Title: DNA, Genes and Chromosomes.
Content:
Nucleic acids are polymers of nucleotide monomers, each of which consists of a
heterocyclic base, a pentose, and phosphate. Biological nucleotides are
predominantly nucleoside 5'-phosphates. In nucleic acids, monomers are joined
by 3',5'-phosphodiester links. Nucleases are enzymes that catalyse the breaking
of these links. In the DNA double-helix, two polydeoxyribonucleotides, each a
right-handed helix, wind about a common axis. The structure involves specific
base complementarity and an antiparallel arrangement of the two polymers, and
is stabilised by hydrogen-bonding between paired complementary bases and by base-stacking
forces. DNA carries genetic information in unique sequences of bases. The
Central Dogma of Molecular Biology summarises the way in which these sequences
direct the sequences of bases in RNA and the sequences of amino-acids in
polypeptides. The DNA of virus particles is highly condensed, and, in various
viruses, has a coding potential of from less than 10 to about 200 genes. DNA of
bacterial cells is circular, and, highly condensed as a nucleoid, is attached
to the cell membrane. That of Escherichia coli has a coding potential of about
4 x 103 genes. Bacterial cells contain small extrachromosomal DNA molecules
called plasmids, which are much used in genetic engineering. DNA of eukaryotic
cells is linear and contained within the nucleus. That of human cells has a
coding potential of about 105 genes. Mitochondria and chloroplasts contain
small circular DNA molecules. In eukaryotic cells, some genes are repeated many
times, and, in some genes, coding sequences (exons) are interspersed with
non-coding sequences (introns); further, coding regions of the DNA (containing
genes) are interspersed with large non-coding regions, the sequence of which is
often repetitive. Such non-coding regions are particularly concentrated in the
centromere and the telomeres. Condensation of DNA in cells involves supercoils,
which are produced and removed by the action of enzymes called topoisomerases.
Eukaryotic chromosomal DNA is highly supercoiled around bundles
("nucleosomes") of basic proteins called histones.
Key words:
Nucleic acid, Nucleoside,
Nucleotide, Polynucleotide, Purine, Pyrimidine, Adenine, Cytosine, Guanine,
Thymine, D-ribose, 2-deoxy D-ribose, 3',5'-phosphodiester link, Nuclease,
Double-helix, Specific base complementarity, Antiparallel arrangement, A-T and
G-C base-pairs, Base-stacking forces, Major and minor grooves, Central Dogma of
Molecular Biology, Transcription, Translation, Genome, Chromosome, Chromatin,
Gene, Nucleus, Nucleoid, Plasmid, Mitochondrial and chloroplast DNA, Exon,
Intron, Repetitive base sequence, Centromere, Telomere, Supercoiling,
Topoisomerase, Nucleosome, Histone.
Textbook reference:
Lehninger et al. Chapters 12, 23.
Title: DNA Replication.
No. of lectures: 3.
Content:
In replication of DNA, a balance is required between extreme accuracy and
sufficient laxity to allow mutations for the genetic variability required for
adaptive flexibility. Specific base complementarity permits DNA of a specified
sequence to be synthesised accurately on a template of pre-existing DNA. Most
replication is semi-conservative, begins at particular origin(s) on the DNA
template, and proceeds from the origin(s) bi-directionally. Proteins required
at the origin for initiation of DNA replication in Escherichia coli include
DnaA, DnaB, DnaC, HU, SSB, primase and gyrase. Synthesis of DNA (both during
replication and repair of DNA) is catalysed by DNA polymerases. The precursors
of the monomers are 2'-deoxyribonucleoside 5'-triphosphates. All DNA
polymerases require a pre-formed primer with a 3' end, onto which monomers may
be added. During DNA replication, this priming function is performed by short
stretches of RNA, the synthesis of which is catalysed by particular
DNA-dependent RNA polymerases (enzymes able to catalyse polymer synthesis without
a primer) called primases. DNA synthesis during replication occurs in a 5' to
3' direction, the DNA template being read in a 3' to 5' direction. There are
three DNA polymerases in E. coli - DNA pols I, II, and III - and several in
eukaryotic cells. DNA synthesis during DNA replication is semi-discontinuous, a
leading strand being synthesised continuously, and a lagging strand being
synthesised discontinuouly as segments sometimes called "Okazaki
fragments", each of which is started by an RNA primer. In E. coli, the
major polymerising enzyme for both leading and lagging strands is DNA pol III.
