
H2 H2 H2 H2 H2 H2 H2
Fatty acid:
(Palmitic acid)
Palmitic acid is a saturated fatty acid. Monosaturated fatty acids have a

single C=C double bond, and polyunsaturated fatty acids have more than one
C=C double bond.
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Triglyceride:
H2C O
O H
C O
H2
Triglycerides are not water-soluble. They are used as energy stores in

adipose tissue. Nonpolar molecules like the triglycerides are called lipids.
Lipids other than triglycerides occur in biological membranes. They include the
phospholipids, glycolipids and cholesterol.
2. Carbohydrates are made from monosaccharides. These are polyalcohols
containing an aldehyde group (aldoses) or keto group (ketoses).
Monosaccharides may have:
3 carbons: Trioses 6 carbons: Hexoses
4 carbons: Tetroses 7 carbons: Heptoses
5 carbons: Pentoses etc.
Examples:
O O O O
O
OH OH
OH HO HO
OH HO OH
HO OH
OH HO OH HO
OH HO
HO OH O
HO
OH HO OH
HO
Galactose Glucose Mannose Fructose

Ribose
(Gal) (Glc) (Man)
Only D-isomers are shown as these are prevalent in nature.

Gal, Glc and Man are not enantiomers but diastereomers because they are
not mirror images. Monosaccharides that are distinguished by the orientation of
substitiuents around a single asymmetric carbon are called epimers.
Most monosaccharides form ring structures by a hemiacetal or hemiketal
bond between the carbonyl group and one of the hydroxy groups:
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O
O OH
HO HO
OH
HO HO OH
OH OH
OH
O OH
HO
HO OH
OH
D-Glucose β-D-Glucose or α-D-
Glucose
Glucose spends 99.9975% of its time in the ring form. C-1 in the ring form is
asymmetric. It gives rise to α and β isomers which are called anomers.
Because the ring can open and close spontaneously, α and β equilibrate until
34% is α and 66% is β. This is called mutarotation.
Disaccharides (from 2 monosaccharides), oligosaccharides (from "a few"
monosaccharides) and polysaccharides (from “many” monosaccharides) are
formed by glycosidic bonds. These are acetal or ketal bonds that involve at
least one anomeric carbon (aldehyde or keto carbon). Glycosidic bonds
involving the anomeric carbon of a carbohydrate can also be formed with non-
carbohydrate components.
Important disaccharides:
Maltose: Glucose+ Glucose (α – 1, 4 bond)
Lactose: Galactose + Glucose (β – 1, 4 bond)
Sucrose: Glucose + Fructose (α, β – 1, 2 bond)
Important polysaccharides:
Starch: Glucose, with α – 1, 4 bonds and some α – 1, 6 bonds.
Glycogen: Like starch, but with more α – 1, 6 bonds
Cellulose: Glucose, with β – 1, 4 bonds
Monosaccharides and disaccharides are water soluble. Polysaccharides are

hydrated, but not all are water soluble. Monosaccharides have reducing
properties because of the presence of the carbonyl carbon.
VII. AMINO ACIDS
Amino acids contain a carboxy group, an amino group, a hydrogen atom and
a variable side chain R (“residue”). These four groups are bonded to a central,
asymmetric carbon called the alpha-carbon. Only L-amino acids are found in
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proteins. The carboxy group has a pK close to 2 while the amino group pK
ranges from 9 to 10. Thus, amino acids can exist in different protonation states:
COOH COO- COO-
NH3+ H NH3+ H H2N H
R R R
pH = 1 pH = 7 pH = 11
At pH = 7, amino acids exist as zwitterions – molecules that possess both a

positive and a negative charge.
Also some of the amino acid side chains are ionizable:
Group Amino Acid pK*____

Carboxy Glutamate, aspartate 4.0
Sulfhydryl Cysteine 8.0
Phenolic OH Tyrosine 10.0
Guanidino Arginine 12.5
Amino Lysine 10.8
Imidazole Histidine 6.0
______________________________________________
* pK values depend on the solvent environment, and vary widely in active
sites of enzymes and unique protein environments.
The number and pK values of ionizable groups in a molecule can be
determined experimentally by a titiration curve, which plots solution pH as a
function of increasing quantity of added base (or acid). Each titration plateau
indicates an ionizable group and its position shows the pK to that group.
Ionizable groups “buffer” the pH of a solution because they release or absorb
protons at pH values close to their pK.
You have to know the (approximate) structures of the 20 amino acids (consult
Meisenberg & Simmons, Page 27). The amino acid side chains can engage in a
number of nonconvalent interactions and convalent bonds:
Hydrophobic Hydrogen Salt Covalent

Amino Acid interaction bond bond bond
Glycine (Gly, G) (+) - - -
Alanine (Ala, A) + - - -
Valine (Val, V) ++ - - -
Leucine (Leu, L) +++ - - -
Isoleucine (Ile, I) +++ - - -
Serine (Ser, S) + + - Ph., CHO
Threonine (Thr, T) ++ + - Ph., CHO
Cysteine (Cys, C) + (+) + Disulfide
Methionine (Met, M) ++ - - -
Phenylalanine (Phe, F) +++ - - -
Tyrosine (Tyr, T) ++ + - Ph.
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Tryptophan (Trp, W) ++ + - -
Aspartate (Asp, D) - ++ +++ -
Asparagine (Asn, N) - +++ - CHO
Glutamate (Glu, E) + ++ +++ -
Glutamine (Gln, Q) + ++ - -
Lysine (Lys, K) ++ +++ - -
Arginine (Arg, R) + +++ +++ -
Histidine (His, H) - +++ + -
Proline (Pro, P) ++ - - -
Ph. = phosphate ester; CHO = glycosidic bond.

The isoelectric point (pI) of a molecule is the pH at which the number of
positive charges exactly equals the number of negative charges. While pK is a
property of an individual ionizable group, pI is a property of an entire molecule.
VIII. PROTEIN STRUCTURE
Polypeptides range in size from a few amino acids to hundreds or even

thousands. Proteins consist either of a single polypeptide chain, or they are
formed from separate polypeptide chains called subunits. Some proteins
contain a nonpolypeptide component, a so-called prosthetic group.
Peptide bonds are formed between the α-carboxy and α-amino groups of
two amino acids. Each peptide has an amino terminus, conventionally written on
the left, and a carboxy terminus, written on the right side.
R' O R''' O R[n-1]

H H H H
C N C N N C N COO-
NH3+ C N C N C
H H
O R'' O O R[n-2] O Rn
The peptide bond is rigid. Although technically a C-N single bond, it has
“partial double bond character”. Like the C=C bond, it is planar and cannot
rotate. The H and O of the peptide bond are in trans.
The other two bonds in the polypeptide backbone can rotate. Therefore the
polypeptide can fold, bend and twist itself into a variety of shapes. The resulting
̀higher order structure ́ is stabilized by noncovalent interactions. Levels of protein
structure:
1. The primary structure of a protein is its convalent structure. It is defined

by the amino acid sequence and any other convalent structures (such as
disulfide bonds) that may be present.
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2. The secondary structure is any regular, repetitive folding pattern in the
molecule. Only a few secondary structures are energetically possible:
In the α-helix, the polypeptide is wound in a right-handed spiral, with 3.6

amino acids per turn. This compact, rod-like structure is maintained by hydrogen
bonds between the components of the peptide bonds: Each peptide bond C=O
forms a hydrogen bond with the peptide bond N-H amino acid residues ahead of
it. Amino acid side chains face outward from the helix axis. Proline and glycine
don’t fit well into the α−helix. The α-helix is the most common secondary
structure in proteins. It occurs both in many fibrous (long, stretched-out) proteins
such as myosin and keratin, and in many globular (compact-shaped) proteins.
In the β-pleated sheet, the polypeptide backbone is stretched apart.
Different portions of the protein are aligned either parallel or antiparallel, forming
hydrogen bonds between their peptide in the β−pleated sheet, giving rise to
large, blanket-like structure.
3. The tertiary structure is the overall folding pattern of the polypeptide

chain. In globular proteins it consists of short sections of secondary structure
alternating with irregularly folded sequences. Tertiary structures are formed
primarily by hydrophobic interactions of amino acid side chains. Typical globular
proteins have a core of hydrophobic side chains, while hydrophilic side chains
are on the surface where they interact with water or with other proteins.
4. The quaternary structure is described by the subunit composition and

inter-subunit interactions of oligomeric proteins (proteins with more than one
subunit). Monomeric proteins have no quaternary structure.
Disulfide bonds are formed by an oxidative reaction between 2 Cys residues
after folding of the protein into its higher-order structure. They may be between 2
Cys residues in the same polypeptide (intra-chain), or between 2 different
polypeptides (inter-chain). Disulfide bonds are uncommon in cytosolic proteins.
IX. NON-POLYPEPTIDE COMPONENTS OF PROTEINS
Many proteins contain a non-polypeptide component. In these proteins the

polypeptide is called the apoprotein, and the non-polypeptide is called the
prosthetic group.
1. Glycoprotein contain mono- or (more commonly) oligosaccharides

convalently bonded to Ser, Thr or Asn side chains. Most plasma membrane
proteins and secreted proteins are glycoproteins.
2. Phosphoproteins contain phosphate bound by esterification of Ser, Thr
or Tyr side chains. These proteins become phosphorylated by protein kinases
and dephosphorylated by protein phosphatases. Phosphorylation changes
protein conformation. It is a very important mechanism for the physiological
regulation of enzyme activity and other biological processes.
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3. Lipoproteins are non-covalent aggregates of protein and lipid
(triglyceride, phospholipid, cholesterol). Most important are the plasma
lipoproteins, which ferry lipids through the bloodstream. Other proteins contain
sites of myristic acid or phosphatidylinositol plasma membrane anchors.
4. Nucleoproteins are non-covalent aggregates of protein with DNA or
RNA. Examples are chromatin (DNA + histone proteins), ribosomes and
spliceosomes (RNA + proteins).
5. Hemoproteins contain a heme-group bonded either covalently or non-
covalently. Heme is a ring structure with a coordinated Fe ion. Hemoglobin and
the cytochromes (oxidation/reduction catalysts) contain heme.
6. Flavoproteins contain flavin adenine dinucleotide (FAD) or flavin
mononucleotide (FMN) bonded covalently or non-covalently. Flavins are ring
structures which transfer electrons (+ protons) in many membrane associated
redox reactions.
7. Metalloproteins contain a metal ion. Many enzymes contain metal ions
which are involved in catalysis. There are many metal carrier and storage
proteins as well.
Several other groups can occur in enzymes. Nonpolypeptide components of
enzymes are called coenzymes.
X. PROTEIN DENATURATION
The normal interactions that maintain the higher-order structures of proteins

are weak and can be disrupted easily. Heat denaturation occurs when the
protein is heated to more than 50 – 70o C. It results in loss of biological activity
and precipitation. Renaturation is sometimes possible with small proteins
(ribonuclease, lysozyme) under laboratory conditions, but denaturation is
irreversible in the real world (example: boiled egg). Also other insults can
denature proteins:
Strong acids and bases denature proteins by disrupting ionic interactions.
Organic Solvents can denature proteins by disrupting hydrophobic
interactions. Proteins are not soluble in organic solvents.
Detergents also disrupt hydrophobic interactions. They can denature
proteins without precipitating them.
Small hydrophilic substances, such as urea, can denature proteins when
they are present in very high concentration.
Heavy metal ions (lead, mercury) bind to carboxylate or sulfhydryl groups of
proteins. That’s why they are toxic!
The covalent bonds in proteins are more robust, but peptide bonds are
hydrolyzed by heating in strong acids and bases, and by proteolytic enzymes.
Disulfide bonds are cleaved by reducing and oxidizing agents.
XI PROTEINS IN THE LABORATORY
1. Protein concentration in solution:
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a. Absorbance: Proteins do not absorb visible light (380-760 nm) and are
uncolored unless they contain either a flavin (FAD or FMN) or heme as
prosthetic group. However, almost all proteins contain tyrosine and/or
tryptophan. The aromatic rings of these amino acids absorb UV-light,
with
a maximum at 280 nm.
b. The biuret assay: In alkaline solution, copper salts form a violet
complex with substances containing two or more peptide bonds.
c. The Folin-Lowry Method: Similar to biuret, but more sensitive. Very
commonly used.
d. The ninhydrin reaction: Ninhydrin reacts with primary amino groups to
yield a purple product. Used for free amino acids, also proteins.
2. Separation and purification of proteins:
a. Precipitation: Proteins can be precipitated without denaturing them by
-Adjustment pf pH. Protein solubility is minimal at the isoelectric

point.
-Adjustment of salt concentration. A modest salt content enhances
solubility because intermolecular salt bonds are disrupted. High
salt concentrations (> 10%) reduce the solubility because the
salt competes with the protein for solvent.
-Organic solvents. The addition of water-miscible organic solvents
(ethanol, acetone) at low temperature precipitates proteins,
usually without denaturing them.
b. Dialysis: This method uses a porous cellophane membrane to separate

molecules by size: salt and small molecules are removed from the protein
solution.
c. Gel filtration: This method separates proteins (and other
macromolecules) by molecular size using small porous beads of a crosslinked
gel in a column chromatographic procedure. Big molecules come out first.
d. Ion exchange chromatography: The stationary phase contains charged
groups which interact with oppositely charged groups on the protein. Proteins
are eluted by increasing the salt concentration and/or changing the pH.
e. Electrophoresis: This method is based on the movement of charged
solutes in an electrical field. At a pH above its isoelectric point, a protein
migrates to the anode, below the isoelectric point to the cathode.
Electrophoresis can be done in gels, on thin layer, cellulose acetate foils, etc. It
is used in clinical laboratories to separate plasma proteins or diagnostically
important isoenzymes. It separates molecules by their charge/mass ratio.
However, if done in a cross-linked gel (polycrylamide, agarose), it separates by
size!
18
f. Isoelectric focusing: a pH gradient is set up in a gel. In the electrical
field, proteins stop migrating when the local pH equals their pI. Often now
combined with the use of electrophoresis to produce separation in two-
dimensions (2D-gels).
g. Affinity chromatography: A ligand (substrate, antibody, inhibitor, etc.) is
covalently linked to the stationary phase. The target protein binds and is then
eluted with free ligand.
h. HPLC = High Performance Liquid Chromatography: A lab work-horse
for the separation of drug-molecules, amino acids, peptides and small proteins.
This method normally separates based on the selective retention of analytes on a
silica-gel stationary phase. An organic solvent like acetonitrile is used to chase
off the retained proteins one at a time.
3. Protein detection
a. Immunological detection is done in ELISA (Enzyme-linked

Immunosorption Assay), and even more sensitive radioactive detection is done in
RIA (Radioimmunoassay). These methods can measure proteins at picomolar
and lower concentrations.
b. High resolution mass spectrometry (MS) is now used to sensitively
determine the presence of trace amounts of proteins in biological fluids.
4. Protein structural analysis
Amino acids sequences are known for many proteins. Steps in primary
structural analysis include:
Amino acid analysis. Complete acid hydrolysis of the protein, followed

by HPLC separation of the amino acids for quantitative analysis.
Fragmentation of the protein into shorter peptides, usually with
proteolytic enzymes.
Amino-terminal determination using chemicals which selectively react
with the terminal amino group.
Sequencing using Edman degradation chemistry.
Secondary structure analysis is usually done using circular dichroism
which measures the α-helix and β-sheet content.
Tertiary and quaternary structure analyses are now done using X-ray
crystal structure determination and nuclear magnetic resonance
(NMR) spectroscopy.
19
HANDOUT 1: OBJECTIVES IN SUMMARY
A. Molecular Structure
1. Recognize in chemical formulas the important functional groups and bond types, including
hydroxy, carbonyl, carboxy, sulfhydryl and amino groups, and ester, ether, glycosidic, thioester
and amide bonds.
2. Know the relative electronegativities of O,N,S,C and H.
State that chemical bond formation is usually endergonic while hydrolytic bond cleavage is
exogernic.
3. Define the term “energy rich bond”.
4. Describe the principal types of non-covalent interaction between biomolecules, including
hydrogen bonds, salt bonds, van der Waals interactions and hydrophobic interactions.
5. Describe structural features of biomolecules, which tend to increase or decrease water
solubility, including the effect of pH and charge pattern.
6. Know the definition of terms, such as, redox reaction, anion, cation, zwitterions, pH value,
isoelectric point and buffering capacity.
7. Application of the Henderson Hasselbalch equation to problems in which either the pH, pK or
the ratio of protonated/unprotonated form has to be calculated.
8. Recognition of asymmetric carbons in chemical structures and predict existence of optical
isomers.
9. Recognize the general structures of glycerol, fatty acids, and triglycerides in the chemical
formula.
10. Describe the properties of fatty acids and triglycerides with respect to water solubility,
chemical stability of the ester bond and effects of pH changes on protonation state water
solubility.
11. Recognition of the general structure of monosaccharides both in open –chain form and the
Harworth projection.
12. Define the terms aldose, ketose, triose, pentose, hexose, hemiacetal, hemiketal, and
glycosidic bond.
13. Name the chemical building blocks and bond types in nucleic acids.
B. Amino acids and protein structure.
1. Recognize the structures of the 20 major amino acids.

