Cell and Molecular Biology

STEM MODULE: CELLULAR & MOLECULAR BIOLOGY
TABLE OF CONTENTS
INTRODUCTION ........................................................................................................................ 2
GOALS AND OBJECTIVES......................................................................................................... 5
TOPIC 1: DNA AND RNA ........................................................................................................ 7

TOPIC 2: FROM DNA INTO PROTEIN ...................................................................................10
TOPIC 3: CELL DIVISION......................................................................................................14
TOPIC 4: MOLECULAR EVENTS IN CELL GROWTH .............................................................16
TOPIC 5: CELL CYCLE AND THE REGULATION OF CELL DIVISION.....................................19
TOPIC 6: APOPTOSIS ...........................................................................................................20
TOPIC 7: MOLECULAR BASIS OF CANCER..........................................................................25
TOPIC 8: GENETIC DISORDERS ..........................................................................................31
TOPIC 9: LABORATORY TESTS ...........................................................................................37
TOPIC SUB-HEADINGS INCLUDE:
KEY ISSUES
SELF-TEST TASKS
CASE SCENARIOS
MULTIPLE CHOICE QUESTIONS
REFERENCES
 Royal Australasian College of Surgeons – April 2000 Page 1 of 45

INTRODUCTION
This module introduces the key concepts which underpin our current knowledge of mammalian biology.
Therefore, as well as providing an overview of our current knowledge in this area, it also will help prepare
you for all of the other modules in this first section, with the probable exception of regional and systematic
anatomy!
The key concepts and detailed knowledge for this module will be largely covered in the
following specific references (note they are currently the most recent editions):
th
Cotran, Kumar and Robbins, Pathologic Basis of Disease, 6 Edition, WB Saunders
International, 1999; Chapters 1-8.
th
Ganong WF, Review of Medical Physiology, 19 Edition, Appleton & Lange, 1999; Chapter 1.
th
Roitt, Essential Immunology, 9 Edition, Blackwell Scientific Publications, 1997; Chapters 1, 2,
4, 7, 8, 9, 17,18.
In particular, you will find that this module will prepare you for and overlaps with those that address
neoplasia, wound healing, immune response to infection, trauma, cancer, transplantation and the blood
coagulation system and its diseases.
Although not in your recommended reading list, access to a current pharmacology

textbook is highly advisable. Two appropriate textbooks are:
th
Katzung, Basic and Clinical Pharmacology, 7 Edition, Appleton and Lange, 1998; Chapters 2,
5, 43 – 56.
th
Rang, Dale and Ritter, Pharmacology, 4 Edition, Churchill Livingston, 1999; Sections 1, 2 & 3.
The key concepts in this module relate to the structure and function of genes and the structure and function
of the cell. The following are intended as a guide to your study in this area, with the proviso that unifying
concepts and key molecular and cellular interactions are far more important at this stage than
memorising detailed biochemical pathways.

The Structure and Function of Genes
• The structure of DNA, mitosis and meiosis.

• The control of cell cycling, cyclin-dependent kinases, the P53 and Rb tumour-suppressor genes, cell
cycle checkpoints and apoptosis.
• Translation of DNA into RNA, exons and introns, RNA splicing.
• Control of DNA transcription, regulatory genes and promoter elements, DNA repair genes.
• Ribosomes, RNA and protein synthesis.
• Chromosomal translocations and genetic mutations in the causation of disease (in particular cancer),
base deletions, point mutations, frameshift mutations, replication error repair (RER) disorders.
• Mendelian dominant and recessive disorders.
• Polygenic diseases.
The Structure and Function of the Cell
• Major ultrastructural features of mammalian cells and their functions, with special reference to the
actions of antibiotics, hormones which bind to both membrane and intracellular receptors, growth factors
and cytotoxic chemotherapy agents.
• The anatomy of the immune response (Roitt Chapter 8) with particular reference to lymphocyte
trafficking, vascular addressins, selectins, integrins and other adhesion molecules, cell-cell and cell-
matrix interactions.
• Antigen processing and presentation, dendritic cells, T-lymphocytes, B-lymphocytes, the T-cell receptor,
the B-cell receptor, the major histocompatibility complex (MHC), lymphokines, cytokines, accessory cell-
cell interactions.
• The molecular basis of cellular motility and cell-matrix interactions, with particular reference to the
synthesis, structure and degradation of the intercellular matrix, collagens, fibronectin, collagenases and
metalloproteinases.
• The cellular biology and molecular biology of wound healing with special reference to angiogenesis and
the molecular events in cell growth.
• Receptor-ligand interactions at the cell membrane and their consequent intracellular signalling pathways,
with special reference to the role of the ras proto-oncogenes in oncogenesis.