5'-exonuclease activity of DNA pol I is used to remove RNA primers, the gaps in
the newly synthesised DNA being filled by the action of the polymerase activity
of DNA pol I. Both DNA pol I and III have 3'-exonuclease activity which is used
to proof-read the last monomer incorporated during the polymerising activity of
these enzymes.
Key words:
Semi-conservative replication, Meselson-Stahl experiment, Origin of replication,
Bi-directional replication, Cairns' experiment, Theta structures, Dna A, Dna B,
Dna C, SSB protein, Primase, Gyrase, DNA polymerase, DNA pols I, II, III, RNA
primer, Template, 2'-deoxyribonucleoside 5'-triphosphate precursor,
Semi-discontinuous replication, Leading strand, Lagging strand, Okazaki
fragment, Proof-reading (Editing), DNA ligase.
Textbook reference:
Lehninger et al. Chapter 24.
Title: DNA Sequencing and Amplification.
No. of Lectures: 1.
Lecturer: WFL.
Content:
The sequence of thousands of bases in a DNA polymer may be rapidly
determined using the 2', 3'-dideoxynucleotide chain termination method of
Sanger. This facility has enabled research like the Human Genome Project. The
polymerase chain reaction is a powerful tool allowing rapid amplification of
base sequences present in very small quantities of DNA. It depends on the
production of two primers that bind to sequences flanking the sequence to be
determined, and the use of a heat-stable DNA polymerase. Applications of the
process include the detection of very small amounts of virus or bacteria in
biopsy samples and, the screening for genetic diseases. It is also used in
forensic medicine and has created the new fields of molecular palaeontology and
archaeology.
Key words:
2', 3'-dideoxynucleotide; chain termination; polyacrylamide gel
electrophoresis; polymerase chain reaction; synthetic oligonucleotide primers;
Taq DNA polymerase.
Textbook reference:
Lehninger et al. Chapters 12, 28.
Title: Transcription.
No. of Lectures: 2.
Lecturer: KIJS.
Content:
Transcription is the process in which an enzyme catalyses the transfer of
the genetic information contained in a stretch of DNA into an RNA strand that
has a base sequence complementary to the DNA template strand. There are three
major forms of RNA, each with a different function. mRNA (messenger RNA)
contains the information to encode a protein; tRNA (transfer RNA) is the
adaptor that reads the information in the mRNA and transfers the appropriate
amino-acid onto the growing polypeptide chain during protein synthesis; and
rRNA (ribosomal RNA), together with a large number of proteins, forms the
ribosome which carries out protein synthesis. The enzymes that catalyse
transcription are DNA-dependent RNA polymerases. They catalyse addition of
nucleotides (selected by Watson-Crick base-pairing interactions) to the 3'-OH
of the RNA chain, thus building RNA in a 5' to 3' direction. In contrast to
replication, only selected parts of the genome are transcribed at any one time,
so transcription needs to be tightly regulated to ensure that only the genes
for the protein products required at a particular time are transcribed at that
time. This requires regulatory sequences in order to indicate the beginning and
end of the segments (genes) to be transcribed. Initiation of transcription
begins at the promoter, and DNA is unwound, allowing addition of
ribonucleotides and formation of an RNA-DNA hybrid. Termination occurs at
specific sequences - best understood in Escherichia coli - where termination can
be either r (rho)-dependent or r (rho)-independent. E. coli has a single
DNA-dependent RNA polymerase consisting of five core sub-units and a sixth
(called the s-factor) which is responsible for initiation at the promoter.