2. Name the non-covalent interactions that can be formed by the different amino acid side chains.
State the approximate pK values of alpha-amino and alpha-carboxy groups of amino acids and
the approximate side chain pK values of histidine, lysine, arginine, aspartate and glutamate.
3. Describe the structure of the peptide bond, including its partial double bond character, planarity
and ability to engage in hydrogen bonding.
4. Define the terms primary structure, secondary structure, tertiary structure, quaternary structure,
fibrous protein, globular protein and globulin.
5. Describe the dimensions of the α-helix and the β-pleated sheet, their presence in globular and
fibrous proteins and the role of hydrogen bonds in their formation.
6. Name the components of glycoproteins, lipoproteins, nucleoproteins, phosphoproteins, heme
proteins, flavoproteins and metalloproteins and describe the interaction between the polypeptide
and prosthetic group in each case.
7. Describe the process of heat denaturation of proteins and its biological consequences.
8. List various types of denaturing agents and specify the mechanism by which they cause
denaturation.
9. State the susceptibilities of peptide bonds and disulfide bonds to acids, bases, oxidizing and
reducing agents.
10. Know the use of UV absorbance, the biuret and Lowry methods for the measurement of
protein concentrations.
11. Name the principles by which proteins are separated in different types of chromatography and
electrophoresis.
20
BIOCHEMISTRY and GENETICS
Handout 2
ENZYMATIC REACTIONS
Thermodynamic properties are those features of a chemical reaction that

are related to its energy balance and equilibrium. Kinetic properties are those
related to the rate (velocity) of the reaction. Only the kinetic properties of
reactions are changed by enzymes.
I. EQUILIBRIUM REACTIONS
In theory, all chemical reactions are reversible. If only noncovalent

interactions are involved, as in protonation-deprotonation, antigen-antibody
binding and hormone-receptor binding, the reactions are always found at
equilibrium concentrations. Chemical reactions in which covalent bonds are
formed and broken are also reversible, but there is an energy barrier to the
reaction. Therefore the reactions are not always found at equilibrium
concentrations. The equilibrium is described by the equilibrium constant (Keq).
For the reaction
A B
[B]
Keq =
[A]
For the Reaction A + B C + D
[C] x [D]
Keq =
[A] x [B]
[A], [B], [C] and [D] are the molar concentrations of reactants at equilibrium.
Although the equilibrium constant is a thermodynamic characteristic, it is
related to the rate constants of forward (k+1) and reverse reactions (k-1) :
k1
A B
k-1
A rate constant is the proportion of a substrate transformed into a product
per unit of time. In this example, the actual rates (V, velocity, or speed) of the
forward and backward reaction are:
21
Vforward = k1 x [A]
Vreverse = k-1 x [B]
At equilibrium, the rates of the forward reaction and the backward reaction
are equal.
k1 x [A] = k-1 x [B]
k1 [B]
=
k-1 [A]
Enzymes do not change the equilibrium constant. They modify k+1 and k-1
in exactly the same proportion.
II. ENERGETICS
Change in Enthalpy H:
∆H = ∆E + (P x ∆V)
∆ E is the change in the energy content of the reacting molecules in the

system, P is the pressure, and ∆V is the volume change. Px∆V is the work done
by the system. It is near-zero in biological systems, therefore ∆H≈∆E. ∆H
corresponds to the overall change in chemical bond energies during the reaction.
Change in Free Energy (Gibbs Energy) G:

∆G = ∆H – T x ∆S
T is the absolute temperature (in Kelvin), and ∆S is the change in entropy.

Entropy is a measure for the randomness of a system. ∆G is the force that
drives chemical reactions: the reaction can proceed only when ∆G is negative.
Note that the preferred states in nature are low enthalpy and high entropy.
Physical processes in which chemical bond energies don’t change (∆H = 0),
for example diffusion across a membrane, are also driven by ∆G. These
processes are driven by an increase in entropy (i.e. a negative value for [Tx ∆S]).
The body has to use chemical bond energy to combat the tendency for
increasing entropy. That’s what metabolic energy is good for!
Exothermic reaction: ∆H < 0, heat is released.

Endothermic reaction: ∆H > 0, heat is absorbed
Exergonic reaction: ∆G < 0, reaction can proceed
Endergonic reaction: ∆G > 0, reaction cannot proceed.
When ∆G = 0, the reaction is at equilibrium.

∆G is affected by the concentrations of the reactants. The standard free
energy change, ∆G0’, is the free energy change under biological standard
conditions:
- Reactant concentrations are 1 mol/L, except H+ and H20.
22
- pH = 7.0
- Water is the solvent
- Gas concentrations are 1 atmosphere (760 mm Hg) partial pressure
In calculations of ∆G0 and Keq (the equilibrium constant in water at pH 7),
[H2O] has a value of 1. The proton concentration of 10-7 M is given a value of 1.
The actual free energy change (∆G) for the reaction {A + B = C + D} is:
[C] x [D]
∆G = ∆Go' + 2.303 RT Log
[A] x [B]
R = gas constant (1.98 x 10-3 kcal/mol x K)
At 25o C this becomes:

[C] x [D]
∆G = ∆Go' + 1.36 Log
[A] x [B]
When we are at equilibrium,

[C] x [D]
Keq =
[A] x [B]
∆G = 0 at equilibrium, therefore there is a logarithmic relationship between
Keq and ∆G0’:
Keq ∆G0’
10-5 + 6.82
10-4 + 5.46
10-3 + 4.09
10-2 + 2.73
10-1 +1.36
1 0
101 - 1.36
102 - 2.73
103 - 4.09
104 - 5.46
105 - 6.82
III. ORDER OF REACTIONS
Zero-order reaction: The reaction rate ν is independent of the substrate

concentration. Zero-order reactions are observed only in catalyzed reactions
when the catalyst is saturated with substrate. The decrease of the substrate
concentration is linear. Formula (k = rate constant, units are mol-sec-1):
ν=k
First-order reaction: The reaction rate is proportional to the substrate
concentration (units of k are sec-1):
23
ν = k x [A]
The decrease of the substrate concentration is asymptotic, and its half-life
can be determined.
Second-order reaction: The reaction rate depends on the
concentrations of 2 substrates:
ν = k x [A] x [B]
Pseudo-first-order reaction: There are 2 substrates, but one is present in

large excess and is not rate-limiting. kap describes the apparent rate constant for
these conditions. Example: Hydrolysis-reactions.
A + H20 B + C
ν = kap x [A]
IV. PRINCIPLES OF ENZYMATIC CATALYSIS
All ‘true’ chemical reactions, catalyzed or uncatalyzed, go through a short-

lived unstable transition state ():

G
Free No catalyst
∆Gact
Energy
With catalyst
S
Progress of Reaction
The transition state has a higher free energy content than the substrate and
the product, creating an energy barrier between S and P. This energy barrier,
known as the free energy of activation (∆Gact), is the difference in the free
energy contents of S and T. A condition that is stable kinetically, but is not the
most favorable state thermodynamically, is called metastable. The human body
is metastable.
Enzymes increase the rate of the reaction by decreasing the free energy of
activation. The transition state of the catalyzed reaction is different from that of
the uncatalyzed reaction. It has a lower free energy content. The equilibrium of
the reaction remains unchanged.
The active site is the portion of the enzyme molecule that binds the
substrate and catalyzes the reaction:
- It occupies only a small portion of enzyme molecule.
- It binds the substrate by noncovalent interactions
- It includes amino acids which are widely separated in the primary structure of
enzyme but come close together in its higher-order structure
- If the enzyme contains a prosthetic group, it is in the active site.
24
- Substrate binding may trigger a conformational change in the enzyme molecule
which brings catalytically active groups to the substrate. This is called
induced fit, as opposed to the lock-and-key model
- Catalysis is effected by direct interactions between the substrate and the active
site of the enzyme.
Substrate specificity is determined by the ability of the substrate to bind

properly to the active site of the enzyme. Enzymes are steroselective.
V. MICHAELIS-MENTEN KINETICS
An enzymatic reaction with a single substrate and a single product can be

broken down into the sequence:
k1 k2 k3
E +S E-S E-P E + P
k-1 k-2 k-3
E = enzyme; S = substrate; P = product.
E-S = enzyme-substrate complex; E-P = enzyme-product complex;
In Michaelis –Menten Kinetics, we make a few assumptions:

1. The reaction has only one substrate
2. The product concentration [P] is very low, therefore we neglect the
backward reaction of P to E-S.
3. The concentration of E-S is constant for the duration of the reaction.
This is the 'steady-state' assumption.
4. We consider only the initial time period, assuming that the substrate
concentration [S] remains in excess.
5. k2, also called the catalytic rate constant kcat, is rate-limiting for the
overall reaction. Therefore k1 and k2 only influence [E-S] at steady-state.
Therefore the steps in enzyme catalysis simplify to:
k1 kcat
E +S E-S E + P
k-1
The rate of the reaction is:
V = kcat x [E.S]
The reaction rate is maximal when all enzyme molecules are present as
enzyme-substrate complex (Note: Etotal = ET = E-S + Efree):
Vmax = kcat x [ET]
ET is the total enzyme concentration. kcat corresponds to the turnover

number: the maximal number of substrate molecules converted to product by
one enzyme molecule per second. It is a constant for any given combination of
substrate, temperature and pH. Vmax is measured at very high substrate
concentrations when essentially all enzyme molecules are present as enzyme-
substrate complex, and [E-S] = [E]Total.
25
The Michaelis constant (Km) is the substrate concentration at which the
reaction rate is half-maximal. The Km is the substrate concentration at which one
half of the enzyme molecules are present as enzyme-substrate complex, and
reaction velocity is half-maximal. Km is also a ratio of rate constants:
k2 + k-1
Km =
k1
Km and Vmax can be determined experimentally by varying the substrate

concentration. Enzymatic reactions show saturation kinetics. This can be
shown graphically, either directly or in a Lineweaver-Burk plot:
In the Lineweaver-Burk plot, the intersection with the y-axis is 1/Vmax, and
the intersection with the x-axis is –1/Km.
Limiting conditions:
1. If [S] >> Km: V ≈ Vmax = kcat x [ET]
The reaction rate is proportional to the enzyme concentration but

independent of the substrate concentration: the reaction is zero-order.
kcat
2. If [S] << Km: V ≈ Km x [E] x [S]
The reaction rate is proportional not only to the enzyme concentration but
also the substrate concentration. To determine enzyme activities in the clinical
laboratory, very high substrate concentrations are used.
The relationship between [S], Km, V and Vmax is described by the Michaelis-
Menten equation:
V = Vmax x [S]
[S] + Km
26
Note that in bisubstrate reactions, each of the substrates has its own Km.
VI. DETERMINANTS OF THE REACTION RATE
1. Temperature
Within a certain temperature range, the reaction rate increases with
temperature. The Q10 value is the factor by which a reaction is accelerated by a
10-degree rise temperature. Typical Q10 values:
Q10 = 2.0 -5.0 for uncatalyzed reactions.
Q10 = 1.7 - 2.5 for enzymatic reactions.
At high temperature ( > 50 –60o C), enzyme activity falls off quickly because
the enzyme is denatured.
2. pH, The hydrogen ion concentration

The pH-dependence of enzymes is bell-shaped. This is because the groups
in the active site have to be in the proper protonation state for catalysis, and
because reversible conformational changes and finally irreversible denaturation
occur at extreme pH valves.
3. Enzyme inhibition
Some drugs and toxins are enzyme inhibitors.
- Reversible inhibition: the inhibitor binds to the enzyme noncovalently.

Enzyme activity can be restored by removing the inhibitor.
- Irreversible inhibition: The inhibitor forms a covalent bond with the
enzyme protein, frequently in the active site. In the body, this kind of
inhibition can be overcome only by the synthesis of new enzyme.
Types of reversible inhibition:

In competitive inhibition, the inhibitor competes with the substrate for
the active site. In most cases, the inhibitor is a close structural analog of
the normal substrate. Competitive inhibition increases the Km while
leaving the Vmax unchanged. Examples:
1. The enzyme succinate dehydrogenase catalyzes the reaction:

OOC-CH2-CH2-COO + FAD OOC-CH=CH-COO + FADH2
The enzyme is inhibited competitively by malonate:

OOC-CH2-COO
Malonate binds to the active site of the enzyme like succinate, but cannot
be converted to a product.
27
2. Methanol (CH3-OH) is dangerous because it is converted to the toxic
metabolite formaldehyde by the liver enzyme alcohol dehydrogenase (ADH).
Methanol poisoning can be treated by ethanol (H3C-CH2-OH) because ethanol
competes with methanol for the enzyme. In this case, the inhibitor (ethanol) is
converted to a product (acetaldehyde).
In noncompetitive inhibition, the inhibitor binds to a site distinct from the

substrate binding site. If the inhibitor binds equally well to the free
enzyme and the enzyme-substrate complex, Km remains unchanged but
Vmax is reduced.
In uncompetitive inhibition, the inhibitor binds to the enzyme-substrate

complex but not the free enzyme. This type of inhibition reduces both
Km and Vmax.
Example for irreversible inhibition: Organosphosphate compounds are used

as pesticides and nerve gases. They react convalently with a serine residue in
the active site of acetylcholinesterase, the enzyme that destroys the
neurotransmitter acetylcholine at cholinergic synapses:
O CH3 O CH3
Enzyme-Ser-OH + F P O C
H CH
Enzyme SerO P O C
H CH
+ HF
CH3 3 CH3 3
VII. ALLOSTERIC ENZYMES
Allosteric enzymes switch back and forth between different conformations

that differ in their catalytic properties. They are usually multi-subunit. If there is
more than one active site, there may be cooperativity. Most regulated enzymes
in metabolic pathways are allosteric. They are regulated by:
- Allosteric effectors which bind reversibly outside the active site.
- Phosphorylation and dephosphorylation by regulatory enzymes called
protein kinases and protein phosphatases.
Both the Km and the Vmax of the enzyme may be affected. Allosteric
enzymes do not obey Michaelis-Menten kinetics. Note that allosteric regulation
may be positive or negative.
Substrate concentrations in the living cell are, in most cases, well below the
Km of the enzyme. Therefore an increased supply of substrate leads not to a
buildup of the substrate but to its enhanced metabolism.
VIII. REACTION TYPES

Most enzymes are named according to their substrate and reaction type,
with "ase" attached to the end. They are classified by their reaction type:
1. Oxidoreductases 4. Lyases
2. Transferases 5. Isomerases
28
3. Hydrolases 6. Ligases
1. Oxidoreductases catalyze electron transfers. Most of these reactions

involve hydrogen or oxygen. Dehydrogenases transfer hydrogen with
electrons. These reactions require a coenzyme as hydrogen donor or
acceptor. Example:
COO- COO-
OH + NAD+ O + NADH + H+
CH3 CH3
Lactate Pyruvate
Oxygenases use molecular oxygen as a substrate.

Monooxygenses incorporate one of the O atoms into an organic
molecule, and dioxygenases both. Peroxidases use hydrogen
peroxide as a substrate.
2. Transferases transfer a group from one molecule to another.
3. Hydrolases cleave bonds by the addition of water. They are named
according to their substrates:
Peptidases (proteases) cleave peptide bonds
Glycosidases cleave glycosidic bonds in carbohydrates
Esterases cleave carboxylic ester bonds
Phosphatases cleave phosphate (ester) bonds
Lipases cleave ester bonds in triglycerides
Phospholipases cleave bonds in phospholipids
Nucleases cleave phosphodiester bonds in nucleic acids
4. Lyases remove groups nonhydrolytically, leaving a double bond.

5. Isomerases interconvert isomers
6. Ligases form a bond while cleaving an energy-rich phosphate bond
(usually in ATP).
IX. REACTION MECHANISMS
Catalysis is effected by:
1. The entropy effect: The substrates of a 2-substrate reaction are

brought together in an active site of the enzyme. This increases the probability of
collisions between the reactive groups tremendously.
2. Transition state stabilization: The enzyme makes favorable
noncovalent interactions with the transition state of the reaction, thereby
stabilizing it and decreasing its free energy content.
3. Acid-base catalysis: The enzyme exchanges protons with the
substrate during the reaction. This requires ionizable groups in the active site of
29
the enzyme. Also electron pair acceptors and electron pair donors (Lewis acids
and Lewis bases) in the active site can participate in the reaction.
4. Covalent catalysis: The enzyme forms a transient covalent bond with
substrate during catalysis.
Example: The serine proteases (trypsin, chymotrypsin, thrombin etc.)

Catalyze their reactions by a combination of acid-base catalysis, transition state
stabilization, and covalent catalysis. They form a covalent acyl-enzyme
intermediate which is then cleaved hydrolytically.
X. COENZYMES
Some (not all) enzymes require a coenzyme for their reaction. There are 2
types of coenzymes:
1. Some coenzymes are bound permanently to the active site of the

enzyme, either covalently or noncovalently. These coenzymes are true
prosthetic groups.
2. Some coenzymes are soluble molecules which associate with the
enzyme active site only during the reaction. They function as cosubstrates.
Like other substrates, they become changed chemically during the reaction and
have to be regenerated in a different reaction.
1. Adenosine Triphosphate (ATP)

ATP is a cosubstrate in reactions of energy metabolism. Chemically, it is a
nucleotide:
NH2
N N
N
} Adenine
}
O O O O
O P O P O P O Ribose
O O O HO OH
ATP can serve as the energetic currency of the cell because it contains 2
energy-rich phosphoanhydride bonds, each with a free energy content of 7.3
kcal/mol. ATP is synthesized from ADP + phosphate during catabolic reactions,
most of this by oxidative phosphorylation in the mitochondria.
ATP hydrolysis drives:

- Biosynthetic (anabolic) reactions.
- Active transport across membranes
- Cell motility and muscle contraction
30
There are 2 modes of ATP hydrolysis:
(1) ATP + H2O --------Æ ADP + Phosphate

(2) ATP + H2O --------Æ AMP + Pyrophosphate
The second mode provides more energy (~2x) because pyrophosphate is

quickly hydrolyzed to phosphate to pyrophosphatase. ATP is used for:
1 Nucleic acid synthesis. ATP is an immediate precursor for RNA

synthesis and an indirect precursor for DNA synthesis.
2. Phosphorylation reactions. ATP can transfer its last phosphate to
an acceptor molecule. These reactions are catalyzed by kinases, a type of
transferase. Example:
H2C OH H2C OH
HO CH + ATP HO CH O + ADP
C OH C O P O
H2 H2
O
Glycerol Glycerol 3-phosphate
3. Coupling to endergonic reactions. Endergonic reactions cannot
proceed in the cell unless the enzyme couples the endergonic reaction to ATP
hydrolysis. Example:
Palmitic acid + Coenzyme A Palmitoyl-CoA + H2O ∆G0’ = +8.0 kcal/mol
This reaction cannot proceed in the indicated direction. The reaction that
actually takes place in the body is:
Palmitic acid + CoA + ATP Palmitoyl-CoA + AMP + Pyrophosphate ∆G0’ =

+0.7 kcal/mol
This reaction proceeds in the indicated direction because its ∆G0’ is close to
zero and one of the products (pyrophosphate) is rapidly removed from the
equilibrium.
Important for the reaction equilibrium:
1. The reaction proceeds whenever the actual ∆G (not the ∆G0’!) is

negative. Therefore the reaction can be driven in the right direction when the
concentration of a substrate is very high and/or that of a product is very low. In
our example, pyrophosphate is very low. Also, the [ATP] / [AMP] ratio in the cell
is about 100, and this lowers the real ∆G by about 2.73 kcal/mol.
31
2. When ATP is hydrolyzed in the reaction, the ∆G0’ is reduced by 7.3
kcal/mol. This is usually sufficient to drive the reaction.
In the cell, the adenine nucleotides are in equilibrium through the adenylate
kinase (myokinase) reaction:
ATP + AMP 2 ADP
The energy status of the cell can be described by the cellular [ATP] /[ADP]
ratio, or by the energy charge:
Energy charge = [ATP] + ½ [ADP]