The Human Genome Project
The Human Genome Project began with a project to sequence all transcribed genes (the EST project), and
partial sequences of at least 15,000 transcribed genes are currently in databases worldwide. This project
would have only sequenced the 5-10% of the Human Genome (all human DNA) which contains the exons
(transcribed portions) of the 70,000-110,000 genes which are the instruction set for a human being.
The latter half of the 1990s saw the progressive rapid automation of DNA sequencing, which has changed
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the focus to the sequencing of all human DNA (3 x 10 base pairs), the Human Genome Project. The Human
Genome is expected to be entirely sequenced by December 2000! The change in focus from RNA to DNA
means that introns in transcribed genes will be sequenced as will all other DNA outside of the human genes.
There may be sequences which are important in the control of those genes and/or there may be viral inserts
into the human genome. These could be inserts of DNA viruses or DNA copies of RNA viruses. In this
context it is important to note that virtually all lymphohaematopoietic tumours in many animals, which include
cats and rodents, are due to the activation of vertically transmitted DNA copies of RNA viruses (retroviruses),
within the lifetime of the animal. Ionising radiation and chemicals are known activators of these latent viruses
in animals.

GOALS AND OBJECTIVES
The remainder of the module will concentrate predominantly on the structure and function of genes, rather
than on the structure and function of the cell.
Suggested references include:
th
Cotran RS, Kumar V and Collins T, Robbins Pathologic Basis of Disease, 6 edition, WB
Saunders International, 1999. Specifically Chapter 6, part of Chapter 1 – covering apoptosis,
and parts of chapter 8, concerning the molecular basis of cancer.
th
Ganong WF, Review of Medical Physiology, 19 edition, Appleton & Lange, 1999; Parts of
Chapter 1 are suggested.
th
Guyton and Hall, Human Physiology and Mechanisms of Disease, 6 edition, 1997.
Particularly Part I, Chapter 3, “The Genetic Control of Protein Synthesis, Cell Function and
Cell Reproduction”.
For an overview of how some molecular genetic tests are performed, the following
references are suggested:
Ferrari M et al: Molecular diagnosis of genetic diseases. Clinical Biochem 29: 201-208, 1996.
Koreth J et al: Microsatellites and PCR genomic analysis. J Pathol 178: 239-248, 1996.
Shibata H et al: DNA-based diagnostics in the study of heritable and acquired disorders. J Lab
Clin Med 125: 421-432, 1995.
Werner M et al: Interphase cytogenetics in pathology: principles, methods, and applications of

fluorescence in situ hybridisation (FISH). Histochem Cell Biol 108: 381-390, 1997.
For an overview of apoptosis:

Cummings, MC et al: Apoptosis. Am J Surg Path 21: 88 – 101, 1997.
For a list of genetic disorders that can be diagnosed in Australia, using DNA
technology, the following web site is suggested: http://murdoch.rch.unimelb.edu.au,
through the VCGS link.

In the outline that follows, key points from Robbins have been emphasised, and a structure provided with
which to study this area. The principles in each topic section are all important; the detail can be complex and
somewhat daunting. The module is linked with curriculum objectives Pathology II. 1. Genetics and
Molecular Biology.