Eukaryotic cells have three DNA-directed RNA polymerases: Pol I catalyses the
synthesis of rRNA, Pol II primarily the synthesis of mRNA, and Pol III the
synthesis of tRNA and of some other specialised, small RNAs. Some RNA molecules
undergo extensive post-transcriptional processing. This is particularly true
for mRNAs in eukaryotes, which acquire a 5'-cap and a poly(A) tail, to help
stabilise the mRNA. Intron sequences are removed in a process called splicing.
Key words:
Transcription, mRNA, tRNA, rRNA, DNA-directed RNA polymerase, s-factor,
Template strand, Non-template strand, Transcription bubble, r-protein,
r-dependent termination, r-independent termination, 5' cap, Poly(A) tail,
Splicing.
Textbook reference:
Lehninger et al. Chapter 25.
Title: The Genetic Code and Translation.
No. of lectures: 3.
Lecturer: WFL.
Content:
Translation refers to the synthesis of a polypeptide on a template of messenger
RNA (mRNA). Three bases read in sequence along a DNA template are called a
"triplet" code-word. The complementary three bases on mRNA are called
a "codon". Each of twenty coded amino-acids has at least one codon.
The genetic code was solved by using synthetic RNA molecules as mRNAs in a
test-tube protein-synthesising system. The code is degenerate, non-overlapping
and comma-less. Knowledge of the nature of the code helped to elucidate the
mechanism of frame-shift mutations. Degeneracy minimises deleterious effects of
mutations. Codewords are assigned to amino-acids non-randomly. The code is
near-universal. Transfer RNA (tRNA) acts as an adaptor between an amino-acid
and the mRNA codon that encodes it. Each tRNA has an anticodon (at least
partially) complementary to a codon of the amino-acid to which it is assigned.
Amino-acyl tRNA synthetases are enzymes having functions crucial to the accurate
translation of genetic information. Each very specifically catalyses the
activation of a particular amino-acid and attaches it to the 3' end of an
appropriate tRNA. The amino-acid, once loaded, is anonymous, and recognition
thereafter is between tRNA anticodon and mRNA codon. The "Wobble
Hypothesis" enunciates the idea that some bases in the third position of
an anticodon may pair with more than one codon base. Some amino-acyl tRNA
synthetases are able to "proof-read" the loaded amino-acid and remove
it if it is incorrect. Ribosomes are the site of codon-anticodon recognition.
They consist of two sub-units of unequal size, both containing RNA (rRNA) and
protein. During translation, polypeptide is synthesised in an N terminus to C
terminus direction, and the mRNA template read in a 5' to 3' direction. AUG is
an initiator codon in Escherichia coli. At the start of a message, it is
recognised by a methionyl tRNA loaded with N-formylmethionione. This is the
only tRNA able to bind directly to the peptidyl site of the 50S ribosome
sub-unit. In E. coli, initial interaction between mRNA and ribosome involves
base-pairing between four-to-nine bases of the 16S RNA of the 30S ribosome
sub-unit, and complementary bases to the 5' side of the AUG initiator codon. Peptide
bond formation involves the activity of an rRNA molecule, which acts as a
ribozyme. Occurrence in frame of one of three termination codons (UAA, UAG,
UGA) terminates polypeptide synthesis.
Key words:
Translation, Messenger RNA (mRNA), Central Dogma of Molecular Biology,
Genetic Code, Triplet, Codon, Degeneracy, Non-overlapping, comma-less,
near-univerasal nature of code, Frame-shift mutation, Point (single-base)
mutation, Silent mutation, Conservative mutation, Open reading frame,
Overlapping genes, Transfer RNA (tRNA), Adaptor function, Clover-leaf
structure, Anticodon, Amino-acyl tRNA synthetase, Wobble hypothesis,
Proof-reading during translation, Ribosome, Ribosomal RNA (rRNA),
N-formylmethionine, Initiator codon (AUG), Peptidyl site, Amino-acyl site, 70S
sub-unit, 30S sub-unit, GTP, Ribozyme, Termination codons (UAA, UAG, UGA).
Textbook reference:
Lehninger et al. Chapter 26.
Title: Regulation of Gene Expression.
No. of Lectures: 3.
Lecturer: KIJS.