[ATP] + [ADP] + [AMP]
The nucleotides guanosine triphosphate (GTP), uridine triphosphate (UTP),

and cytidine triphosphate (CTP) are also used as coenzymes in some energy-
dependent reactions.
2. Redox Coenzymes
Some coenzymes transfer either naked electrons or electrons + protons
(hydrogen atoms) during redox reactions. Nicotinamide-adenine dinucleotide
(NAD+) and its phosphorylated derivative NADP+ are cosubstrates in
dehydrogenase reactions. Structures:
O NH2
{ }
C NH2 Adenine
Nicotinamide N N
+
N N N
}
O O O O
Ribose
O P O P O
O O HO OH
O O
The flavin nucleotides flavin mononucleotide (FMN) and flavin-adenine
dinucleotide (FAD) are also hydrogen carriers, but they function as tightly bound
prosthetic groups of the flavoproteins. Structure of FAD:
32
O
{
H3C N
Riboflavin NH
H3C N N O
NH2
CH2
OH
OH
N
N N
N
} Adenine
OH
}
O O O
H2C Ribose
O P O P O
O O HO OH
Straight electrons (without protons) can be transferred by many iron-

containing proteins. In the iron-sulfur proteins the iron is bound to cysteine side
chains, and in the heme proteins it is part of the heme group. The iron switches
between the ferrous (Fe2+) and the ferric (Fe3+) state during the electron transfer.
Also other metal ions are common in enzymes, including zinc, copper and
manganese. In some enzymes the metal ion participates in electron transfers,
but in others it acts as a Lewis acid (electron pair acceptor).
3. Other Coenzymes.
There are many other coenzymes, each with its own special function.
Examples:
1. Coenzyme A (CoA) is a cosubstrate. It contains a sulfhydryl group

which forms thioester bonds with organic acids. Example:
GMP + Pyrophosphate
GTP
O
CoA SH + H3C COO- CoA S CH3
Coenzyme A Acetate Acetyl-CoA
2. S-Adenosylmethionine (SAM) is a cosubstrate that contains a methyl

group. This methyl group is transferred to an acceptor during enzymatic
reactions.
Many, but not all, coenzymes contain a vitamin as part of their structure:
Nicotinamide (niacin) in NAD+ and NADP+, ribloflavin in FMN and FAD,
pantothenic acid in coenzyme A.
XI. MEMBRANE TRANSPORT
Regulated transport across membranes is required for the exchange of

nutrients, metabolic intermediates, and waste products; to maintain ion gradients;
and to transfer nutrients, electrolytes, and water across absorptive epithelia.
33
Passive diffusion across the lipid bilayer depends on the physical
properties (size, lipid solubility) of the diffusing molecule. No energy required.
Carrier-mediated transport requires an integral membrane protein
(“carrier”) that interacts with the solute.
Carrier-mediated transport shows:
- Substrate specificity
- Saturability
- Specific inhibition and regulation
The carrier forms a gated channel that undergoes a conformational change
during the transport cycle.
Passive transport does not require an energy source and is always down
the electrochemical gradient. It includes passive diffusion (no carrier involved)
and facilitated diffusion (through a carrier).
Active transport is always carrier-mediated. It can accumulate a solute
against its electrochemical gradient. Primary active transport hydrolyzes ATP
while secondary active transport transports a second solute down its
electrochemical gradient.
Uniport: The carrier transports at least one solute.
Cotransport: The carrier transports at least two different solutes.
- Symport: Two solutes transported in the same direction.
- Antiport: Two solutes transported in opposite directions.
Note: secondary active transport is always cotransport.
Electroneutral transport: No net transport of electrical charges.
Electrogenic transport: Net transport of electrical charges.
1. Examples of Passive Diffusion
- Most membranes are relatively permeable to water, although there are

also water channels in some cells.
- Osmosis is the passive diffusion of water from a compartment with lower
solute concentration to one with higher solute concentration.
- Biological membranes are permeable to various extents to some very
small hydrophilic molecules: urea, methanol, ethanol.
- Blood gases (O2, N2, CO2) are permeable, but the bicarbonate ion is not.
- Membranes are permeable for small lipid-soluble substances (fatty acids,

steroid hormones, some drugs). If the molecule is ionizable, only the
uncharged form diffuses.
2. Examples of Facilitated Diffusion

- Glucose uptake by liver, erythrocytes, etc. is effected by an
electroneutral uniport carrier.
- The blood-brain barrier transport of glucose and amino acids.
3. Examples of Contransport
34
Symport:
- Sodium contransport of amino acids and glucose in the intestinal
mucosa and the renal tubules, and of amino acid neurotransmitters into the
nerve terminal.
Antiport (=” exchange diffusion”).
- Sodium-calcium exchange across plasma membrane of many cells.
- Shuttles in the inner mitochondrial membrane.
4. Examples of Active Transport
- The Na+, K+ATPase (sodium-potassium ATPase) is in the plasma

membrane of all cells with highest activity in brain and muscle. The sodium-
potassium ATPase is an electrogenic antiporter, with 3 sodium pumped out of
the cell and 2 potassium pumped into the cell. The transport cycle requires the
phosphorylation of an aspartate side chain in the carrier by ATP. It transports
Na+ out of and K+ into the cell.
- A Ca2+ -ATPase is present in the sarcoplasmic reticulum of muscle fibers
and in the plasma membrane of many cells. It pumps calcium out of the
cytoplasm and works by a mechanism similar to that of the Na+, K+ ATPase.
Membrane ATPases consume 10 – 30% of the basal metabolic rate.
Cardiotonic steroids inhibit the sodium-potassium ATPase, thereby
reducing the sodium gradient across the plasma membrane. This impairs the
sodium-dependent calcium-extrusion mechanism. Increased intracellular
calcium leads to an increased force of contraction (positive inotropic effect).
XII. Homeostasis Of The Intracellular Environment
Cellular processes depend on temperature, pH, and in many cases, the

correct concentrations of inorganic ions. Differences between intra-and
extracellular environment:
1. Ion concentrations:
Ion Extracellular (mM) Cytoplasmic (mM)
Na+ 140 10
K+ 4.7 140
Mg2+ 1.4 30
Ca2+ 2.5 0.001
Cl- 113 4
HPO42- 2.0 11
HCO3- 2.8 10
35
The calcium concentration is very low in the cytoplasm but much higher in
mitochondria and ER. Phosphate is high in the cell because of its role in energy
metabolism (ATP synthesis!). The transmembrane gradients of sodium and
potassium are required for cell excitability.
2. The pH is about 7.4 in blood plasma and extracellular fluid, but
near-neutral in the cytoplasm: pH = 6.8 at 370C, pH = 7.0 at 250C. The pH
difference across the plasma membrane favors the transfer of carbon dioxide out
of the cell, and it facilitates the removal of acidic products such as lactic acid.
3. As far as possible, reducing conditions are maintained inside the
cell. This is needed to protect proteins, lipids, and nucleic acids from oxidative
damage.
4. Most of the cellular metabolites are charged at pH 7. This is
because the important acidic groups (carboxy and phosphate) have pKs below 7,
and the important basic groups (amino groups) have pKs above 7. It means that
most metabolites require carriers to cross membranes.
Physiological buffer systems include:
- Phosphate
- Bicarbonate
- Protein
Of these three systems, proteins are considered the most important

because of the large quantity of protein present in the body (about 12 kg). The
amino acid histidine is most important because its side chain pK is not too far
from the physiological cellular pH. Also, unlike the phosphate system, the
histidine side chain dissociates with a temperature dependence similar to that of
water.
The buffer systems can be overwhelmed in disease states, leading to
acidosis or alkalosis. Types:
Respiratory acidosis: Retention of carbon dioxide which forms carbonic
acid. In lung diseases.
Respiratory alkalosis: Abnormality low carbon dioxide and carbonic acid
because of hyperventilation
Metabolic acidosis: Overproduction of organic acids (lactic acid, ketone
bodies …), or failure of the kidneys to excrete excess protons.
Metabolic alkalosis: Abnormal loss of acid, for example by vomiting acidic
stomach contents.
36
A. Biochemical reactions.
1. Define the equilibrium constant of chemical reactions and the terms enthalpy,
entropy and free energy.
2. Describe in qualitative terms the relationship between free energy change
and equilibrium constant.
3. Describe the difference between thermodynamic and kinetic properties of
reactions.
4. Compare the effect of enzymes on thermodynamic and kinetic properties of
biochemical reactions.
5. Describe the role of enzyme-substrate complex in enzymatic catalysis, and
point out the reversible, non-covalent nature of the initial interaction between enzyme and
substrate.
6. List the major classes of enzymes: Oxidoreductases, transferases, ligases,
lyases, hydrolases and isomerases.
7. Identify the difference between the cosubstrate and the prosthetic group of
an enzyme.
8. Describe the structure of ATP, with emphasis on its energy rich bonds and
list the reaction types in which ATP participates.
9. Define the energy charge and state the relative abundances of ATP, ADP,
and AMP in healthy and metabolically stressed cells.
10. Relate the coenzymes NAD, NADP, FAD, heme and SAM to the reaction
types in which they participate.
B. Enzymatic catalysis.
1. Recognize the difference between zero-order and first-order reaction, and

state the conditions leading to zero of first order kinetics in catalyzed reactions.
2. State the importance of the transition state in chemical reactions, its relative
free energy content, and the effect of enzymes on the transition state.
3. Describe the active site concept in enzyme catalysis and list the basic
assumptions made in Michaelis-Menten kinetics.
4. Describe the relationship between substrate concentration and reaction rate
for typical enzymatic reactions.
5. State the importance of Km and Vmax for the rate of enzymatic reactions,
and Vmax and Kcat/Km for the rate of enzymatic reactions at high and low substrate
concentrations.
6. Describe the effects of temperature and pH for the rates of enzymatic
reactions.
7. Describe the effects of competitive, noncompetitive and irreversible inhibition
on the kinetic parameters of enzymatic reactions.
8. Name the four principal mechanisms in enzymatic catalysis and describe the
reaction types catalyzed by the six principal classes of enzymes.
37
BIOCHEMISTY and GENETICS I
Handout 3
DNA AND GENE EXPRESSION
I. DNA STRUCTURE.
1. Bases, Nucleosides and Nucleotides

DNA contains the two purine bases adenine (A) and guanine (G), and the
pyrimidine bases cytosine (C) and thymine (T). RNA contains uracil (U) instead
of thymine.
NH2 O
N N N N
N N H2N N N
H H
Adenine Guanine
O NH2 O
CH3
HN N HN
O N O O
H N N
H H
Thymine Cytosine Uracil
Nucleosides consist of a base linked by an N-glycosidic bond to the 1’ –

carbon of ribose or 2'-deoxyribose.
Examples:
Adenosine = A + ribose
Guanosine = G + ribose
Cytidine = C + ribose
Uridine = U + ribose
2'-deoxyadenosine = A + 2'-deoxyribose
2'-deoxythymidine = T + 2'-deoxyribose
Nucleotides are nucleoside derivatives carrying 1, 2 or 3 phosphate

groups at the 5’-carbon of ribose or 2-deoxyribose. They are named as
derivatives of their corresponding nucleosides. Examples:
A + ribose + 1 phosphate = Adenosine monophosphate (AMP)

U + ribose + 3 phosphates = Uridine triphosphate (UTP)
38
2. DNA structure.
DNA is an unbranched polymer of 2-deoxyribonucleoside
monophosphates. These nucleotides are linked by phosphodiester bonds
between the 3’ end of one 2-deoxyribose and the 5’ end of the next 2-
deoxyribose. The phosphate groups are negatively charged. The molecule has
a polarity, with a 5’ end and a 3’ end. Conventionally, the 5’ end is written left
and the 3’ end right. The primary structure of the DNA strand can be described
by its base sequence.
The Watson-Crick double helix (= B-DNA) is the principal higher-order
structure of DNA. Important features:
1. The two strands of the double helix are antiparallel.
2. The bases face inward to the helix axis while the sugar-
phosphate backbone forms two ridges.
3. There are a major groove and a minor groove which are
lined by the edges of the bases.
4. In each strand, the bases are stacked flat one on top of the
other.
5. The bases interact by hydrogen bonds, forming A-T and C-G
base pairs
6. The two strands form a right-handed helix with about 10
bp's per turn.
This structure has several important implications:

. A large number of unique DNA sequences can be
generated by permutations of the 4 bases.
. The edges of the bases are exposed in the major and minor
grooves. Therefore the base sequence of the DNA can be recognized
by DNA-binding proteins.
. The two strands can be separated easily
. The base sequence of one strand predicts exactly the base
sequence of the complementary strand.
3. Chemical stability
The covalent structure of DNA is quite stable. Rigorous conditions (boiling
in strong acid) are required to break the covalent bonds. The double helix,
however, is destroyed by heat-denaturation (“melting” of DNA).
1. Short DNAs melt more easily than long DNAs.

2. A-T-rich DNA melts more easily than C-G-rich DNA.
3. Low ionic strength and alkaline pH favor melting.
The viscosity of the DNA solution decreases and the UV- absorption
(measured at 260 nm) increases. Denatured DNA renatures when cooled slowly.
This process is called annealing. Short DNAs anneal much faster than long
DNAs.
39
4. Supercoiling
DNA can be overwound, with less than the canonical 10 base-pairs/turn.
This is called a positive supertwist. Or it can be underwound, with more than
10 bp/turn. This is called a negative supertwist. Over- and under-winding can
occur when the DNA duplex is circular (in prokaryotes), or when the ends of the
duplex are firmly attached to structural proteins (in eukaryotes). The
supertwisting is regulated by topoisomerases. Usually, the DNA in cells is
moderately underwound. This facilitates strand separation for transcription and
DNA replication.
II. DNA REPLICATION
1. Semiconservative replication
Mechanism of DNA replication:
. The double helix unwinds. The point where unwinding occurs is called
the replication fork
. New DNA is synthesized in the replication fork, using the old strand as
a template
. The old strand keeps unwinding until the whole DNA is replicated.
. The replicated DNA consists of one complete old strand and one complete
new strand.
2. DNA polymerases.
New DNA is sythesized by DNA polymerases. Important properties of
bacterial DNA polymerases.
. They require deoxyribonucleoside triphosphate as precursors. dATP,
dGTP, dTTP. Pyrophosphate is released during the polymerization reaction.
. They synthesize DNA from 5’ - - -> 3’
. They require a single-stranded DNA template. Therefore the parental
DNA duplex has to unwind first before the DNA polymerases can start.
. They read their template from 3’ - - -> 5’. Therefore the template
strand and the new strand are antiparallel. All base-paired nucleic acids are
antiparallel.
. They incorporate only nucleotides that make proper base pairing with
the base in the template strand.
. They possess a 3’ –exonuclease activity which is specific for
mismatched bases and which is used for proofreading.
. Many bacterial DNA polymerases have a 5’ –exonuclease activity
which is required for DNA repair.
. The DNA polymerases require a primer with a free 3’ –hydroxy group.
Bacteria have three DNA polymerases:
Poly I: Most important for DNA repair. It has a low binding affinity for
its template, and therefore it makes only short pieces of DNA at a time: Low
processivity.
40
Poly II: Unknown function
Poly III: The essential enzyme for DNA replication. Very high processivity.
Thanks to the 3’ –exonuclease activities of the DNA polymerases, the

error rate is only about one misincorporated base per 100 million nucleotides.
3. Steps in bacterial DNA replication.

1. E. coli has a circular chromosome (≈3 x 106 bp) with a single
replication origin called Ori-c. Initiator proteins associate with Ori-c to start
unwinding.
2. A helicase unwinds the DNA. This requires ATP.
3. The topoisomerase gyrase relaxes positive supertwists and actively pumps
negative supertwists into DNA (ATP-dependent). This prevents overwinding ahead of
the moving replication fork. The gyrase is not in the replication fork.
4. Single-strand DNA binding proteins (SSB proteins) bind the single-
stranded DNA to prevent annealing.
5. A specialized RNA polymerase called primase synthesizes a small
RNA primer.
6. Poly III starts DNA synthesis at the 3’ end of the primer
7. One of the new strands, the leading strand, is synthesized
continuously.
8. The other strand, the lagging strand, is synthesized piecemeal. The
pieces are called Okazaki fragments.
9. The small RNA pieces at the 5’ ends of the Okazaki fragments are
removed by Poly I and replaced by DNA.
10. The fragments of the lagging strand are connected by DNA ligase.
III. RNA AND TRANSCRIPTION.
1. RNA Structure
RNA can form a double helix like DNA, but is usually single-stranded except in
some viruses. Cellular RNAs contain both base-paired segments (“stems”) and unpaired
segments (“loops”). An RNA strand can also anneal with a complementary DNA strand.
Annealing between nucleic acids of different kinds or from different sources, for example
DNA with RNA, or human DNA with chimpanzee DNA, is called hybridization.
2. Types of RNA
There are three major types of cellular RNA, all of them synthesized as
copies of DNA sequences:
a) Messenger RNA (mRNA) carries the information of the linear DNA
sequence to the ribosomes where proteins are synthesized. In eukaryotes each
mRNA is copied from a single gene and encodes a single polypeptide chain
(“monocistronic” mRNA). In prokaryotes most mRNAs are copied from a string
of genes and encode more than one polypeptide (“polycistronic” mRNA). By
definition, each gene codes for one polypeptide.
41
b) Ribosomal RNA (rRNA) is a structural component of the ribosome. There
are 3 prokaryotic and 4 eukaryotic rRNAs, each of them present in a single copy in the
ribosome.
c) Transfer RNA (tRNA) carries amino acids to the ribosome for protein
synthesis.
3. Transcription
RNA synthesis is by transcription from a DNA template. It requires the enzyme

RNA polymerase. Mechanism: like DNA synthesis, but
- The precursors are ATP, GTP, CTP and UTP
- RNA polymerase does not need a primer
- RNA polymerase has no nuclease activities
Steps in transcription (E. coli):