TOPIC 1: DNA AND RNA
1.1. Structure of DNA
DNA comprises two polynucleotide strands, wound around each other forming a double helix. Each
single strand is made up of individual nucleotides. Each nucleotide comprises a phosphate, a sugar
and a base.
Base
Ph.
Sugar
The sugar in DNA is pentose, a 5- carbon sugar arranged in a ring shape and in DNA is ‘deoxyribose’
while that in RNA is ‘ribose’. (Deoxyribose means that the 2´ carbon has no attached ‘OH’ group,
while it does in RNA).
The nucleotides are linked together forming individual chains and 2 complementary chains are also
linked, forming the double helix, arranged like a ladder, with the sugar of one nucleotide linked to the
phosphate of the next nucleotide. The sugar-phosphate linked groups form the sides of the ladder,
and the attached bases point inwards, forming the rungs. Each chromosome (each cell having 23
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chromosome pairs) is composed of approximately 3 x 10 nucleotide pairs. A nucleoside is the
combination of a sugar unit and a base. There are 4 types of bases, each containing nitrogen. They
are ‘pyrimidines’ – thymine and cytosine - and these have a single ring structure, and purines –
adenine and guanine - which have 2 rings. To each nucleoside, 3 phosphate groups are attached
(alpha, beta and gamma*), this forming a nucleotide. Each nucleotide is called by its base, thus dTTP,
dGTP, dATP and dCTP (or abbreviated to T, G, A and C). As mentioned, each individual nucleotide is
joined to the next one with the phosphate group connecting the 5ćarbon of one deoxyribose sugar to
the 3´ carbon group of the deoxyribose of the next nucleotide. This link is referred to as a 5´-3´
phosphodiester bond. (The other 2 phosphates are lost when the bond occurs). The rungs of the
ladder (the bases) of opposite or complementary strands are linked to each other via hydrogen bonds.
Thymine binds to adenine and cytosine binds to guanine. Three hydrogen bonds link guanine and
cytosine and 2 hydrogen bonds link adenine and thymine.

DNA has direction and is described according to the 5énd or the 3énd. The 5´ end is where there
are still 3 phosphate groups attached to the 5ćarbon of the sugar. The other end, the 3’ end, is where
there is a carbon attached to an ‘OH’ group. New DNA is synthesised in the 5´→ 3´ direction. By
convention, the 5´ end of the sequence is written on the left, and this represents DNA sequence close
to the start of the gene, while the 3énd is written on the right, and this is the end of the sequence
closest to the end of the gene.
The 2 individual DNA strands run in opposite directions to each other and with the consistent A → T
link and C → G link, the strands are complementary. Knowing the sequence of one strand thus gives
you the sequence of the other. Each paired strand of DNA is wound up to give a ‘supercoil’ and
topoisomerases are enzymes that can add or remove turns in the DNA helix.
1.2. Types of DNA
The great majority of DNA does not code for genes. DNA that codes for messenger RNA (mRNA) is
called ‘unique sequence’ or ‘non-repetitive DNA’, and each diploid cell has two copies of the sequence
for each gene, one on each of a chromosome pair. The non-coding DNA is present as so-called short
or long repeated DNA sequences (‘short interspersed repeated sequences’ and ‘long interspersed
repeated sequences’).
Satellite DNA (about 10% of human DNA) is in the form of short, tandemly repeated DNA sequences.
These code for ribosomal and transfer RNA. Some of the sequences are grouped around
centromeres. This is called satellite DNA because it separates out on a density gradient as a ‘satellite’
to the main peak of DNA.
Pseudogenes are similar to known structural genes but have minor changes in them – either in their
coding or their regulatory sequences, that prevent their transcription or translation.
1.3. Structure of RNA
RNA is essentially similar to a single strand of DNA, with two main differences. First, the sugar
backbone is ribose rather than deoxyribose, and second, uracil replaces thymine as one of the bases.
The other three bases are the same. As RNA forms from a DNA template, uracil binds to adenine.