Content:
Only a small fraction of the genes of either prokaryotic or eukaryotic
cells are expressed at any one time. Some genes may only be required to be
expressed at a specific point in the cell cycle or when cells are grown in a
particular nutrient, while other cell genes need to be expressed at all times
(housekeeping genes). Since it is energetically costly to make a protein, it is
beneficial to the cell to regulate gene expression tightly. Regulation of gene
expression most often occurs at the level of transcription, but may also occur
at the levels of post-transcriptional processing, translation or
post-translational processing. Initiation of transcription is regulated by
proteins that either activate or repress transcription by binding to specific
sequences in the DNA. DNA-binding proteins are modular: they contain
DNA-binding domains as well as protein:protein interaction domains and domains
involved in activation of transcription. In bacteria, genes the products of
which have related functions are linked into groups called "operons":
this allows them to be regulated together. Most prokaryotic RNAs are
polycistronic (code for more than one protein) and have a single promoter (to
initiate transcription) and other regulatory sequences. Most operons are
subjected to negative regulation by the binding of a repressor protein to the
operator (a DNA sequence near the promoter). When the genes are required, the
repressor is bound by an inducer which causes dissociation of the repressor
from the operator, allowing transcription to proceed. Eukaryotes have
monocistronic RNAs (generally one protein product) and control of transcription
is primarily by positive regulatory mechanisms. In contrast to bacterial RNA
polymerase, eukaryotic polymerases have little intrinsic affinity for their
promoters, and require additional factors in order to bind to DNA. The extent
of expression of a eukaryotic gene is the result of the complex interaction of
a number of transcription factors binding to elements upstream of the
transcription initiation site.
Key words:
Positive regulation, Negative regulation, Transcription repressor proteins,
Transcription activator proteins, Transcription factors, Operator, Operon, lac
operon.
Textbook reference:
Lehninger et al. Chapter 27.
Title: Genetic Engineering.
No. of lectures: 3.
Lecturer: MJT.
Content:
This part of the course deals with the procedures (recombinant DNA
technology or "genetic engineering") that have been developed to
exploit our knowledge of the structure of nucleic acids and the replication and
expression of genetic information. They allow us to identify, isolate, multiply
up, analyse and express virtually any genetic material. These procedures are
usually performed in cells other than those from which the nucleic acids have
been obtained, and have enabled us to sequence genes, locate mutations within a
gene, determine where a given sequence is located on a chromosome, determine
whether or when a gene is expressed in a cell, identify individual organisms by
exploiting chromosomal polymorphism, and introduce genes into organisms in
which they do not occur naturally. Finally, they allow us to express foreign
genes in order to change the properties of a host or to obtain large quantities
of gene product. Cloning, the making of multiple identical copies (of a nucleic
acid, cell or even organism), involves several steps; these include the cutting
of DNA at precise sites by means of restriction nucleases, the covalent joining
of the DNA fragment under study to the vector (a DNA molecule capable of
replication and selection) using a DNA ligase, selection of the hybrid
molecular construct, introduction of the hybrid molecule into a host cell, and
then identification/selection of host cells receiving and maintaining the
construct. Recombinant DNA technology has profoundly transformed many of our
basic concepts of biology and has major applications in the detection and
treatment of disease, in forensic science and in biotechnology. It also raises
important ethical concerns (not dealt with in these lectures) which impinge on
our everyday life.
Key words:
Cloning, Restriction endonucleases, Cloning vectors, Cloning site,
Recombinant DNA, DNA ligase, kinase, phosphatase, terminal transferase,
Exonuclease, Reverse transcriptase, Cohesive (sticky) ends, Blunt ends,
Linkers, Polylinkers, In vitro packaging, Cosmids, Selection, DNA
(gene/genomic) library, cDNA library, Molecular probes, Southern blotting,
Expression vectors, Site-directed mutagenesis, Fusion protein, Shuttle vectors,
Electroporation, Viral vectors, Transgenic plants, Transgenic animals, Vector,
Plasmid, Restriction enzymes, Sticky ends, Blunt ends, Ligase, Phosphatase,
Recombinant DNA, Clone, Positive selection strategies, Antibiotic selection,
Complementation, Indirect detection strategies, Transformation, Host cells, DNA
fingerprinting, DNA sequencing, Southern blotting, Probes, Gene libraries.