1. RNA polymerase, sliding along the DNA, binds to the promoter. This
requires the σ (sigma) subunit of RNA polymerase. Promoters look different in
different genes, with consensus sequences at about –10 (TATAAT, “Pribnow box”) and
-35 bp (TTGACA) from the start of transcription.
2. RNA polymerase, separates a short stretch of DNA duplex.
3. RNA polymerase sythesizes the RNA in the 5’ ----Æ 3’ direction using the
original strand of the DNA strands as a template strand. The template strand is called
the coding strand. The DNA anneals behind the RNA polymerase.
4. There is a termination site at the end of the gene where the RNA polymerase
falls off its template. Most bacterial termination sequences contain a palindrome.
All bacterial RNAs are synthesized by the same RNA polymerase. The rate-
limiting steps in transcription are promoter recognition and strand separation. The error
rate of RNA synthesis about 1 in 10,000. The life span of mRNA is only a few minutes
in bacteria but several hours in eukaryotes. Only 3% of the RNA in E. coli is mRNA.
4. Post-transcriptional processing
Modification of RNA after transcription:
mRNA is usually not modified in prokaryotes but translated immediately. But

extensive processing occurs in eukaryotes.
tRNA has many modified bases, both in prokaryotes and eukaryotes: methylated
bases, pseudouridine, inosine (nucleoside containing hypoxanthine), ribothymidine
(thymine bound to ribose) etc. Most tRNAs are processed from precursor transcripts by
nuclease cleavages.
rRNA is processed by nucleases from a transcript that contains the sequences of
all major ribosomal RNAs (5S, 16S, 23S in bacteria, 5.8S, 18S, 28S in humans). There
are some methylated bases (prokaryotes) or ribose residues (eukaryotes) in rRNA.
IV. PROTEIN SYNTHESIS
42
1. The Genetic Code
The genetic code describes the relationship between the base sequence of the
mRNA (and the DNA) and the amino acid sequence of the protein.
Important features:
- A base triplet called a codon on the mRNA specifies an amino acid.
- There are 61 amino acid coding codons. One of them (AUG, coding for formyl
methionine) is also used as a start codon. There are 3 stop codons: UAA,
UAG and UGA.
- The code is non-overlapping and comma-less. There is no overlap between codons
and no empty spaces in between.
- The code is colinear. The codons on the mRNA are in the same sequence as the
encoded amino acids in the polypeptide.
- The code is unambiguous. Each codon specifies one and only one amino acid.
- The code is degenerate. Most amino acids are specified by more than one codon.
- The code is universal. There are only minor deviations, especially in the small
genomes of mitochondria and chloroplasts.
2. tRNA
tRNAs are small RNAs (80 nucleotides in length) which bring amino acids to the
ribosome. Typical “cloverleaf” structure. The 3’ –terminus, ending in the sequence
CCA, binds the amino acid through an ester bond with one of the hydroxy groups of
ribose. The anticodon base-pairs with the codon of mRNA during translation. There is a
certain freedom of pairing between the third codon base and the first anticodon base
(“wobble”). Therefore cells can do with less than 61 tRNAs.
Aminoacyl-tRNA sythetases are cytoplasmic enzymes which transfer an amino
acid to the 3’ end of the tRNA. ATP is hydrolyzed to AMP + pyrophosphate in this
reaction. Each aminoacyl-tRNA synthetase has a high specificity for “its” tRNA and
amino acid. This specificity is required for the fidelity of the genetic code.
3. Ribosomes
Ribosomes consist of rRNA + protein. Subunits: 30S + 50S = 70S in bacteria, 40S
+ 60S = 80S in eukaryotes. The 16S rRNA (bacteria) or 18S rRNA (eukaryotes) is in the
small subunit, the others in the large subunit. Ribsomes self-assemble from rRNA +
ribosomal proteins. The subunits are separate in the resting state and form the complete
ribosome only when they synthesize proteins. Magnesium is required for ribosome
assembly. The ribosome has 2 binding sites for tRNA: the P site (P = peptidyl) contains
the peptidyl-tRNA during the elongation phase, the A site (A = aminoacyl, or acceptor)
accepts the newly incoming aminoacyl-tRNA.
4. Steps in translation
- The initiation complex is formed when the 30s subunit binds to the 5’-terminal
part of the mRNA and to the initiator-tRNA that carries formyl-methionine (bacteria) or
methionine (eukaryotes). In bacteria (but not eukaryotes), the Shine-Delgarno Sequence
on the mRNA has to base-pair with a complementary sequence in the 16S ribosomal
RNA. Then the 50S subunit is added. The formation of the initiation complex requires
43
soluble cytoplasmic proteins called initiation factors, and one GTP is hydrolyzed to
GDP + phosphate.
- The fMet-tRNA is in the P-site, base-paired with the start codon AUG.
Elongation starts when an aminoacyl-tRNA is placed into the A site by elongation factor
EF-Tu. This requires GTP hydrolysis.
- Next the peptide bond formed by the peptidyl transferase activity of the large
ribosomal subunit.
- Translocation brings the newly formed peptidyl-tRNA from the A site to the P-
site. The mRNA moves 3 nucleotides along the ribosome. This requires GTP hydrolysis.
- Termination of translation occurs when a stop codon is encountered in the
mRNA sequence. The polypeptide is hydrolyzed from the tRNA after the binding of
protein releasing factors to the stop colon.
5. Antibiotics
Many (not all) antibiotics inhibit protein synthesis:
a) Rifampicin: An inhibitor of bacterial RNA polymerase

b) Actinomycin D: Inhibits transcription by DNA-base intercalation.
c) Streptomycin: Inhibits binding of fMet-tRNA and causes misreading
of mRNA in prokaryotes.
d) Tetracycline: Inhibits the binding or aminoacyl-tRNA in prokaryotes.
e) Chloramphenicol: Inhibits the peptidyl transferase of prokaryotes.
f) Cycloheximide: Same as chloramphenicol, but in eukaryotes.
g) Erythromycin: Inhibits translocation in prokaryotes.
h) Puromycin: A structural analog of aminocyl-tRNA. Causes
premature polypeptide chain termination
Clinical note: Most bacteria can survive for considerable periods of time without
protein synthesis. Therefore most drugs inhibiting bacterial protein synthesis are only
bacteriostatic, not bactericidal.
V. REGULATION OF GENE EXPRESSION
Gene expression is regulated, most often at the level of transcription and

sometimes by post-transcriptional mechanisms. Proteins that are synthesized sometimes
and sometimes not are called inducible. Proteins that are synthesized at all times are
called constitutive.
Examples in E. Coli of gene regulation:
1. The lac operon contains genes for the catabolism of the disaccharide lactose. Lactose
is transported into the cell by the carrier lactose permease and then cleaved into glucose
and galactose by the enzyme β-galactosidase. These proteins, together with the enzyme
thiogalactoside transacetylase, are produced in the presence of lactose but not its
absence. The genes for these 3 proteins are clustered in the lac operon. They produce a
44
polycistronic message which is translated into separate polypeptides. Organization of the
lac operon:
No inducer:
P I P O A B C
Repressor mRNA
repressor protein binds to operator
and blocks transcription of A, B, C genes
Protein
With inducer: Presence of inducer prevents repressor

binding to operator
A, B, and C are the genes for β-galactosidase, lactose permease and the
transacetylase. These are the structural genes that are expressed as polycistronic unit. P
is the promoter, where transcription begins. O is the operator. The operator is a
binding site for a repressor protein, next to (and overlapping) the promoter. The
binding of the repressor prevents transcription. I is the gene for the repressor. It is
expressed constitutively. The repressor binds to the operator in the absence of lactose. In
the presence of lactose the repressor binds allolactose (formed from lactose). Allolactose
binding causes a conformational change in the repressor which prevents its binding to the
operator, thus allowing RNA polymerase to transcribe the structural genes. The operon
consists of a promoter, operator and structural genes. The I gene may be next to the
operon (as in the lac operon), but this is not always the case.
2. Other Catabolic Operons are also induced by nutrients, usually by the
removal of a repressor protein from the operator.
3. The Tryptophan Operon contains 5 structural genes which encode
enzymes for tryptophan biosynthesis. These genes are expressed in the
absence but not the presence of external tryptophan. In this case the repressor
protein itself (“aporepressor”) does not bind to the operator, but the binding of
tryptophan to the repressor induces a conformational change which causes the
repressor to bind to the operator: Tryptophan acts as a corepressor.
Tryptophan makes feedback inhibition of its own synthesis.
4. Catabolite Repression is a general regulatory mechanism for carbon
metabolism in E. coli. Because glucose is the favored tasty treat of bacteria (it
enters glycolysis directly), many catabolic operons in E. coli are transcribed only
in the absence of glucose. The level of cyclic AMP (cAMP) is low when glucose
is present and high when glucose is absent. cAMP binds to CAP (Catabolite
Gene Activator Protein). The CAP-cAMP complex binds to the promoter region
of many catabolic operons and stimulates RNA polymerase binding and
transcription.
45
VI. VIRUSES
1. Virus Structure
Viruses are obligatory intracellular parasites: they can replicate only by
infecting a host cell. Outside the cell, the virus exists as an inert particle called a
virion. The virion consists of:
1. Nucleic acid: DNA or RNA , single-stranded or double-stranded, but

only one kind is present. Viruses have anywhere between 3 and 250 genes.
2. The capsid is a protein capsule that surrounds the nucleic acid. It is
formed from a few structural proteins which polymerize into a symmetrical shape.
Nucleic acid + capsid are called nucleocapsid.
3. An envelope is present only in some animal viruses, not in
bacteriophages (=bacteria-infecting viruses). It is a piece of membrane
acquired from the host cell that contains viral proteins called spike proteins.
2. The Lytic Cycle of Bacteriophage T4

Lytic infection by the DNA bacteriophage T4 and related phages proceeds
in the following sequence:
1. Adsorption: The tips of the tail fibers attach to a surface component
of E. coli which serves as a “phage receptor”. This determines the host range:
bacteria without the phage receptor cannot be infected.
2. Penetration: the tail sheath contracts, the phage DNA is injected into
bacterium. The capsid remains outside.
3. Synthesis of phage proteins: The phage DNA serves as a template
for mRNA synthesis, and viral proteins are synthesized on bacterial ribosomes.
Phage proteins produced include:
- A DNAase which degrades the host genome. Viral DNA is protected
because it contains hydroxymethylcytosine instead of ordinary cytosine
- Enzymes for nucleotide biosynthesis
- DNA polymerase, DNA ligase
- Viral capsid proteins
- Lysozyme and phospholipase which destroy the bacterial cell envelope.
Transcription of viral genes occurs in a specific sequence. Immediate-

early genes are transcribed first, followed by delayed-early and late genes. RNA
polymerase of the host is used for immediate-early transcription and later is
modified chemically by viral enzymes. This modified polymerase is specific for
late transcripts.
4. Virion assembly: DNA and capsid proteins self-assemble into virus
particles.
5. Lysis: The host cell is destroyed (lysed) after about 25 minutes.
About 200 virions are released.
3. The Lysogenic Cycle of λ lambda phage.
46
λ –phage has double-stranded DNA with mutually complementary single-
stranded ends (cohesive ends, or “sticky ends”). After viral DNA had entered the
host cell, the sticky ends anneal and are joined by the bacterial DNA ligase. At
this time the λ phage may proceed along the lytic pathway or follow the
lysogenic pathway. In the latter case, it inserts itself into the bacterial
chromosome with the help of a virally-encoded integrase enzyme, always
between the gal and bio operons. The inserted viral genome is called lysogenic.
In the λ –phophage only the gene for the λ –repressor protein is transcribed and
translated. The λ –repressor protein prevents the transcription of the other
phophage genes. When the cellular level of this repressor protein drops too low,
the prophage excises itself from the bacterial chromosome to enter the lytic
pathway. This usually happens when the bacterium is exposed to damaging
environmental influences such as radiation or heat-stress.
4. Animal Viruses
Some typical differences between animal viruses and bacteriophages:
- Some animal viruses replicate in the nucleus and others in the cytoplasm.
- After adsorption to the cell surface the complete virus particle enters the cell,
often by endocytosis.
- Instead of lysing the cell, animal viruses are most commonly shed by budding
from the plasma membrane, not all virions are released at the same time, and
the cell may recover.
- Some viruses acquire an envelope while budding out of the nucleus or through
the plasma membrane.
- The immune system can fight viral infections either by forming antibodies to
capsid proteins or spike proteins (followed by phagocytosis of the antibody-
coated virus), or by destroying the virus-infected cells which display viral
proteins on their surface.
5. RNA Viruses
RNA viruses can be double-stranded, (+) single-stranded or (-) single-
stranded. (+) ss RNA can serve as mRNA while (-) ss RNA is complementary to
the mRNA. All RNA viruses require a virally-encoded RNA-dependent RNA
polymerase (“RNA replicase”). (-) ss RNA viruses have to carry at least one
copy of this enzyme in the virus particle. Viral RNA replicases have no
proofreading ability, therefore the mutation rates are very high.
6. Retroviruses
Retroviruses are enveloped viruses containing 2 copies of their (+) –
stranded RNA genome in the virus particle together with the viral enzyme
reverse transcriptase.
Retroviral Life Cycle:

1. The nucleocapsid enters the host cell by simple fusion of the viral envelope
with the host cell membrane.
47
2. The reverse transcriptase copies the viral RNA into a double-stranded DNA (a
cDNA) which becomes inserted into the host cell genome with the help of a
virally-encoded integrase. The inserted viral cDNA contains the viral genes
flanked by long terminal repeats which contain the viral promoter.
3. After its insertion into the host cell DNA, the viral DNA directs the synthesis of
viral RNA and proteins. The viral RNA is used both for protein synthesis and
as the new genomic DNA
4. New virus particles assemble from viral RNA + protein. These bud out of the
cell continuously.
Like the RNA replicases, the retroviral reverse transcriptases have a high
error rate. Most retroviruses (the AIDS virus is an exception) cannot infect
nondividing cells because they cannot get across the nuclear envelope.
7. Plasmids
Plasmids are semi-independent genetic entities in bacteria, consisting of circular
double-stranded DNA carrying a few genes. The genes are not essential for bacterial
survival under ordinary conditions but confer special abilities: antibiotic resistance, toxin
production, ability to metabolize usual substrates, etc. They also have a replication origin
and genes regulating their own replication. Most important: R-factors are plasmids that
make the bacterium resistant to antibiotics. The plasmid-encoded enzyme B-lactamase
(“penicillinase”) destroys penicillin.
VII. GENETIC RECOMBINATION
1. Types of Recombination
The joining of different DNA molecules is called genetic recombination.
There are 2 types:
- Site-specific recombination requires a specific integrase enzyme which
recognizes sequences on one or both of the DNA molecules to be joined. The
integration of λ–phage and of the retroviral cDNA are examples.
- General recombination (=homologous recombination) requires no specific
sequences, but sequence identity or similarity between the recombining DNAs:
transformation in bacteria, crossing-over during meiosis.
2. Parasexual Processes in Bacteria

Bacteria don’t make “real” sex, but they can exchange genetic information
through parasexual processes:
Transformation is the uptake of foreign DNA. This is random in some

species, but other species take up selectively DNA of their own species. The
DNA integrates into the chromosome by homologous recombination.
Transduction is the transfer of cellular DNA by a bacteriophage.
Conjugation is the transfer of DNA by a self-transmissible plasmid. The
F-factor of E. coli causes the formation of sex pili which can form a cytoplasmic
bridge between the F-factor carrying cell (F+-cell) and a cell without F-factor (F- -
48
cell). The F factor sends a copy of itself into the F--cell. Also some R-factors are
self-transmissible.
1. Describe the covalent structures of DNA and RNA, including their constituent monomers,
bond types, functional groups and ionization state.
2. Describe the structure of the Watson-Crick double helix, with approximate helix parameters,
relevant non-covalent interactions, and the rules of base pairing.
3. Define the process of melting of DNA.
4. Define the different types of nucleases.
5. Name the different types of DNA polymerases of pro- and eukaryotes and identify their
functionally important properties.
6. Define the semiconservative model of DNA replication.
7. Identify the proteins involved in prokaryotic DNA replication, and state the sequence of their
actions.
8. Know the functionally important characteristics of prokaryotic and eukaryotic RNA
polymerases.
9. Define the terms “template strand” and “coding strand”.
10. List the important properties of the genetic code.
11. Describe the post-transcriptional processing of tRNA, rRNA, and mRNA in prokaryotes and
eukaryotes and the general structure of tRNA and the formation of aminoacyl-tRNA.
12. Identify the steps in ribosomal protein synthesis, their energy requirements and requirements
for soluble protein factors.
13. Locate the sites of action for some common antibiotics on transcription or translation.
14. Describe the operon model for the regulation of gene expression in prokaryotes.
15. Define the principles of positive and negative control of transcription by DNA binding proteins.
16. Describe the principle events in the life cycles of DNA viruses, RNA viruses and retroviruses
and identify the required viral enzymes.
17. Describe the properties of histones and their interaction with DNA in nucleosome structure.
18. Know the approximate proportions of protein-coding, non-coding, unique, moderately
repetitive and simple-sequence DNA in the human genome.
19. Define the terms “promoter” and “enhancer”.
20. Outline the importance of histones, chromatin structure, DNA methylation and sequence-
specific DNA-binding proteins for the regulation of eukaryotic gene expression.
21. List the types of repetitive elements in the human genome, and describe the mechanism for
the mobility of Alu and LINE-1 sequences.
22. Define the terms solitary gene, duplicated gene, gene family, pseudogene and processed
pseudogene.
23. Describe the importance of telomere erosion and of telomerase for the mortality of somatic
cells.
24. List the mechanisms by which transcription factors can be regulated.
25. List the principal types of mutations, and predict their likely effects on the functionality of the
encoded protein.
26. Describe the causes of mutations, including replication errors, radiation and the major types
of chemical mutagens.
27. Define the nature and functions of the major DNA repair systems, including the 3’-
exonuclease activities of replication complexes, methyl-directed mismatch repair, direct repair,
base excision repair and nucleotide excision repair.
28. Describe the major DNA repair defects, including chromosome breakage syndrome,
xeroderma pigmentosa, and Cockayne syndrome.
29. State the importance of mutations for genetic diseases and carcinogenesis.
30. Describe the diploid nature of the human genome and its implications for the expression of
genetic disorders.
49
31. Know the terms “homozygous”, “heterozygous”, “dominant” and “recessive”.
32. Describe the molecular defect of sickle cell disease, its clinical expression, and the
relationship between the two.
33. Define the terms “α-thalassemia”, “β-thalassemia”, thalassemia major and thalassemia minor.
34. Describe the clinical presentation in different types of thalassemia.
35. State the reason for the beneficial effect of increased HbF-expression in patients with sickle
cell disease and β-thalassemia, and the beneficial effect of concurrent α-thalassemia minor on
the clinical source of sickle cell disease.
36. List the treatment options for the major hemoglobinopathies.
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Handout 4
THE HUMAN GENOME AND MUTATIONS
I. The Human Genome
The human genome is the complete sequence of DNA bases in the

human organism. The finalized drafts of the complete human genome are now
being compiled. The human genome was completed after sequencing smaller
genomes of model organisms. The entire genome sequences of about 1000
different viruses and 100 microbes can be found in the Entrez Genome Browser.
The genomes represent both completed genomes and those for which
sequencing is still in progress. The three domains of life - bacteria, archaea, and
eukaryota - are represented, as well as many viruses and organelles.
Compared with model organisms the human genome is very large:
E. coli (4.6 Mb-Megabase) prokaryote
S. cerevisiae (12.1 Mb) primitive eukaryote
A. thaliani (100 Mb) plant
D. melanogaster (140 Mb) fly
M. musculus (3.3 Gb-Gigabase) mouse
H. sapiens (3.0 Gb) human
T. aestivum (17.0 Gb) wheat
The human sequence and its variation is also vital in its importance in
medicine. It provides the basis for understanding human disease inheritance and
susceptibility.
The genome is organized in chromosomes of differing size. Human
diploid somatic cells have 22 pairs of autosomes. In addition, females have two
X chromosomes, males one X and one Y. Mitochondria also contain DNA which
makes up a small fraction of the entire human genome.
The unit of DNA which encodes polypeptides is called the gene. In the
human genome, there are approximately 30,000 genes. As for most eukaryotes,
human genes are not tightly arranged with their nearest neighbors. Very long
stretches of DNA occur between genes and also within genes themselves. DNA
in humans can be categorized as follows:
Unique DNA sequences:

Protein Coding Regions of Genes (Exons) 1.5%
Non-coding Regions (Introns) 25.0%
Other UNIQUE DNA sequences 12.0%
Repetitive DNA sequences:
All repetitive DNA sequenced to date 53.0%
Not sequenced 8.0%
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II. CHROMATIN STRUCTURE
1. Histones and Nucleosomes

Chromatin is about 50% DNA and 50% histones. Histones are small,
basic proteins (lots of Lys and Arg!) that come in 5 varieties: histones H1, H2A,
H2B, H3 and H4. Histones are well-conserved in all eukaryotes. Nucleosomes
are formed from a core of histones (2 copies each of histones H2A, H2B, H3 and
H4), with about 140 bp of DNA wound around. About 60bp are in the linker DNA
between the nucleosomes. H1 sits on the linker DNA. Higher-order structure:
the nucleosomes coil up into a solenoid (diameter 30 nm). In the condensed
chromosomes, the 30 nm fibers are attached to scaffold proteins. The DNA in
condensed chromosomes consist of DNA:histones:non-histone proteins in 1:1:1
ratio. Euchromatin is dispersed chromatin in which the genes can be
transcribed. Heterochromatin is condensed chromatin that cannot be
transcribed. The mitochondrial DNA has no histones.
2. Repetitive DNA
There is about 3 billion bp of DNA in the human genome, with about 40-
80,000 genes. Only 1.2% of the DNA codes for proteins. The rest is junk DNA
between the genes and in the introns of the genes. Introns are sequences
within the genes that are transcribed but not translated. Some of the noncoding
DNA is repetitive.
Tandemly repeated sequences are most abundant in the centromeric and
telomeric regions (“satellite DNA”). Simple-sequence DNA consists of short,
tandemly repeated sequences of between 2 and few dozen nucleotides in the
repeat unit. Minisatellites and microsatellites are tandem repeats outside the
centromeres and telemeres.
Interspersed elements are sequences that occur, with variations, in
different locations in the genome. Alu sequences are about 300 bp long and
are present in about 500,000 copies in the genome (6-8% of the total genome),
with about 80% sequence homology between different copies. LINE-1
sequences are 6000 bp long. The complete sequence is present in only a few
thousand copies, but truncated LINE-1 sequences are very common.
3. Mobile DNA
Alu sequences and LINE-1 sequences can shuffle their location. These
sequences end in an oligo-A tract, and they are framed by short direct repeats.
They can jump to new locations by reverse transcription:
1. The element is transcribed into an RNA by RNA polymerase III. This

RNA becomes polyadenylated, like an mRNA.
2. Reverse transcriptase makes a cDNA from this RNA. The complete
LINE-1 elements contain a gene for a reverse transcriptase.
3. The cDNA is inserted into a new genomic location.
52
These mobile elements are considered selfish DNA. They can cause
mutations when they jump into a gene. Another name for mobile element is
transposon or less commonly retroposon.
4. Genes
Genes occur mostly in one copy per haploid genome. Duplicated genes
are present in 2 or more copies which are close together on the chromosome.
Gene families with similar, but nonidentical functional genes are common,
usually with closely-related members clustered in the same chromosomal region.
Example: globin genes. Pseudogenes are nonfunctional copies of functional
genes. Usually they are close to their functional counterpart. Duplicated genes,
gene families and pseudogenes arise by gene duplication, usually from
crossing-over (homologous recombination) between misaligned chromosomes
during prophase of meiosis I. Individual exons can duplicate (or be deleted) by
the same mechanism.
Processed pseudogenes are nonfunctional sequences that duplicate the
exon sequences of a functional gene. They are non-viral retroposons that are
derived by the reverse transcription of a cellular mRNA (or sometimes a partially
processed hnRNA) and the insertion of the resulting cDNA into the genome e.g.,
reverse transcribed by the Line-1 reverse transcriptase. They are not necessarily
close to their functional counterpart. Viral retroposons are the remnants of
retroviral genomes that have mutated into non-functionality.
5. Telomeres
Telomeres are the end pieces of the chromosomes. They consist of the
repeat sequence TTAGGG, which is repeated hundreds of times to a total length
of several thousand bp. They tend to shorten during repeated rounds of DNA
replication because lagging strand replication cannot go all the way to the end.
Excessive shortening is prevented by the enzyme telomerase, which adds to the
repeat sequence by synthesizing new DNA on an internal RNA template.
Telomerase is active in the germline and in embryonic tissues, but not in most
adult tissues. Therefore the telomeres tend to shorten during a lifetime. This is
important for the mortality of cells, both in the body and in cell culture. Cancer
cells have telomerase, and they are immortal.
6. DNA Replication
There are many origins of replication in human DNA, about one every
100,000 bp. There are several DNA polymerases:
Polymerase α and ζ replicate the lagging and the leading strand,
respectively. Polymerase β and ε are involved in DNA repair and/or
recombination. Polymerase γ is in the mitochondria.
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III. MUTATIONS
Mutations are heritable changes in DNA structure. They arise as errors

during DNA replication or are caused by DNA damage. The basal mutation rate
is the mutation rate in the absence of external mutagens. Mutations in the
germline lead to genetic diseases. Mutations in somatic cells cause cell
dysfunction and, sometimes, cancer. All mutagens are also carcinogens.
1. Types of Mutation
- Base substitutions are the most common type.
Transition: A purine replaces another purine or a pyrimidine
replaces another pyrimidine.
Transversion: A purine replaces a pyrimidine or a pyrimidine
replaces a purine.
- Deletions: One or several nucleotides, or a large piece with up to millions of
nucleotides, is lost.
- Insertions: One or several nucleotides, or a big chunk of DNA, is added.
- Translocation: A piece of DNA is transferred to another location in the
genome.
2. Other Definitions
Point mutations are single-base substitutions.
Silent mutations are base substitutions that do not change the amino
acid sequence because of the degeneracy of the genetic code.
Nonsense mutations change a codon for an amino acid to a stop-codon
(UAA, UAG, UGA) resulting in inappropriate termination of chain
elongation during translation.
Missense mutations change a codon for an amino acid to a codon for a
different amino acid.
Frameshift mutations are small insertions or deletions that change the
reading frame of the mRNA
Splice-site mutations result in abnormal splicing. These lead to anything
from changes in protein length to frameshifts, which again often lead
to truncated proteins.
3. Causes of Mutations
1. The basal mutation rate is caused by spontaneous tautomeric shifts

and hydrolytic reactions. Tautomeric shifts occur with thymine (keto < --- >
enol shift) and adenine (amino < --- > imino shift). Spontaneous depurination is
the most important type of hydrolytic reaction.
2. UV radiation is a component of sunlight. It causes the formation of
pyrimidine dimers and other photoproducts. UV does not penetrate the skin,
but it causes sunburn and skin cancer.
54
3. Ionizing radiation (X-rays, radioactive radiation) is more energy-rich
than UV radiation. It causes many kinds of DNA damage, but especially double-
strand beaks. Ionizing radiation can penetrate the whole body.
4. Base analogs are “false” bases that are incorporated into DNA.
Example: 5-bromouracil can replace thymine.
5. Deaminating agents deaminate adenine to hypoxanthine and cytosine
to uracil. Example: nitrous acid.
5a. Deamination of methylated cytosine leads to thymidine, which is not
recognized as an unnatural base in DNA, and therefore the repair of T::G
mismatches often is erroneous. This is the most common type of point mutation
in the genome.
6. Alkylating agents alkylate N and O atoms in the bases. Examples:
methyl bromide and ethylene oxide.
7. Intercalating agents are flat, hydrophobic molecules that insert
themselves between stacked bases. Examples: acridine dyes and benzene.
Ethidium bromide used to visualize DNA in electrophoresis is another
intercalating agent.
8. Viruses may insert their own DNA into the host cell chromosome,
either habitually (retroviruses) or by accident (DNA viruses).
Mutagens are most effective when they hit the cell immediately before or
during DNA replication (during S phase of the cell cycle), because there is no
time for repair. Cancer cells are more easily killed by radiation than normal cells
because they divide more rapidly and spend more time in S phase.
4. Mutagenesis Testing
The Ames test uses bacteria that are dependent on a particular nutrient as a
result of a mutation (auxotrophic bacteria). After exposure to the chemical, the
bacteria are screened for back-mutations which restore their ability to grow in the
absence of the nutrient. Mutagens increase the rate of back-mutations.
IV. DNA REPAIR
All cellular organisms have repair systems for many different kinds of DNA
damage. DNA repair is possible because only one strand is damaged, therefore
the undamaged second strand can supply the sequence information for the
repair enzymes.
1. Nucleotide Excision Repair

Nucleotide excision repair removes bulky lesions including thymine
dimers, alkylations and bulky adducts. The mechanism of excision repair:
- A repair crew consisting of several specialized proteins scans the DNA.
- The repair complex binds to a bulky lesion.
- Endonucleases make 2 incisions in the damaged strand, one 5’ and
the other 3’ of the lesion.
- Helicases in the complex separate the two strands.
55
- The damaged DNA piece is removed.
- The resulting gap is filled by DNA polymerase.
- The last phosphodiester bond is formed by DNA ligase.
- Nucleotide excision repair reaches all parts of the genome, but there is
a subsystem for the preferential repair of transcribed genes.
2. Other Repair Systems

Depurination (loss of a purine base by spontaneous hydrolysis) is
repaired by an AP endonuclease (AP = apurinic), followed by DNA polymerase
and DNA ligase.
Deaminated bases are removed by base excision repair: hypoxanthine
(formed from adenine) and uracil (from cytosine) are clipped off by cleavage of
the N-glycosidic bond with D-ribose. The next steps are as in the repair of
apurinic sites.
Post-replication mismatch repair corrects base mismatches and small
insertions and deletions after DNA replication. The repair enzymes can
distinguish between the strands because the old strand is methylated on specific
sequences (GATC in E. coli, CMG in eukaryotes). The repair system cuts the
unmethylated new strand either 5’ or 3’ of the mismatch. The damaged piece is
removed by exonucleases and replaced by DNA polymerase followed by DNA
ligase.
3. Repair Defects
Xeroderma pigmentosum is a recessively inherited skin disease with
premalignant and malignant lesions on sun-exposed skin. There are 9 different
types (“complementation groups”), caused by deficiencies of different repair
proteins.
Cockayne syndrome is also caused by defective nucleotide excision
repair, but only the preferential repair of transcribed genes is affected. No skin
cancer, but poor growth, neurological problems, and early senility.
Hereditary non-polyposis colon cancer (HNPCC) is an inherited cancer
susceptibility syndrome caused by defects in post-replication mismatch repair.
Chromosome breakage syndromes include Bloom syndrome, ataxia-
telangiectasia, and Fanconi anemia. These are multi-system disorders with an
increased incidence of chromosome breakage.
V. EUKARYOTIC GENE EXPRESSION
1. Transcription
There are 3 nuclear RNA polymerases in eukaryotes:
RNA polymerase I makes rRNA (in the nucleolus).
RNA polymerase II makes mRNA.
RNA polymerase III makes small RNAs: tRNA, 5S rRNA.
α-amanitin is a mushroom poison (from Amanita phalloides) that inhibits
RNA polymerase II.
56
2. mRNA Processing
Eukaryotic mRNA is processed in the nucleus before it is sent into the
cytoplasm for translation. Steps:
1. Capping is the addition of a methylguanosine residue to the 5’ end of

the mRNA (Figure 8.11 in the Meisenberg book). It occurs co-transcriptionally.
The cap protects the 5’ terminus from the action of nucleases.
2. Polyadenylation is the addition of a poly-A tail to the 3’ end of the
transcript. Transcription proceeds beyond the polyadenylation signal
(consensus: AAUAAA). This is followed by a nuclease cleavage and the
enzymatic addition of the adenine nucleotides (no template required!).
3. Splicing is the removal of intron sequences from the primary
transcript. It requires spliceosomes which consist of small nuclear
ribonucleoproteins (snurps). The intron-exon junctions have consensus
sequences that are recognized by the spliceosome.
3. Translation
The important steps are the same as in prokaryotes, but:
1. Eukaryotic proteins start with methionine, not formylmethionine.
2. There is no Shine-Delgarno sequence, but translational initiation
depends on interactions of mRNA with ribosomal proteins.
3. Eukaryotic mRNAs are not polycistronic, i.e., they normally
encode only one protein. This is normally positioned as the 5’
open reading frame in the mRNA.
Eukaryotic translation is inhibited by diphtheria toxin. This toxin modifies

an elongation factor covalently, thereby preventing translocation.
VI. REGULATION OF EUKARYOTIC GENE EXPRESSION
1. Global Effects
- Histones inhibit transcription nonselectively by tightly binding the DNA
double-helix. Histones are modified by acetylation, phosphorylation and
methylation at specific sites.
- DNA methylation inhibits transcription. Cytosine bases in the
palindrome CG are methylated on both strands, and methylation patterns are
maintained during DNA replication. Specific proteins interact with the methylated
regions. Not all CG palindromes are methylated: This is regulated.
2. Regulatory DNA Sequences.

Promoter for RNA polymerase II extend to about –200 bp from the
transcriptional start site. Highly variable sequence, but most contain a TATA
box 25-30 bp upstream of the start site. Most promoter elements have to be on
the correct strand and in the correct 5’ ---> 3’ orientation.
57
Enhancers can be present up to some thousand bp upstream or
downstream of the transcriptional start site. Enhancers increase the rate of
transcription. They need not be in correct 5’ ---> 3’ orientation to be effective.
Silencers are like enhancers, but they reduce rather than enhance the
rate of transcription.
Promoters, enhancers and silencers contain binding sites for regulatory
proteins. The individual binding sites are often called response elements. Note
that in eukaryotes the inhibitory effects of histones and DNA methylation have to
be overcome by sequence-specific DNA binding proteins that bind to promoter
and enhancer sequences.
3. Transcription Factors
The regulatory proteins that bind to promoters and enhancers are called
transcription factors. General transcription factors are promoter-binding
proteins that are required for the transcription of all genes by a particular RNA
polymerase. They are the functional equivalents of the bacterial σ-subunit.
Others affect the transcription of some genes but not others.
Many transcriptional regulators bind DNA in a dimeric form, either as
homodimers or as heterodimers of 2 slightly different polypeptides. Recognition
occurs in the major groove where the 'side-on' steric features of base-sequences
can be recognized by proteins.
Types:
Zinc-finger proteins contain between 2 and about a dozen zinc fingers in
their DNA-binding region: zinc complexed between 4 Cys or 2 Cys and 2 His
residues, with an intervening loop.
Leucine-zipper proteins contain a DNA-binding basic domain, a
dimerization domain with leucine residues spaced 7 amino acids apart in an
amphipathic α-helix, and a transcriptional activator (or repressor) domain.
Helix-loop-helix proteins have a dimerization domain with 2 amphipathic
α-helices separated by a loop.
Helix-turn-helix proteins have 2 α-helices separated by a B-turn. One of
the α-helices fits into the major groove of the DNA.
4. Regulation of Transcription Factors

1. The synthesis of transcription factors is controlled often in a cell type-
specific manner: most transcription factors (except the “general”
transcription factors) are present only in certain cell types and at certain
stages of cell differentiation.
2. They can be controlled by the reversible binding of small molecules.
Example: receptors for steroid and thyroid hormones.
3. They are controlled by interactions with other proteins.
4. They are controlled by phosphorylation and dephosphorylation,
frequently in response to growth factors or the second messengers of
hormones.
58
5. Post-transcriptional Controls
1. The use of alternative promoters or alternative polyadenylation
signals can create mRNAs which differ at the 5’ end or the 3’ end,
respectively.
2. Alternative splicing of a single mRNA transcript can produce different
polypeptides from the same gene.
3. RNA editing is the post-transcriptional chemical modification of a base
in the mRNA. The example, an amino acid coding codon can be
modified into a stop codon to produce a shorter protein.
4. mRNA stability varies in different cell types and under different
conditions. mRNA stability can be controlled by specific mRNA
sequence binding-proteins that impair the action of nucleases.
6. Translational Controls
1 Start-site variation occurs when alternative start codons can be used
by the ribosome. The resulting polypeptides differ at their N-terminus.
2. Initiation factor control works through the phosphorylation of the
initiation factor eIF-2 which slows all translation. This occurs during the M phase
of the cell cycle, and when growth factors are not present
3. Cap-binding proteins can inhibit translation by binding to the 5’ end of
the mRNA.

1. Know the proportion and characteristics of DNA sequence elements in the human genome.
2. Describe the properties of histones and their interaction with DNA in nucleosome structure.
3. Define the terms promoter, enhancer, and silencer.
4. Explain how histones, chromatin, DNA methylation, and transcription factors can influence the
rate of eukaryotic transcription.
5. Name and describe the principal types of mutations, and explain their effects on the structure
and function of proteins.
6. Describe the causes of mutations.
7. Name and describe the systems for repair of DNA lesions.
8. List inherited human diseases which result from faulty DNA repair systems.
9. Name the different types of eukaryotic DNA polymerases, and describe replication.
10. Know the different types of eukaryotic RNA polymerases, and accessory factors.
11. Describe the processes of post-transcriptional processing of tRNA, rRNA and mRNA in
eukaryotes.
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Handout 5
CHROMOSOME ABERRATIONS
I. THE HUMAN KARYOTYPE
Chromosomes become visible only during mitosis and meiosis. They are
classified as:
Metacentric: The centromere is in the middle.

Submetacentric: Intermediate between metacentric and acrocentric.
Acrocentric: Centromere near the end, forming a stubby short arm and a
long arm. Only the long arm has genes.
Telocentric: The centromere is at the end; only one arm is present.
Does not occur in humans.
The short arm of a chromosome is designated by the letter p (petite,

French for small, the long arm by q. The acrocentric chromosomes have small
satellites attached to their short arms, which contain clusters of genes for
ribosomal RNA; losing one or two of these clusters will not affect the cell in any
adverse way.
Diploid somatic cells have 22 pairs of autosomes. In addition, females
have two X chromosomes, males one X and one Y. The karyotype (=
chromosome constitution) can be examined during mitotic metaphase.
Techniques:
1. Bone marrow biopsy (no culture required).

2. Leukocyte culture
3. Fibroblast culture from the skin
4. Amniotic cells, cultured like skin fibroblasts.
Leukocyte culture is most important. Phytohemagglutinin (a lectin from

beans) is used as a mitogen, and colchicine is used to arrest mitosis in
metaphase.
Human chromosomes can be grouped into:
Group A (1-3): Large, metacentric to submetacentric.