1.4. Types of RNA
There are three main types of RNA: messenger or mRNA, accounting for about 5% of the RNA in the
cell, transfer or tRNA, accounting for 15%, and ribosomal or rRNA, which accounts for 80%.
A. mRNA
This is produced in the nucleus and is then transported into the cytoplasm. It decodes the genetic
information of the DNA and directs protein synthesis. Most mRNA is unstable in the cell and
short-lived and this instability is one way that the amount and duration of gene expression can be
controlled. mRNA undergoes various modifications after it is produced. At the 5´-end (upstream)
a cap structure is added and towards the 3´-end (downstream) a poly (A) tail is added. This is an
area rich in adenine bases and this sequence acts as a stop signal to terminate transcription. The
addition of the cap structure and the poly (A) tail help to stabilise the mRNA. Once in the
cytoplasm, mRNA becomes associated with ribosomes and there forms the template for producing
a specific sequence of amino acids. The ribosome moves along the mRNA, rather like a zipper,
as the amino acids are linked up to form a chain.
B. tRNA
Transfer RNAs capture the correct amino acid, bring it to the ribosome and insert it into the
growing amino acid chain. Each amino acid is first activated by ATP and it is then attached to one
end of the tRNA. The other end of the tRNA comprises three bases which are complementary to
the bases on the mRNA. The tRNA thus forms the link between the code on the mRNA and the
particular amino acid. The end of the tRNA containing the three nucleotides which decode the
genetic information is called the ‘anticodon’.
C. rRNA
Ribosomal RNA is one of the components of ribosomes, the other main component being protein.

TOPIC 2: FROM DNA INTO PROTEIN
2.1. DNA Replication
DNA replication during cell division is called ‘semiconservative replication’. The 2 strands of the
double helix of the DNA separate and 2 new complementary strands are synthesised, following the
A:T, C:G rules of base pairing, one new DNA strand being formed on the template of one of the
original separated DNA strands.
To begin with, the original DNA helix is unwound and that is through topoisomerase enzymes. Next
the 2 DNA strands are separated, and this begins at the replication origin. Here the hydrogen bonds
between the paired bases are broken, using a helicase enzyme. Single-strand binding proteins then
bind to the single-stranded DNA at these replication forks and that stops reannealing between the
bases. DNA synthesis then occurs using a DNA polymerase enzyme. The process is started with the
use of a primer and this is a short length of RNA.
DNA synthesis along the ‘leading’ strand occurs in the 5´ to 3´ direction. The polymerases catalyse
the formation of new phosphodiester bonds, as the α (alpha*) phosphate group of the new nucleotide
attaches to the free 3´ OH group on the sugar of the nucleotide already in place. Synthesis is thus
continuous in the 5´-3´ direction on the leading strand. 5´-3´ synthesis on the lagging strand is
discontinuous, as this lagging strand is being unzipped backwards. Small lengths of DNA are
synthesised and these are called Okazaki fragments. Each of these fragments is started by its own
RNA primer. DNA polymerase I then eventually removes these RNA primers and allows DNA to
continue to fill in the gaps. All the small newly synthesised DNA fragments are then joined together
with a DNA ligase enzyme.
2.2. Genes
A gene is a length of DNA that contains the information required to produce one polypeptide
sequence. Most (over 70%) of DNA is non-coding or intergenic. A gene can be present on one or
other of the paired DNA strands, though only one strand is used for any one gene. An individual gene
sequence is always read from the 3’ end to the 5’ end, though usually it is the complementary strand
(in the 5’ to 3’ direction) which is shown. This complementary strand is comparable to the
corresponding RNA sequence for that gene.