Textbook reference:
Lehninger et al. Chapter 28.
Title: Protein Structure.
No. of lectures: 5
Lecturer: NAB.
Content:
Proteins fulfil a diversity of functions, for example as enzymes, as
structural elements of cells and tissues, as carriers of gases and nutrients,
as contractile elements in muscle, as antibodies, and as hormones. All this
diversity comes from relatively simple building-blocks, L-amino-acid residues.
Amino-acids act as zwitterions, and may therefore be used as buffers for
biological studies. Buffering ability is an important property of proteins, the
charge of which may alter as the pH changes.The pH at which a particular
protein has no net charge is called its isoelectric point. Four levels of
structure in a protein molecule may be distinguished. The primary structure is
the sequence of individual amino-acid residues, which is always written with
the N-terminus on the left and the C-terminus on the right. The terms secondary
and tertiary structure describe features of the three-dimensional folding of
the polypeptide chain; they determine the final shape of the molecule and the
juxtaposition of individual amino-acids within the folded structure. Secondary
structural features such as the a-helix and the b-sheet occur in varying
proportions in different proteins. Tertiary structure relies on a number of
different types of force, including hydrogen bonds, ionic bonds, hydrophobic
interactions and disulphide bonds. Quaternary structure describes the
aggregation of several polypeptide chains, with specific interactions between
the polypeptide sub-units; the sub-units are held together mainly by
hydrophobic interactions. Different types of protein structure are required for
different functions. All proteins fall into two broad classes: globular and
fibrous proteins. Globular proteins include the hormone insulin, the precursor
of which is polymerised as a single polypeptide chain, which is then processed
to a form containing two chains, held together by disulphide bonds. Myoglobin,
another globular protein, contains a haem prosthetic group. Haemoglobin is a
member of the same family, but is more complex in its structure. It contains
four sub-units, held together by hydrophobic forces. It shows co-operative
binding of oxygen and allosteric regulation by several ions. Most enzymes and
most circulating proteins are globular. Fibrous proteins have structural roles.
An example is keratin, which is made up of a-helices. Collagens contain an
unusual triple helix that is quite distinct from the a-helix. These helices
form only when there are repeat structures in the polymer, in which glycine
occurs at every third monomer position. Collagen is also rich in proline and
lysine residues, both of which may be hydroxylated; this is an example of a
post-translational modification.
Keywords:
Amino acid, Buffering, Henderson-Hasselbalch equation, Three-dimensional
structure, a-helix, b-sheet, Globular protein, Insulin, Myoglobin, Haemoglobin,
Fibrous protein, a-keratin, Collagen, Elastin.
Textbook Reference:
Lehninger et al. Chapters 5, 6, 7.
Title: Enzymes.
No. of lectures: 4.
Content:
Nearly all biochemical reactions are catalysed by globular proteins called
enzymes. Enzymes may enhance reaction rates by factors of 107-1014. They show
great specificity for particular substrates. Some require a non-protein
component, or cofactor, for activity. There are six classes of enzymes:
oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. An
enzyme increases the rate of a spontaneous reaction by lowering the activation
energy of the reaction. Spontaneous reactions do not occur instantaneously
because of an energy barrier, represented by the requirement for the reaction
to pass through an unstable transition state. Enzymes lower the activation
energy required to attain the transition state. The major source of free energy
used to lower the activation energy comes from the decrease in free energy
occurring when multiple, non-covalent interactions are made between the
substrate(s) and the enzyme: this is sometimes called "binding
energy." Such interactions occur maximally between enzyme and the
transition state. Enzyme kinetics (that is, experimentation on rates of reactions
catalysed by purified enzymes under a variety of controlled conditions,)
provides insight into enzyme mechanism. Many enzyme-catalysed reactions exhibit
a hyperbolic curve when a plot of initial reaction rate (Vo; that is, the rate
measured before the rate of the back-reaction becomes significant) against
substrate concentration is made. Michaelis and Menten proposed a simple model,
which involves the production of an enzyme-substrate complex, to account for
this behavior. Km, the Michaelis constant, is defined in terms of rate
constants for the reaction catalysed, and is numerically equal to the
concentration of the substrate when Vo is equal to one-half the maximal rate of
the reaction (Vmax) seen at high substrate concentrations. A transposition of the
Michaelis-Menten equation, called the Lineweaver-Burk equation, predicts that a
1/Vo against 1/[Substrate] plot should give a straight line and enable an
accurate graphical measurement of Km and Vmax. Under particular conditions, Km
provides information about the affinity of an enzyme for a substrate: under
such conditions, affinity is inversely proportional to Km. A knowledge of Vmax
provides information about the catalytic efficiency of the enzyme. Temperature,
pH and exogenous inhibitors (which may be reversible or irreversible, and
competitive or non-competitive) may all affect the rate of a catalysed
reaction. Transition state analogues are often particularly potent inhibitors.