Group B (4-5): Submetacentric, smaller than group A.
Group C (6-12): Submetacentric, smaller than group B.
Group D (13-15): Acrocentric
Group E (19-18): Submetacentric
Group F (19-20): Small metacentric
Group G (21-22): Small acrocentric
X chromosome: Like a C-group chromosome
60
Y chromosome: Small submetacentric
Banding techniques are staining procedures that distinguish between
different types of chromatin within the chromosomes: Ǫ (quinacrine mustard)
banding, G (Giemsa) banding, and R (reverse) banding. They can distinguish
different chromosomes within each group, and they can detect structural
abnormalities. Karyotype with banding costs about twice as much as a regular
karyotype. High-resolution banding with prophase chromosomes is not often
done in clinical routine, but is an important research technique.
Chromosome painting makes use of fluorescent-labeled probes that
bind to one of the chromosomes (as in FISH = fluorescent in-situ hybridization;
FISH is discussed in the section on recombinant DNA methods). Different-
colored probes are used to distinguish between chromosomes. Fluorescent
probes can even be used to detect aneuploidy in the interphase nucleus. This
method tends to supplement or even replace classical karyotyping.
Heteromorphisms are variations in the karyotype that are in most cases,
not associated with disease:
- Length variations of the long arm of the Y chromosome (Yq) are

present in 10% of all males. Yq is mostly junk DNA, and most of these variants
are innocuous.
- The satellites of the acrocentric chromosomes are variable. Also these
heteromorphisms are generally asymptomatic.
- There are fragile sites on many chromosomes, which are visible as
small constrictions. Chromosome breakage at these sites can be induced by
culturing in a folate-deficient medium. Many fragile sites are variable, and most
(but not all) of them are harmless.
- Centromeric heterochromatin, and also heterochromatic bands outside
the centromeres, can be variable.
Question: What should you do when you pick up a heteromorphism
incidentally during prenatal diagnosis?
II. SEX CHROMATIN
In interphase nuclei, one of the two X chromosomes of a female is

frequently visible as a heterochromatic mass, the Barr body. Number of Barr
bodies in diploid somatic cells = number of X chromosomes minus 1: Normal
females have one, normal males none. Sex chromatin in buccal smear cells is
an important screening test for sex chromosome aberrations.
The Lyons hypothesis states that only one of the X chromosomes in a cell
is in the dispersed, genetically active form during interphase. Additional X
chromosomes become heterochromatic and genetically inactive. This involves
DNA methylation. Inactivation occurs about 15 –16 days post-conception and is
random. Inactivation can affect either the maternally-derived or the paternally-
derived X chromosome: normal females are mosaics of cell clones in which
either one or the other X chromosome is active. This is important for the
expression of X-linked diseases in heterozygous females. The genes in the
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pseudoautosomal region on the short arm of the second X chromosome
escape inactivation, and also some other genes remain active. Therefore people
with an abnormal number of X chromosomes do have clinical abnormalities.
Only the few genes in the pseudoautosomal region have an equivalent on the Y
chromosome.
The chromatin of the Y chromosome can be demonstrated in the
interphase nucleus by Ǫ-banding. This method can be used to distinguish X-
and Y-bearing sperm cells. Most of the long arm of the Y chromosome is
heterochromatic and noncoding. The short arm carries the male-determining
SRY gene, which directs the development of the testis. It codes for a
transcription factor. There are also a few Y-linked genes that are required for
spermatogenesis, and mutations in these genes can lead to male infertility.
III. TYPES OF CHROMOSOME ABERRATIONS
Gross chromosomal aberrations are present in 1 out of every 160 live

births and 35% of spontaneous abortions. More than half of all pre-implantation
embryos are mosaics with at least one abnormal cell line. These chromosome
aberrations are a major reason why even for young women the risk of pregnancy
is never higher than 25% to 39% per month. About 25% of women with primary
amenorrhea, 11% of infertile males and 10% of the institutionalized mentally
impaired have chromosomal abnormalities.
Numerical aberrations:
a) Aneuploidy: One chromosome is present in an abnormal copy

number: trisomy, monosomy.
b) Polyploidy: The whole set is present in an abnormal number:
triploidy (69 chromosomes total), tetraploidy (92 chromosomes).
Consequences:
- Polyploidy results in fetal death.
- Monosomy for an autosome results in a nonviable embryo,
trisomy is better tolerated.
- Trisomy of large autosomes causes death, only trisomies of some
of the smaller autosomes are viable.
- Aneuploidy of sex chromosomes is well tolerated, but at least one
X chromosome has to be present.
- People with a Y chromosome are usually male, those without a Y
chromosome are usually female.
Aneuploidy results from non-disjunction during meiosis, or during mitosis

in the germ line. If it happened in a mitotic division of spermatogonia or oogonia,
there is germline mosaicism: presence of a whole tribe of abnormal cells in the
gonad. This means there is an increased recurrence risk after the birth of an
affected child. Aberrations of chromosome number generally are not inherited
from an affected parent, but arise de novo.
62
Mosaicism is caused by non-disjunction during the first mitotic divisions
after fertilization: different somatic cells of the patient have different karyotypes.
Chromosomal rearrangements are aberrations of chromosome
structure, such as large deletions or translocations. They often arise de novo,
but can also be inherited.
IV. AUTOSOMAL TRISOMIES
Only 3 autosomal trisomies are observed in live-births:

Trisomy 21: Down syndrome. About 1 in 800 births.
Trisomy 18: Edward syndrome. About 1 in 8000 births
Trisomy 13: Patau syndrome. About 1 in 20,000 births.
1. Trisomy 21 (Down syndrome)

95% of Down syndrome patients have trisomy 21, 3% a Robertsonian
translocation (“translocation-type Down syndrome”), and 2% are mosaics with
trisomic and normal cells. In 80% of the cases non-disjunction occurred in the
mother, usually in meiosis I. About 60% of trisomy 21 conceptuses abort
spontaneously. Phenotype:
Hypotonia (infants)
Short stature
Short, stubby hands and feet
Palpebral fissure, epicanthic fold
Large, protruding tongue
Small or malformed ears
Flat occiput
Short, broad neck
Simian crease
IQ typically between 25 and 50
Infertility in males
There is an increased risk of:

Congenital heart defects (30 –40%): Endocardial cushion defect,
atrial and ventricular septal defects.
Childhood leukemia is 10 – 20 times more common than in euploid
individuals. Both myelogenous leukemia (infants) and
lymphoblastic leukemia (older children) are increased.
Old patients often develop dementia. Life expectancy is reduced, but
many patients live into their 50s and 60s. The neuropathologic changes of
Alzheimer’s disease (plaques and tangles) are present in patients dying at >35
years. The β-amyloid precursor protein (APP) gene encoding one of the
components of Alzheimer plaques is located on chromosome 21.
Maternal Age
Very young mother: 1 in 1500
Mother 35 years: 1 in 365
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Paternal Age has only a very slight effect (if any, the effect is delayed 20
years relative to the maternal age effect).
Prenatal diagnosis: Karyotype analysis after chorionic villus sampling or
amniocentesis. Also maternal serum α-fetoprotein (reduced in Down syndrome).
The recurrence risk after the birth of a trisomy 21 child to a young mother is
about 1% because of possible germline mosaicism.
2. Trisomy 18 (Edward syndrome)

80% of patients are female
Birth-weight under 6 pounds
Failure to thrive
Hypertonicity, flexed fingers
Prominent occiput
Low-set, malformed ears
Micrognathia
Short sternum, small pelvis, rocker-bottom feet
Cardiac and renal malformations
90% die within a year
Profound mental deficiency in survivors
3. Trisomy 13 (Patau syndrome)
Apparent deafness
Minor motor seizures
Hypertonia/flexed fingers
Sloping forehead, microcephaly
Eye abnormalities
Cleft lip, cleft palate
Agenesis of the olfactory bulb, arhinencephaly, holoprosencephaly
Cardiac/colon/uterine malformations
Polydactyly
Rocker-bottom feet
90% die within a year
Profound mental deficiency in survivors
V. SEX CHROMOSOME ABERRATIONS: MALE.
1. Klinefelter syndrome
Incidence: 1 in 1000 males, mild maternal age effect.
Chromosomes: 80% are 47, XXY. The others are mosaics (46,
XY/XXY most common), 48, XXXY, or 49, XXXXY.
Phenotype (47, XXY): Small testes are the most consistent physical
finding. Infertility. Height is average or above average.
Undervirilization, with poor growth of beard and body hair, penis small
or normal, 25% develop gynecomastia. Testosterone is low, estrogen
variable, pituitary gonadotropins high. IQ is decreased by about 15
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points. More severe abnormalities (with mental deficiency) in 48,XXXY
and 49, XXXXY.
Diagnosis: Most patients present either with poor sexual development or
infertility. Buccal smear and/or karyotype is indicated in these
situations. Klinefelter is the single most important cause of severe
male infertility.
Treatment: Testosterone improves virility but not fertility.
2. XYY constitution (“murderer chromosome”).

Incidence: 1 in 1000 males. No parental age effect.
Phenotype: No consistent abnormalities, except:
- Increased height
- IQ reduced by10 –15 points
- Increased risk of criminal conviction
XYY males are over represented in prisons (3 in 1000), institutions for the
mentally defective (3 in 1000) and institutions for mentally defective
criminals (20 in 100), but most are never diagnosed. Normal fertility;
children are normal.
3. XX Males
Rare (1 in 20,000). Usually caused by a small translocation of the testis-
determining gene to the X chromosome. Klinefelter-like phenotype.
VI. SEX CHROMOSOME ABERRATIONS: FEMALE
1. Turner’s syndrome (gonadal dysgenesis)

Incidence: 1 in 5000 females
Chromosomes: 50-60% are 45, XO. Others are mosaics (46, XX/45,
XO; 47, XXX/45, XO) or structural rearrangements. 99% of XO fetuses
abort spontaneously.
Gonads: “Streak ovaries”, consisting of connective tissue without
oogenesis. Degeneration of the ovaries starts during fetal
development.
At birth: Peripheral lymphedema (first few days only, webbing of neck,
20% have aortic coarctation.
Later: Short stature, final height is 140 – 150 cm. Broad chest, widely-
spaced nipples, cubitus valgus, low posterior hairline.
At puberty: There is no puberty.
Mental development: is about normal.
Diagnosis: Buccal smear, karyotype. Some are diagnosed before
puberty because of slow growth, others are diagnosed later during
workup of primary amenorrhea.
Treatment: Estrogen after puberty, growth hormone in younger patients.
2. Triple-X (47, XXX, “superfemale”)

Incidence: 1 in 1000 females, maternal age effect.
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Phenotype: No consistent abnormalities, but IQ is decreased 10 – 15
points, and some have impaired fertility or menstrual problems. Their
children are usually normal. 48, XXXXX are rare, with mental
deficiency and dysmorphic features.
3. XY females
Rare (1 in 20,000), caused by deletion of the testis-determining gene.
Turner-like phenotype.
VII. Abnormal Sexual Development With Normal Chromosomes
1. True hermaphroditism
Definition: Presence of ovarian and testicular tissue, either as separate
gonads or combined in an ovotestis.
Incidence: Very rare.
Phenotype: A variable mix of male and female structures.
Chromosomes: Most are 46, XX. Some are mosaics with a Y-containing
cell line, some are chimaeras (created by the fusion of two embryos).
2. Mixed Gonadal Dysgenesis
Definition: Presence of testis and streak ovary.
Incidence: Rare
Phenotype: Variable. Often virilization at puberty.
Chromosomes: Most are mosaics 46, XY/45, XO.
3. Female Pseudohermaphroditism.
Definition: Virilized phenotype in a female. Only ovaries are present.
Causes: Prenatal androgen or progesterone treatment. Or a recessively
inherited deficiency of 11 – or 21 – hydroxylase in the adrenal cortex:
congenital adrenal hyperplasia (“adrenogenital syndrome”,
incidence 1 in 10, 000). The synthesis of corticosteroids is blocked
while androgen synthesis is possible.
Pituitary
ACTH
+
Cholesterol Progestins
Corticosteroids Androgens
The lack of corticosteroids causes excessive ACTH secretion and

stimulation of the adrenal cortex overproducing progestins and adrenal
hyperplasia. With corticosteroids synthesis blocked, the progestins are
converted to androgens. These adrenal androgens make virilization in
females and precocious puberty in males. If the enzyme deficiency is
complete, there is dangerous hyponatremia and hyperkalemia because
66
mineralocorticoids are missing. Cortisol treatment permits normal
development both in males and females. Early diagnosis is important.
4. Male Pseudohermaphroditism
Definition: Feminized phenotype in a genetic male.
Causes: Defective androgen action.
Androgen insensitivity (testicular feminization) is a rare X-linked recessive
disorder. The external phenotype is female. Female body proportions,
breast development, feminine psychosexual development. No uterus
and fallopian tubes, primary amenorrhea, no Wolffian duct structures,
non-functional testes are present in abdomen. Sometimes an “inguinal
hernia” turns out to be a testis. Otherwise after puberty when
amenorrhea or infertility is the presenting problem. The testes are
prone to malignancies and should be removed. There is a whole
spectrum of incomplete androgen insensitivity, ranging from infertile
male to phenotype female.
5α–reductase deficiency, a rare autosomal recessive condition, prevents
the formation of dihydrotestosterone from testosterone.
Dihydrotestosterone is the most potent androgen. The external
genitalia of a genetic male are mostly female at birth but internal
Wolffian duct structures are present. Virilization occurs at puberty.
VIII. Chromosomal Rearrangements
Chromosomal rearrangements are caused by chromosome breakage,

often followed by faulty healing of the fragments. To be stable during mitosis, the
rearranged chromosomes must have one centromere and the telomeres.
Balanced rearrangements do not change the copy number of genes,
therefore their phenotype is normal. Genes can still be expressed, even on the
wrong chromosome, as long as they still have their promoter and enhancers.
Unbalanced rearrangements lead to a net excess or deficiency of genes,
therefore the phenotype is abnormal.
1. Deletions
Both terminal and interstitial deletions occur, and they come in all sizes.
A) Turner syndrome. Deletions in the short arm of the X
chromosome (Xp-) make typical Turner syndrome. Deletions of the long arm
(Xq_) make only streak gonads and primary amenorrhea.
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B) Autosomal deletion syndromes. Usually with a combination of
physical abnormalities and mental deficiency. Larger deletions (>10 million bp)
can be seen in the banded karyotype, and smaller deletions can be diagnosed
with FISH. Examples:
- Cri-du-chat syndrome is caused by deletion of part of the short arm
of chromosome 5 (5p-). Physical stigmata are mild, but there is
severe to profound mental retardation. Infants have a cat-like cry.
80% of patients are female.
- Williams syndrome is caused by an interstitial deletion in the long
arm of chromosome 7 (7q-). Patients are pixie-faced, with short
stature, transient hypercalcemia, and mental retardation but spared
verbal ability.
2. Ring chromosomes
This is a rare type of abnormal chromosome in which end pieces of the
chromosome are lost while the ring is formed:
3. Isochromosomes
Isochromosomes consist of two identical chromosome arms. They are
formed by transverse rather than lengthwise cleavage of the centromere during
the metaphase -to- anaphase transition in meiosis II or mitosis.
68
IsoXq causes Turner’s syndrome. Isochromosomes of autosomes (other
than acrocentrics) are not compatible with life.
Normal Isochrome formation
4. Inversions
Inversions are either pericentric or paracentric. They are usually
asymptomatic, but unbalanced offspring can be produced if crossing-over occurs
in the inverted segment during meiosis.
Paracentric Inversion Pericentric Inversion
5. Reciprocal Translocations
Breaks occur in two non-homologous chromosomes. The fragments unite
to form two abnormal chromosomes:
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Carriers of a reciprocal translocation are normal, but gametes with
abnormal chromosomes may be produced, resulting in unbalanced
translocations in the offspring: High incidence of stillbirths and spontaneous
abortions.
6. Robertsonian Translocation (=centric fusion).

This is a fusion of the long arms of two acrocentric chromosomes after
breakage near the centromere.
The small fragment is lost. This consists of genetically inactive

heterochromatin, as well as rRNA genes. Normally, other rRNA loci are present
and functional and therefore no phenotype is expected.
The carriers of a Robertsonian translocation have only 45 chromosomes.
They are phenotypically normal, but can produce abnormal offspring. Example:
Translocation-type Down syndrome is present in 3% of unselected
Down syndrome patients. Typical features:
70
- Clinical severity is the same as trisomy 21
- There is no maternal age effect
- The cases are often familial
- The propositus has 46 chromosomes
- In many cases one of the parents (usually the mother) has 45
chromosomes.
Offspring derived from a gamete carrying both the translocation

chromosome and a normal chromosome 21 (but not the normal copy of the
chromosome to which chromosome 21 is attached) will have translocation Down
syndrome. In an asymptomatic carrier of a Robertsonian t(21;21) translocation
(isochromosome 21), all children will have Down syndrome.
1. Define polyploid, aneuploid, monosomy, trisomy, deletion, translocation, balanced

rearrangement, unbalanced rearrangement, ring chromosome and isochromosome.
2. Recall the approximate incidence values of chromosome aberrations.
3. Describe the causes of aneuploidy, mosaicism and chromosomal rearrangements.
4. Describe the normal function of the X and Y chromosomes in sex determination.
5. Provide examples of clinical situations in which sex chromatin determination should be
performed.
6. Describe methods for karyotyping and give examples of when a karyotype should be
performed.
7. List the phenotypic features associated with major chromosome aberrations and also the more
common trisomies and sex-chromosome ploidies.
8. Predict Down syndrome incidence based on maternal age.
9. Given a patient with a reciprocal (balanced) translocation, state the likely consequences for the
translocation carrier and future children.
10. Describe the clinical definitions of hermaphroditism, male/female pseudohermaphroditism,
mixed gonadal disgenesis.
11. Describe the pathogenesis in testicular feminzation and adrenogenital syndrome.
12. Identify clinical signs and symptoms suggesting unbalanced chromosomal rearrangements.
71
Handout 6
METHODS IN MOLECULAR MEDICINE
I. RESTRICTION ENDONUCLEASES
The DNA in human chromosomes is too big to be manageable in the test

tube. The chromosomes have to be broken down into smaller fragments of some
hundred or thousand bp. This is done with restriction endonucleases. These
enzymes cleave double-stranded DNA very selectively at palindromic
sequences. The palindromes are 4-8 bp long. The longer the recognition
sequence of the enzyme, the greater is the average size of the fragments
generated. Some restriction endonucleases cleave in the middle of their
recognition sequence and produce blunt-ended fragments. But most make
staggered cuts, producing fragments with short single-stranded overhangs
(“cohesive ends”).
Note that the cohesive end of a restriction fragment can anneal (= base-
pair) with the end of any other fragment generated by the same restriction
endonuclease. You can even link two restriciton fragments covalently. Mix the
fragments, let them anneal, then add DNA ligase.
Restriction endonucleases are bacterial enzymes that are used by the
bacteria as a defense against DNA viruses. Bacteria protect senstitive sites in
their own DNA by methylation. There are lots of restriction endonucleases and a
few hundred of them are commercially available.
II. PROBES
One of the main tools in working with DNA, is a probe that recognizes the
DNA of interest while ignoring everything else. A probe is a labelled (fluorescent
or radioactive) single-stranded nucleic acid that is complementary to the nucleic
acid you are looking for. Most important:
- A cDNA probe represents the exon sequences of a gene. It will
identify any restriction fragment that contains expressed sequences of the gene.
A cDNA is the reverse transcript of an mRNA.
- An oligonucleotide probe is a synthetic DNA that is complementary
to a specific DNA sequence. If used on a restriction digest of genomic DNA, it
has to be at least 17 or 18 nucleotides long because otherwise the target
sequence may be present in multiple sites in the genome. Oligonucleotides of
this length can be synthesized at moderately high cost, when 32P- or
fluorescently-labelled.
The probe hybridizes with single-stranded DNA (or RNA) that is
complementary to its own base sequence. The assay conditions are said to be
of high stringency when the probe anneals only with the correct sequence but
not with closely related sequences. High temperature, low ionic strength and
72
alkaline pH interfere with annealing and therefore represent conditions of high
stringency. Under proper stringency conditions, a single mismatch can prevent
the hybridization of an oligonucleotide probe with its target sequence.
III. SOUTHERN BLOTTING
A restriction digest contains thousands to millions of restriction fragments.