Individual genes, though continuous stretches of DNA, are divided into exons and introns. Exons are
sequences of DNA that code for proteins, while the intervening introns are DNA sequences that are
non-coding. There may be no introns in a gene, but often there are several, eg 10 or 20. Like the
exons, the introns are transcribed into RNA but the introns are later removed. GT is present at the
start of an intron and AG at the end.
An operon is a cluster of related genes and this is generally genes where the protein products are all
required at about the same time in the cell. Operons are found in bacteria. In humans and other
higher species, groups of similar genes are ‘multigene families’. Some produce very similar proteins,
such as ones that are quickly required in bulk. Other multigene families include related but distinct
genes, for example, for the globin genes or for the HLA major histocompatibility genes.
2.3. Transcription
DNA acts as a template for mRNA (messenger) RNA production and transcription is the
production of mRNA complementary to the DNA in the gene. Transmitting genetic information
from mRNA into protein is translation. Transcription is divided into initiation, elongation and
termination steps. Initiation of transcription occurs just upstream of the required gene and there are
short DNA sequences called promoters or start sites, where polymerases bind. This alpha2
betabeta´sigma 5 polymerase DNA complex is called a closed promoter complex. The DNA strands
are then separated from each other at this point by the polymerase and the region is then called an
open promoter complex. Once the DNA strands are opened, the sigma* subunit leaves the
polymerase complex and the residual polymerase enzyme begins to synthesise the new RNA
molecule in the elongation step. After synthesis has begun, the polymerase moves along the DNA
strand. Ribonucleotides are added to the 3´ end of the new RNA. The DNA is progressively unzipped
from its complementary DNA strand, and after transcription has occurred, it is progressively rejoined or
zipped up again. Termination of RNA synthesis occurs at specific sites and these are called
complementary palindromes or invariable inverted repeats (the sequence has symmetry around a
certain point). After these is a run of adenosine bases and these bind relatively weakly to uracil. After
that complementary palindromic region, the weakly bound polymerase comes off the DNA strand.
A large RNA is produced initially and this is unstable. It is modified at its 5´ end (with a cap structure
added) and at its 3´ end (with a poly (A) tail added), producing the more stable mRNA. The introns
(non-coding sequence) are then removed and the exons spliced together forming the mature mRNA.
This is then exported from the nucleus to the cytoplasm where translation occurs.

2.4. The Genetic Code
The genetic code is composed of three bases, known as a codon, and this codon determines
which amino acid is incorporated into the growing protein. As there are 64 possible codons and
only 20 amino acids, several codons may code for a single amino acid. The first and second bases
within an mRNA triplet are critical for the specific amino acid, while the base in the third position is
often less important. For example, GUU, GUC, GUA and GUG each specify valine. The order of
amino acids in a protein corresponds directly to the order of the nucleotides in the DNA. All proteins
begin with the amino acid methionine, coded for by AUG, and this start region is preceded by a purine-
rich region (as methionine is also found elsewhere in proteins). As well as a start codon, there are
also three stop codons: UAA, UAG and UGA; and the open reading frame is the region between the
starting methionine and first stop codon. TATA boxes are A-T regions about 20-30 bases long,
occurring in most genes and present at the 5´ end (upstream) of the start of the open reading frame
(ATG). TATA boxes help localise enzymes to the correct initiation site for the start of transcription.
CCAAT boxes also help to regulate transcription and they are present at about 80 bases proximal to
the ATG starting point.
2.5. Translation
Translation occurs on ribosomes in the cytoplasm. Information encoded by mRNA is translated in

the 5´ to 3´ direction into a chain of specific amino acids, that chain constituting a polypeptide. The
mRNA moves over the surface of the ribosome, bringing the successive codons into position.
tRNA transfers the correct amino acid to the site of protein synthesis and makes sure that the right
amino acid is incorporated at the right place. The amino acid required is activated with ATP, forming
the intermediary aminoacyl-AMP. This then reacts with its particular tRNA, forming so-called charged
tRNA and this is catalysed by aminoacyl-tRNA synthetases. Having the correct amino acid is
obviously critically important. The anticodon part of tRNA contains three bases and these are
complementary to those on the mRNA. Ribosomes normally exist in the cytoplasm as discrete units
and before translation occurs these are assembled into a catalytic unit.
Translation then begins at the correct point on the mRNA, just upstream of the initiation codon (ie for
methionine). The 30 S subunit of rRNA then moves along the mRNA and finds the initiation codon.
Here the correct charged tRNA joins the complex. Its anticodon forms base pairs with the codon of
the mRNA. Protein synthesis then continues progressing from the amino to the carboxyl end of the
protein. A number of initiation factors are involved in this process. The 50 S rRNA subunit joins the
initiation complex and then various elongation factors contribute to elongation of the protein. The