Some antibodies directed against such analogues are catalytic. Competitive and
non-competitive inhibition may be distinguished graphically by means of
Lineweaver-Burk plots. Flow rates through metabolic pathways may be altered by
the activation or inhibition of one or more of the pathway enzymes. Feed-back
inhibition of an early enzyme by a pathway product is an example of this. The
two main ways in which the activity of an enzyme may be altered in a cell are
by allosteric effects and through covalent modification of R groups of
particular amino-acid monomers of the enzyme protein. Allosteric effectors are
cell metabolites that bind to some site on the enzyme other than the active
site. Allosteric enzymes often show sigmoid rather than hyperbolic kinetics;
many have quaternary structure. The concerted (symmetry) and sequential models
of allosteric behaviour seek to account for the co-operativity between enzyme
sub-units implied by the sigmoid kinetics . Allosteric effectors are used to
"fine-tune" flow rates in response to altering intracellular
conditions. Covalent modification of enzymes is often used to alter flow rates
in response to changing extracellular conditions. Some enzymes are synthesised
as inactive precursors called "zymogens".
Key words:
Enzyme, Catalyst, Cofactor, Coenzyme, Prosthetic group, Apoenzyme, Holoenzyme,
Substrate, Active site, Spontaneous reaction, Activation energy, Transition
state, Bonding energy, Enzyme kinetics, Initial reaction rate (Vo), ES complex,
Hyperbolic curve, Michaelis-Menten kinetics, Michaelis constant (Km), Vmax,
Double-reciprocal (Lineweaver-Burk) plot, Competitive and non-competitive
inhibition, Transition state analogue, Catalytic antibody, Feed-back
inhibition, Allosteric effector, Sigmoid kinetics, Co-operativity, Concerted
(symmetry) and sequential models, Zymogen.
Text-book reference:
Lehninger et al. Chapter 8.
Title: Experimental Studies on Proteins.
No. of Lectures: 3.
Lecturer: NAB.
Content:
Proteins must be isolated in order to study their structure and function.
Separation methods depend on the properties that distinguish the particular
protein required from all others present. These include charge and size, which
are used to isolate proteins by ion-exchange and gel permeation chromatography
respectively. The particular protein required may have a specific binding
interaction; this allows affinity chromatography to be exploited. Crude and
purified preparations of protein are analysed by techniques like
electrophoresis (including SDS-PAGE) and isoelectric focusing. Proteins may be
identified by determining their amino-acid composition and, especially, their
N-terminal sequence. The specificity of antibodies is used in many laboratory
procedures to provide fast and sensitive assays in many different applications.
Among these are the enzyme-linked immunosorbent assay (ELISA), immunoblotting
(or western blotting) and immunohistochemistry.
Key-words:
Electrophoresis, SDS-PAGE, Assay of protein concentration, Protein
purification, Specific activity, Ion-exchange chromatography, Gel filtration
chromatography, Affinity chromatography, Amino-acid composition and sequence.
Use of labelled antibodies, Antibody detection, Enzyme-linked immunosorbent
assay (ELISA), Polyclonals and monoclonals.
Text-book reference:
Lehninger et al. Chapter 6.