Southern blotting combines electrophoretic separation and probing to identify
interesting restriction fragments.
1. Treat the DNA with a restriction endonuclease.

2. Separate the restriction fragments by electrophoresis on a cross-linked
gel (agarose or polyacrylamide). These methods separate the
restriction fragments by size: small fragments move fastest. This
gives you a pretty accurate estimate for the size of the restriction
fragments.
3. Dip the gel in a NaOH solution to denature the restriction fragments.
4. Transfer (“blot”) the denatured DNA to a nitrocellulose filter.
Nitrocellulose binds single-stranded DNA very tightly. Blotting
produces a replica of the electrophoretic separation on the
nitrocellulose.
5. Incubate the nitrocellulose filter in a solution of the probe and wash off
excess probe.
6. Visualize the bound probe by autoradiography or fluorescence
scanning.
This procedure answers two questions:

1. Is a sequence complementary to the probe in your DNA extract?
2. How long is the restriction fragment detected by the probe?
Northern blotting is a similar procedure for RNA: the RNA (usually

mRNA) is separated by gel electrophoresis, blotted to nitrocellulose, and probed.
Restriction enzyme digest and NaOH treatment is not included in Northern blot
as opposed to Southern blot.
Western blotting is a method for the separation of proteins. The proteins
are separated by two-dimensional gel electrophoresis, blotted to nitrocellulose,
and “probed” with a monoclonal antibody.
IV. THE POLYMERASE CHAIN REACTION (PCR).
PCR is a method for the enzymatic amplification of double-stranded DNA

in vitro. This is the second major tool in the DNA laboratory. It can be done on
crude DNA extracts, but only a selected segment is amplified. You have to know
at least the flanking sequences of the DNA you want to amplify. You need:
73
1. The extracted DNA.
2. Substrates for DNA synthesis: dATP, dGTP, dCTP, dTTP.
3. A heat-stable DNA polymerase (usually Taq polymerase from the
bacterium Thermus aquaticus). This enzyme polymerizes DNA at
600C and survives repeated heating to 950C. Like other DNA
polymerases, it requires a primer to start DNA synthesis.
4. A pair of oligonucleotide primers that hybridize with complementary
sequences on each of the DNA strands. They mark the ends of the
amplified sequence.
Procedure:
1. The DNA is mixed with deoxyribonucleotides, Taq polymerase, and a

very large (>million fold) excess of the primers.
2. The DNA is denatured by heating to 950C.
3. The solution is cooled to 600C. The primers anneal and Taq
polymerase synthesizes new DNA starting from the 3’ ends of the
primers. The exact temperature depends on the primer sequences.
4. After 2 or 3 minutes, when the Taq polymerase has worked its way
from one primer to (and beyond) the level of the next, the solution is
heated again to separate the strands.
5. The solution is run through 20 – 30 cycles of heating and cooling. In
theory, the amount of DNA between the primers doubles in each cycle.
PCR generates a PCR product. This is a blunt-ended double-stranded

DNA whose ends are marked by the primers. Indeed, the primers are
incorporated in the PCR product. PCR products can be probed, but it is usually
more convenient to simply electrophorese the product and stain the gel with
ethidium bromide. There is so much more PCR product than other DNA that you
74
can always recognize the product as a band and you are rarely able to see the
starting material in the gel.
Because of its sensitivity (only a few DNA molecules are needed), PCR is
the workhorse for all those applications where only minute amounts of DNA are
available, including prenatal diagnosis, preimplantation diagnosis, forensic
applications, and the study of fossil DNA. Limitations:
- Taq polymerase does not proofread its product, therefore the error rate
is high. In part for this reason, the method is suitable only for the
amplification of short pieces of DNA, up to 3 kbp.
- Because of its high senstivity, contamination with extraneous DNA is a
big problem.
V. DNA SEQUENCING AND DEDUCED FUNCTIONS
Historically, there were two important methods of DNA sequencing, the

Maxam-Gilbert Method and the Sanger Dideoxy Method. Today, only the
Sanger method is in use. Both methods require a single-stranded DNA, and they
determine the sequence of up to several hundred bases in one sitting.
We know our chromosomal DNA sequences, but we still need to
determine which sequences encode functional gene products, and what those
gene products accomplish in the body. Genes can be recognized in newly-
sequenced DNA by telltale signs like the TATA box (DNA = TATAAA, start codon
(mRNA = AUG), stop codon (mRNA = UAA, UAG, UGA), polyadenylation signal
(mRNA = AAUAAA) and splice sites (Intron-exon junctions; Introns begin with
RNA = GU and end with AG; there are longer but degenerate consensus
sequences for these boundaries).
If you find a new gene, you can sometimes deduce its function.
- Genes, and the proteins they encode, come in families whose members
are structurally related.
- If the amino-terminus of the encoded protein is a signal sequence, the
protein is most likely a membrane protein or secreted protein.
- Hydrophobic sequences of about 20 amino acids in the encoded
polypeptide suggest membrane-spanning α-helices.
- The presence of specific amino acid sequences may suggest
glycosylation or phosphorylation of a gene product.
VI. GENE MAPPING.
Even with the complete genome sequence in hand, the mapping of genes
is a major task in modern genetic research. It is used to map:
- Genes for Mendelian disorders
- Genes for normal polymorphisms (blood groups, etc.).
- “Susceptibility genes” for multifactorial diseases (diabetes, Alzheimer).
- Genes affecting continuously variable traits (blood pressure,
intelligence, divorce risk……)
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1. Fluorescent in-situ Hybridization (FISH).
A strongly fluorescent cDNA probe is hybridized to a prophase or
metaphase chromosome spread. A banding technique is used, and this permits
the assignment of the gene to a specific chromosome band. This method can be
used whenever a larger genomic probe (>25,000 bp) is available. Sometimes, a
cDNA probe can be used.
2. Deletion Mapping
Most disease-causing mutations are loss-of-function mutations that lead to
a nonfunctional or absent gene product. Occasionally, a very large deletion that
obliterates the gene itself and a variable amount of neighboring DNA (including,
perhaps, some neighboring genes) is the cause of an apparent single-gene
disorder. If the deletion is large enough for detection by high-resolution banding,
the locus can be assigned to a chromosome band with an accuracy of + a few
million base pairs. In other cases, a single-gene disorder is caused by a
chromosomal rearrangement when the breakpoint is within the gene.
4. Linkage Analysis
Genes that are close together on the same chromosome are genetically
linked: They segregate together during meiosis. With increased distance of the
loci, however, there is an increased chance of crossing-over. A distance on the
chromosome with a recombination frequency of 1% is called one centiMorgan.
It corresponds to about 1 million base pairs of DNA.
Genes can sometimes be mapped if the locus of an unknown gene is
close to that of another gene with known position. Example: The genes for
hemophilia and color blindness are about 10 centiMorgans apart on the X-
chromosome; and the hemochromatosis gene is closely linked with the genes for
the HLA antigens on chromosome 6. The most important genetic markers,
however, are not genes but different anonymous DNA markers
- A classical restriction fragment length polymorphism (RFLP): is
caused by a cleavage site for a restriction endonuclease that is sometimes
present and sometimes absent. Two fragments of different length are generated
that can be analyzed by Southern blotting. These are rarely used.
- A variable number of tandem repeats (VNTR) is a short (2 to some
dozen base pairs) sequence that is tandemly repeated many times. Most VNTRs
are not in the coding regions of genes but in untranscribed spacers or introns.
Good VNTRs are highly polymorphic, with many “alleles” in the population.
Ideally, most people in the population should be heterozygous for the VNTR.
VNTRs are analyzed by Southern blotting or PCR. These are the markers most
often used. "DNA Fingerprinting" methods involve analysis of VNTR analysis.
Mathematics:
The recombination fraction τ is the probability of recombination between
two loci (for example a disease gene and an RFLP). It has a numerical value
between 0 and 1 and corresponds to the distance in centimorgan.
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The likelihood ratio can be calculated for different recombination
fractions. It is defined as: The probability that the observed association occurs if
there is linkage (with an arbitrarily assumed value of τ) divided by the probability
for this result if there is no linkage. A high likelihood ratio means that the
observed results are likely to be caused by linkage rather than the random
coninheritance of two unlinked traits.
The lod score is the logarithm of the likelihood ratio. A lod score of >3 is
taken as reasonably good evidence for linkage. Formally, it corresponds to a
likelihood ratio of 1000; in reality, a better description is that a LOD score of >3
corresponds to the usual p<0.05 limit familiar from statistical analysis.
Linkage studies require large families with multiple affected family
members. They can be used even if absolutely nothing is known about the
structure of the gene and its product, but you need to know the inheritance
pattern. If you don’t know on which chromosome the gene is, you may have to
make a genome-wide scan, tracking the inheritance of your gene with a few
hundred VNTRs scattered all over the 22 autosomes. Linkage studies can only
determine the approximate genomic location of a disease gene. The gene itself
has to be identified by the study of candidate genes in the relevant area and by
DNA sequencing.
5. Candidate Genes
Some diseases give clues about the nature of the gene product. Many
connective tissue diseases, for example, are caused by abnormalities of
extracellular matrix proteins (collagen, elastin etc). If you have narrowed down
the position of a disease gene by linkage studies, you can obtain sequence
information for genes in the region from DNA data banks. You can then
tentatively identify “suspicious” genes and see if your patients have mutations in
these candidate genes. This involves mutation scanning and/or the actual
sequencing of the candidate gene in the patients. However, not all mutations
cause disease. You may stumble on a normal polymorphism that has nothing to
do with the disease!
VII. CLONING AND GENOMIC LIBRARIES
DNA can be amplified by PCR. It can also be amplified by incorporation

into a bacterium where it is replicated along with the bacterial DNA. This is
called cloning. A clone is a “family” of cells that are descended asexually from a
common ancestor. Foreign DNA (for example human genomic DNA) is not
incorporated into the bacterial chromosome but into a cloning vector.
The cloning vector may be plasmid: a circular, double-stranded DNA that
has a replication origin and that carries genes. Most bacteria naturally contain
plasmids. Those plasmids that are used as cloning vectors are R-factors that
contain at least one gene for antibiotic resistance. Also temperate
bacteriophages, like λ phage, can be used as vectors. Many cloning vectors are
artificial constructs that were patched up from bits and pieces of plasmids and
bacteriophages. The foreign DNA is ligated into the vector DNA in vitro. The
77
recombinant vector is then spirited into the bacterial host cell where it replicates
along with the bacterial DNA.
Cloning can be used to make a genomic library. This is a large
collection of bacteria, each containing a piece of human genomic DNA. In order
to be useful, a genomic library has to have every genomic DNA sequence
represented in at least one bacterium. Simplified procedure for constructing a
genomic library with a plasmid vector:
1. Take an R-factor plasmid that contains a gene for ampicillin (type of
weak penicillin) resistance, and cleave its DNA with a restriction
endonuclease. You have to use a very selective restriction enzyme
that cleaves the plasmid at only one site, outside the resistance gene,
creating a linear DNA with single-stranded cohesive ends.
2. Extract human genomic DNA and cleave it with the same restriction
enzyme that was used for the plasmid.
3. Mix the cleaved human and plasmid DNA, let them anneal with their
sticky ends, then add DNA ligase: human DNA and plasmid DNA
become covalently linked into a circle.
4. The engineered plasmid is brought into the bacterium by
transformation. This is a low-efficiency process but can be achieved in
satisfactory yield in the presence of high calcium concentration.
5. The selection of transformed clones is achieved by plating the bacteria
on agar plates in the presence of ampicillin. Only transformed bacteria
can survive. Each colony is a clone that contains a plasmid, hopefully
with a piece of human DNA.
6. The colony pattern of the agar plate can be transferred to other agar
plates. This is called replica plating.
7. The colonies of a replica plate are lysed with NaOH. The denatured
DNA is replica-plated to nitrocellulose. After drying, the nitrocellulose
filter is dipped into the solution of a probe which identifies a specific
sequence of human DNA. This step is called screening.
Plasmid vectors are good to propagate small pieces of DNA, up to 5000

bp or 5 kbp. Larger chunks (≈15 kbp) can be cloned in λ phage: the “non-
essential” phage genes are replaced by an insert (= foreign DNA), and the
engineered DNA is packaged into a phage particle in vitro. Only DNA of the right
size is packaged. The engineered phage is brought into the cell and propagated
by lysogenic infection. Very large DNA pieces can be cloned in yeast cells in the
form of yeast artificial chromosomes (YACs), patched up from centromere
sequences, telomere sequences, and cloned DNA.
VIII. cDNA CLONING AND EXPRESSION CLONING
Only 1-2% of the DNA in a genomic library is coding. If you are only
interested in coding DNA, you better make a cDNA library. A cDNA is a double-
stranded DNA copy of a RNA, made by the retroviral enzyme reverse
78
transcriptase. In genome sequencing, cDNAs or fragments of cDNAs are
referred to as expressed sequence tags (ESTs). Recipe:
1. Isolate mRNA by affinity chromatography on an oligo-dT column. The

poly-dA tail sticks to the immobilized poly-dT.
2. Add oligo-dT primers, reverse transcriptase, and deoxyribonucleotides.
The oligo-dT primer hybridizes with the poly-A tail, and the reverse
transcriptase synthesizes the cDNA.
3. Ligate the cDNA into a vector, then proceed as usual.
The tissue of origin doesn’t matter for a genomic library because all cells
have the same genome. But it is important for a cDNA library because different
cells express different genes.
Ordinarily, neither cloned genomic DNA nor cDNA is expressed in
bacteria. For expression cloning, you have to ligate:
- A human cDNA. Genomic DNA won’t do because bacteria cannot
remove introns.
- A bacterial promoter. You need a strong promoter, or one that can be
regulated at will. For example, with the promoter of the lac operon,
the cloned gene is expressed only when the bacteria are kept on a
glucose-free, lactose-rich diet.
- The cDNA of a bacterial ribosome-binding sequence (Shine-Delgarno
sequence) has to be ligated between the cDNA and the promoter.
- A bacterial signal sequence can be included if desired. In this case the
bacteria will secrete the protein.
With expression cloning, you can turn bacteria into lucrative factories for
hormones, clotting factors, cytokines, or any other protein. Only post-
translational modifications (phosphorylation, glycosylation) are problematic
because bacteria don’t have the right processing enzymes. Many genetically
engineered therapeutics are on the market, including human insulin, growth
hormone, interferon, clotting factor VIII, erythropoietin and tissue-type
plasminogen activator, and humanized antibodies.
IX. SITE-DIRECTED MUTAGENESIS AND PROTEIN ENGINEERING
The base sequence of cloned DNA can be changed intentionally. Cloned

DNA is isolated with the help of a restriction endonuclease, modified by
enzymatic methods, and re-inserted into a cloning vector. Several methods are
available for the introduction of targeted mutations.
If you use expression cloning, you can produce proteins with amino acid
substitutions or other minimal changes. You can also make truncated proteins
that are missing part of the polypeptide, for example soluble variants of the spike
proteins of the AIDS virus that interfere with AIDS infection because they jam
virus receptors on the cell surface. Domain shuffling is the patching-together of
parts from different genes to make a new, recombinant gene (and protein).
79
Example: If you link the DNA-binding domain of the androgen receptor with the
retinoic acid-binding domain of the retinoic acid receptor, and engineer this into
your tissues, you can improve your virility by gobbling vitamin A.
X. USE OF DNA DIAGNOSTICS
The molecular diagnosis of disease genes and normal polymorphisms is

the fastest-growing area of modern medical technology. Uses:
- Diagnosis of Mendelian disorders
- Carrier detection in relatives of patients
- Newborn screening
- Population-based screening
- Predictive testing for late-onset diseases
- Susceptibility testing for multifactorial diseases
- Prenatal and pre-implantation diagnosis
- Paternity testing
- DNA fingerprinting of criminal suspects
Technical problems for DNA diagnostics include:
- Most single-gene disorders show substantial allelic heterogeneity.
Therefore, genetic tests must screen for many mutations.
- Common diseases are usually multifactorial. Therefore, genetic testing
allows only a statistical risk estimate.
XI. SOUTHERN BLOTTING WITH ALLELE-SPECIFIC PROBES

Allele-specific probes (ASOs) can be used when both the normal gene
sequence and the mutation are known: there is no allelic heterogeneity and no
locus heterogeneity. Procedure for sickle cell testing:
1. Make (or buy) two synthetic oligonucleotide probes each of the
same length (>18 bp), one complementary to the sequence of
the sickle cell mutation and one complementary to the
corresponding normal sequence.
2. Treat the patient’s DNA with a restriction endonuclease that
cuts left and right of the probed sequence.
3. Divide the restriction digest in two aliquots, and electrophorese
them separately in two lanes of a gel. Then blot to
nitrocellulose.
4. Apply the probe for the normal sequence to one lane, and the
probe for the sickle cell mutation to the other. The stringency
has to be adjusted carefully because the two probes differ by
only one base.
You can distinguish between homozygous normal (only the normal probe
binds), homozygous affected (only the sickle cell probe binds) and heterozygous
(both probes bind). In this example the restriction fragments have the same
length and the same electrophoretic mobility.
80
Example: Adenomatous polyposis coli, an autosomal dominant cancer
susceptibility syndrome. Will the children in generation III get cancer?
II
III
Southern Blots
Example: Prenatal diagnosis of β-thalassemia. Has the fetus II-3

inherited the disease? Is he a carrier? Is the unaffected daughter II2 a carrier, or
homozygous normal?
II
Southern blots
Example: Duchenne muscular dystrophy, a severe X-linked recessive

muscle disease. Are II2 and II3 carriers?
II
Southern Blots
81
In the case of small insertions or deletions, only one probe is needed because
the restriction fragments differ in length. Allele-specific or ogranism-specific
oligonucleotides are probably more often used in Dot blot analysis thereby
saving the time-consuming electrophoresis step.
Limitations: Most single-gene disorders show extensive allelic
heterogeneity i.e., many different mutations. Each set of allele specific
oligonucleotides will only give you information on one specific mutation. You
cannot use allele-specific probes in these cases unless you have identified the
mutation in the affected family first, or are prepared to repeat the experiment with
many different set of oligonucleotides (and then sequencing would probably be
faster and cheaper). Note - A single genetic test sometimes unavailable for
genetic disease testing. See the GeneTest website for available tests and
clinical testing sites for individual conditions. However, see the section on
microarrays.
XII. USE OF PCR.
PCR requires less DNA than Southern blotting. It is therefore the

preferred procedure for prenatal diagnosis and pre-implantation diagnosis.
Example: Cystic fibrosis is a severe autosomal recessive disease that is
most often caused by a 3-basepair deletion. If both parents carry this mutation
(disease risk: 25%), you can do prenatal diagnosis by Southern blotting.
Alternatively, you can use PCR:
1. Amplify a short segment of the fetal DNA (<100 bp) with a
primer pair that frames each potential mutation site.
2. Separate the PCR products by gel electrophoresis. Run
controls with the normal PCR product and the PCR product
with the deletion in separate lanes of the same gel. Then
stain with ethidium bromide.
If the fetal DNA yields only the normal PCR product, the fetus is
homozygous normal; if only the shortened fragment is produced, it is
homozygous affected; if both bands are present, it is heterozygous.
In the diagnosis of point mutations, the two PCR products have the same
length and cannot be distinguished on the gel. Sometimes, a restriction enzyme
recognition site is changed by the mutation; then restriction enzyme digest of the
PCR product will produce fragments of different lengths. Otherwise, allele-
specific probes have to be used.
PCR can also be used in cases where we try to detect presence or
absence of a given DNA piece. First example in HIV infection: primers specific
for the HIV genome will only produce a fragment if the DNA is derived from cells
infected with HIV. Second example is diseases caused by deletion of one or
more exons in a gene. Primers specific for each exon are used in the PCR, and
presence of a fragment will demonstrate presence of the exon in the starting
material. Especially in these last applications is there a major problem in
securing against accidental contamination with extraneous DNA. The product
82
from last time you performed the procedure can sometime float around in the
laboratory in the form of microdroplets.
XIII. DOT-BLOTTING.
(A) Dot blotting with DNA samples: Both Southern blotting and PCR with
electrophoretic separation provide information about the length of a restriction
fragment or PCR product. When this information is not needed, and you can use
dot-blotting. Procedure:
1. Treat the extracted DNA with a rarely-cutting restriction
endonuclease. The probed sites have to remain
intact!
2. Denature the DNA, and apply it in single dots to two
nitrocellulose filters.
3. Probe one filter with a probe for the normal sequence,
and the other one with a probe for the mutation.
Dot blotting takes far less time than the other procedures. The DNA of
many people can be tested simultaneously on the same nitrocellulose filter.
Therefore, it can be used for screening programs. Like other allele-specific
methods, the usefulness of dot-blotting is limited by genetic heterogeneity. Also,
this method tests for only one DNA sequence variant at a time.
(B) Dot blotting with RNA samples: the purpose of this procedure is similar
to the purpose of Northern blot, i.e., to measure the expression level of a gene in
a sample from a tissue. Procedure:
1. Purify total mRNA from the tissues of interest
2. Spot the same amount of mRNA from each sample onto a filter
3. Probe with a cDNA, a single exon, or a long oligonucleotide
4. The resulting signal will be proportional to the concentration of the specific
mRNA in the sample
Because this procedure omits the gel electrophoresis step of a Northern
blot, it is not possible from position information to see that there has been
aberrant hybridization to another mRNA, e.g., from a similar gene (you want to
check beta-hemoglobin; do you get hybridization to alpha-hemoglobin?).
Therefore, the specificity of a probe for Dot blot should be tested in Northern blot
before usage in Dot blot. Advantage of Dot-blot for mRNA is speed, larger
number of samples on a blot, and fewer steps necessary increasing the chance
that the mRNA survives to the time of hybridization (mRNA is a difficult material
to work with, RNAses are rampant).
XIV. MICROARRAY TECHNOLOGY (DNA CHIPS).
1. Limitations of Dot-blotting.
In traditional probing (dot blotting, Southern blotting), the probe is applied
to the immobilized target DNA. To test the target DNA not just for one mutation
but for all theoretically possible mutations (“mutation scanning”), a large number
83
of probes have to be applied successively. For example, a 900 bp PCR product
can be probed with 900 nonanucleotides (9-base oligonucleotides), progressing
stepwise one nucleotide at a time. Theoretically, 9 of these probes will not bind
properly if the cDNA has a single-base substitution. To specify the base change,
the central base in each of the 900 probes can be varied, amounting to a total of
3600 probes. This set of probes can be used if a mutation in the PCR product is
suspected, but we don’t know which mutation to expect, where the mutation is, or
whether there might be more than one mutation. Using such a large number of
probes would be too cumbersome for practical applications, however.
2. Gene Chips.
Gene chips are used for gene scanning, or the simultaneous analysis of
many mutations or polymorphisms: The probes are immobilized on a solid
support, and the target DNA is labeled with a fluorescent tag. In the above
example, all 3600 probes could be bound to different squares on a single glass
or silicon chip about 2 x 2 cm in size. The chip is dunked in a solution of the
fluorescently-labelled target DNA, excess target DNA is washed off, and the DNA
chip is scanned using a laser. Since up to 100,000 probes fit on a single chip,
all scanning and recording has to be computerized. Applications of gene chips,
or microarrays, include:
- Mutation scanning for individual genes in patients, to figure out
whether a clinical problem is caused by a defect in a specified gene.
- The discovery of polymorphisms, especially single-nucleotide
polymorphism (SNPs), in the human genome. There are a few million SNPs in
our genome, but only a few of them (several thousand?) are medically important.
Also, sequence differences between related species (e.g. human and chimp) can
be detected with gene chips.
- The simultaneous testing for multiple mutations and polymorphisms in
genetic screening. Rather than screening for one mutation at a time, people
can be screened for thousands of single-gene disorders, susceptibility genes for
multifactorial diseases, and normal polymorphisms, all in the same procedure.
DNA chips are expected to become the standard method for predictive genetic
screening, also for genetic profiling of fetuses and preimplantation embryos to
achieve better quality control in reproduction.
3. cDNA Chips.
cDNA chips are used in basic research to study patterns of gene
expression. The immobilized probes are not oligonucleotides, but cDNAs; and
they are not used to analyze genes, but mRNA. The mRNA is extracted from the
cells or tissue, labeled with a fluorescent group, and then applied to the cDNA
chip. Since each cDNA on the chip corresponds to one gene, you can see which
genes are expressed. Example: The pattern of gene expression in a tumor can
be compared with gene expression in the normal cells from which the tumor
originated.
84
XV. DNA FINGERPRINTING
This is a set of methods to identify persons by their DNA, used

forensically. VNTRs of various kinds are used:
- Microsatellites are small VNTRs with 2 – 4 bases in the repeat unit.
They are usually analyzed by PCR exactly like the VNTRs used
in linkage studies. This is called single-locus DNA
fingerprinting. Several microsatellites have to be analyzed in
parallel to identify a person reliably.
- Minisatellites are longer VNTRs, with about a dozen or more bases in
the repeat unit. They are usually analyzed by Southern blotting.
If they are identified with a probe for the repeat locations in the
genome, a single test yields a complex pattern. This is called
multi-locus DNA fingerprinting.
DNA fingerprinting has been used in many cases to exonerate wrongfully
convicted people. It is also used for paternity testing, replacing older and less
accurate methods.
XVI. ANTISENSE TECHNOLOGY
Some diseases are caused by the expression of undesirable genes; viral

genes in virus diseases, oncogenes in cancer, and antibody genes in
autoimmune diseases. Antisense technology attempts to prevent the expression
of these genes by means of an oligonucleotide that is complementary to the
gene’s mRNA. The antisense oligonucleotide prevents translation, and it causes
the degradation of the base-paired mRNA by the cellular enzyme RNAse H.
Problems arise from the poor degradation in the cell. Therefore only
oligonucleotide analogs with non-hydrolyzable bonds instead of ordinary
phosphodiester bonds are used:
O O
Phosphodiester O P O Phosphorothioate S P O
Bond O Bond O
Antisense technology can be combined with gene therapy by engineering

an antisense gene into the cells. The RNA transcript of the antisense gene is
complementary to the mRNA of the undesirable gene.
85
Ribozymes are catalytic RNA oligomers which base-pair with specific
mRNA (target) sequences. These drugs also contain a region which cleaves the
target mRNA, thus reducing the concentration of transcripts for protein synthesis.
XVII. SOMATIC GENE THERAPY
Most genetic diseases are caused by inactivating (“loss-of-function”)

mutations. In theory, these diseases can be treated by introducing an active,
functional gene into those somatic cells where it is needed. Problems:
a) Large DNA molecules are not easily taken up by cells.
b) Even if it enters the cell, the gene may be degraded by nucleases.
c) Exogenous genes rarely become integrated into the cellular
genome, although this is theoretically possible by homologous
recombination.
d) The regulation of gene expression is problematic for artificially
introduced genes. Therefore, gene therapy is promising only if
tightly-regulated gene expression is not required.
e) The targeting of a gene to the relevant tissue is difficult.
f) Somatic gene therapy does not affect the germline. The patient
can still transmit the defective gene to his children.
In the United States, some gene therapy trial participants have died as a
result of the therapy given. This has lead to a reconsideration of the ethics of
gene therapy in the treatment of human disease.
Physical methods of gene delivery (liposomes, receptor-mediated

endocytosis) have been attempted, but viral vectors are more promising.
Retroviral vectors are attractive because they insert the gene directly into the
cellular genome. They contain retroviral long terminal repeats, but the gag, pol
and env genes are replaced by the transferred gene, all nicely wrapped in a
retroviral capsid and envelope. The reverse transcriptase + integrase in the virus
particle effect reverse transcription and integration into the genome. Problems:
- Only low titers can be produced.
- Only dividing cells can be transfected by currently used retroviral vectors.
Lentiviral vectors (related to the AIDS virus) are promising because
they can infect nondividing cells. DNA viruses, for example
adenoviruses, can be produced in high titers and infect nondividing
cells, but
- They rarely integrate into the host cell genome.
- They damage the infected cells.
Gene therapies are still experimental. They are not only promising for the
treatment of genetic diseases, but also for some other diseases, for example,
bringing genes for anti-inflammatory proteins into the joints of arthritis patients.
Many gene therapy protocols are ex vivo techniques, and only a small
proportion (Usually less than 1%) of the cells are stably transfected.
86
XVIII. GERMLINE GENE MANIPULATIONS.
Genes can be introduced into the germline, and normal genes can be
artifically disrupted by gene tarrgeting. These methods are used to create
knockout mice and transgenic mice. These animals provide models for human
diseases, and the abnormalities of knockout mice (if any) reveal the functions of
the knocked-out gene. Transgenic animals are created to secrete valuable
therapeutic proteins in their milk or supply human-friendly organs for
transplantation.
Viral vectors are not used for germline gene manipulations. Genetic
engineers prefer gene constructs with flanking sequences that are identical to
“reveal” genomic sequences. These constructs can be incorporated into the
genome by homologous recombination. Techniques:
a) The gene construct is microinjected into the male pronucleus of the
zygote, or the zygote is bathed in the DNA while an electrical pulse
is applied to make the membrane permeable (Electroporation).
The zygote is grown into an embroyo, and the embryo is implanted.
b) Cultured embryonic stem cells are treated with foreign DNA by
microinjection or electroporation. The engineered cell is fused with
an enucleated oocyte, or its nucleus is injected into an enucleated
oocyte. The oocyte is grown into an embryo and implanted. This is
the most important application of cloning technology.
c) Cultured embryonic stem cells are treated with foreign DNA. These
engineered cells are injected into an early embryo. This embryo
will be a chimaera (a composite of two genetically different
individuals), and some of the engineered cells may enter the
germline. The rest is classical breeding.
There are not many indications for germline gene therapy in humans
because most genetic risks are more easily handled by prenatal diagnosis or the
selective implantation of genetically screened embryos. The introduction of
useful genes to diversify the human gene pool, for example genes for vitamin C
synthesis or cobalamine synthesis, or spare copies of tumor suppressor genes to
reduce the cancer risk, or genetical manipulation of immune tolerance would
make more (anti-?) sense.
XIX. PROTEOMICS METHODS
Proteomics entails the study of the complete complement of proteins

encoded by an organism. Separation of proteins is usually accomplished using
two-dimensional (2-D) gel electrophoresis. First proteins are separated by
charge using isoelectric focusing (IEF); the separated proteins are then further
purified by separation in a second dimension based on size differences. The 2-D
gel can then be used as a template to analyze the proteome using antibody-
based technology, or chemical analysis. Mass spectrometry technology allows
87
sensitive and accurate detection of proteins in 2-D gels. Even minor differences
in very large proteins can be analyzed. For instance, the slight mass difference
in the sickle-cell globin protein can be distinguished from the normal globin
protein using a single spot on a 2-D gel analyzed in a mass spectrometer.

1. Outline the procedure of DNA sequencing by the Sanger/Dideoxy and Maxam-Gilbert methods.
2. Define what is meant by the terms palindromic sequence, cleavage specificity, blunt and sticky
ends, cloning site, high/low frequency cutters with respect to restriction endonucleases. Name
and describe the function of DNA ligase, reverse transcriptase, polynucleotide kinase,
topoisomerase, RNAase and DNAase.
3. Outline the procedure of Southern blotting and describe the general use of the method.
4. Outline the procedure of polymerase chain reaction (PCR) and describe the general use of the
method.
5. Define what is meant by "restriction fragment length polymorphism" and provide an example of
when RFLP analysis would be required to make a clinical decision.
6. Provide a rationale for the use of RFLP analysis and analysis of a VNTR site (Variable Number
of Tandem Repeats) in linkage studies.
7. Explain how somatic cell hybrids, in-situ hybridization methods, and linkage studies are useful
in gene mapping.
8. Define the recombination fraction and LOD scores.
9. List the steps in cloning foreign DNA into an R-factor plasmid and in lambda phage.
10 Compare the procedures for generation of a genomic library, a cDNA library and an
expression library; and describe how each library can be screened for a clone of interest.
11. Give examples of proteins produced using genetic technology, and their therapeutic value.
12. Describe procedures for site-directed mutagenesis in cloned DNA.
13. Explain the rationale for using carrier-testing, presymptomatic testing, population-based
heterozygote screening, prenatal diagnosis and pre-implantation diagnosis.
14. Specify examples of when Southern blotting with allele-specific oligonucleotide probes is
useful to diagnose a known mutation.
15. List the advantages and disadvantages of using PCR-based methods for genetic diagnosis.
16. List the advantages and disadvantages of linkage studies for the diagnosis of genetic
diseases.
17. Calculate carrier probabilities and disease risks for Mendelian disorders using linkage studies
and closely linked RFLPs.
18. Describe the advantages and limitations of current gene therapy and antisense protocols,
including vector design, and clinical rationale.
19. Explain how germline gene manipulation is accomplished, and how knockout and transgenic
mice are useful in medical research.
20. Outline the methods used to produce microarrays and 2-dimensional protein gels data from a
clinical sample. Explain how these methods are medically useful for analysis of complex patterns
of gene expression.
88

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Course Handouts 1-13

Biochemistry and Genetics I

The faculty of the Biochemistry Department at Ross University

General Biochemistry Courses:

Enjoy your learning experience in this course! And remember, the

The Biochemistry Faculty

I. ELEMENTS AND MOLECULES

Biomolecules contain only a few of the 92 elements that exist in nature:

Carbon (C): 12 Nitrogen (N): 14

Composition of the human body :

Class of Molecule Content (%) MW (Daltons)

II. COVALENT BONDS AND NONCOVALENT INTERACTIONS

Ions carry either a positive charge (cations) or a negative charge (anions).

Urea Acetamide Acetone Propane

Higher solubility Lower solubility

Polar interactions (ion-ion, ion-dipole, dipole-dipole) are also important for

Functional groups determine chemical reactivity and physical properties

Carbonyl group: H (adehyde), R (ketone)

Amino group: R N H (primary amine)

Carboxylic ester: From carboxy group and hydroxy group.

Phosphate ester: From hydroxy group and phosphate.

Phosphodiester: From hydroxy group and phosphate ester.

Phosphoanhydride: From phosphate and phosphate (-ester).

Ether: From two hydroxy groups.

Thioesters: From sulfhydryl group and carboxy group.

Amides: From (carboxylic) acid and ammonia or amine.

Isomers are chemically different molecules of identical composition. There

Alternative positional, geometric and optical isomers are not equivalent

V. ACIDS AND BASES

A proton can be transferred from one water molecule to another in a

2 H2O H3O+ + OH-

In distilled water, [H3O+] = [OH¯] = 10¯4 M, and

[H3O+] x [OH¯] = 10¯14 M2

pH = 7 neutral ([H+] = 10¯7)

This is a nonenzymatic, instantaneous, freely reversible equilibrium reaction.

The major basic group in biomolecules is the primary amino group:

The protonation/deprotonation reaction.

Can be described by its dissociation constant KD:

KD = [R-COO¯] x [H+] or [H+] = KD x [R-COOH]

We can put this whole equation into the negative logarithm:

Important ionizable groups in biomolecules:

VI. FATS AND CARBOHYDRATES

Palmitic acid is a saturated fatty acid. Monosaturated fatty acids have a

Triglycerides are not water-soluble. They are used as energy stores in

Galactose Glucose Mannose Fructose

Only D-isomers are shown as these are prevalent in nature.

Monosaccharides and disaccharides are water soluble. Polysaccharides are

VII. AMINO ACIDS

At pH = 7, amino acids exist as zwitterions – molecules that possess both a

Group Amino Acid pK*____

Hydrophobic Hydrogen Salt Covalent

Ph. = phosphate ester; CHO = glycosidic bond.

VIII. PROTEIN STRUCTURE

Polypeptides range in size from a few amino acids to hundreds or even

R' O R''' O R[n-1]

1. The primary structure of a protein is its convalent structure. It is defined

In the α-helix, the polypeptide is wound in a right-handed spiral, with 3.6

3. The tertiary structure is the overall folding pattern of the polypeptide

4. The quaternary structure is described by the subunit composition and

IX. NON-POLYPEPTIDE COMPONENTS OF PROTEINS

Many proteins contain a non-polypeptide component. In these proteins the

1. Glycoprotein contain mono- or (more commonly) oligosaccharides