amino acid-tRNA complex lines up next to the codons of mRNA and is joined to the previously
synthesised polypeptide chain, by a peptide bond. The tRNA is released at this point and the mRNA
moves further over the ribosome, bringing another codon into position. This is repeated in the 5´ to 3´
direction until one of the termination codons is reached and the peptide is then released.

TOPIC 3: CELL DIVISION
3.1. Mitosis
This is the type of cell division in which a somatic cell or a germ cell, each with 46
chromosomes, produces two identical daughter cells. Each of the daughter cells also has 46
chromosomes.
The cell cycle has four stages and mitosis is the shortest of the phases. The phases are G1 (G = Gap)
and this phase can vary greatly in length. Next S, for synthesis, when DNA synthesis begins. At this
time, the chromatin of each chromosome has replicated and this results in the formation of two
chromatids. In G2, DNA synthesis is complete and this phase lasts until the beginning of mitosis.
Interphase is the part of the cell cycle between mitosis. Terminally differentiated cells such as
neurons and cardiac muscle fibres do not divide or replicate and are said to be in Go, which is
essentially a very prolonged G1 phase.
Mitosis has been divided into four stages: prophase , metaphase, anaphase and telophase . During
prophase the chromosomes at this stage have been replicated during the S phase and consist of two
parallel strands, called sister chromatids; and these are held together at the centromere region. The
centromere of each chromosome becomes attached to a microtubule of the mitotic spindle. The
nuclear membrane disappears during late prophase, and this allows the chromosomes to spread
around in the rest of the cell. During the next phase, metaphase, the chromosomes are completely
contracted and thus are more readily seen, for example, in histological sections. During anaphase,
the centromere of each chromosome divides and the sister chromatids separate. The sister
chromatids have thus become two daughter chromosomes and they move to opposite poles of the
cell.
The new independent chromosomes have separated completely, forming two complete groups of
daughter chromosomes and each group becomes enclosed in a new nuclear membrane. The
cytoplasm of the original cell also separates at this stage, thus forming two new daughter cells.

3.2. Meiosis
Meiosis is the process of cell division that occurs during the final stages of producing
gametes. Diploid cells have two pairs of each chromosome, thus 46 chromosomes in total,
while haploid cells (gametes) have a single set and thus 23.
There is an initial round of DNA synthesis in meiosis and then there are two phases of cell division.
These are called meiosis I and meiosis II, each of which essentially has its own prophase, metaphase,
anaphase and telophase stages. Meiosis I is called the reduction division and it is this stage which
reduces the number of chromosomes from 46 to 23. At the start of prophase I the chromosomes have
already been split longitudinally into two chromatids and these are joined at the centromere. The
homologous chromosomes form pairs (except for the X and Y chromosomes in male meiosis) and
there is exchange of homologous segments between chromatids. This exchange process is called
crossing-over or recombination. From crossing-over, each chromosome in the gamete will
contain some maternally-derived and some paternally-derived genes. This is as well as the
random separation of homologous chromosomes, with the gamete containing some
maternally-derived and some paternally-derived chromosomes. In metaphase I, the nuclear
membrane disappears, the chromosomes become attached to the spindle and become aligned in the
middle of the cell. During anaphase I, the spindle contracts and the chromosomes separate and move
to opposite poles of the cell. The separation is random – the chromosomes originally derived from the
mother do not go to one pole and those derived from the father to the other. The haploid sets of
chromosomes have separated completely and the original cell divides into two new daughter gametes.
Meiosis II is essentially similar to mitosis, except that in the original cell, there are only 23
chromosomes in the original cell, rather than 46. The two new daughter gametes produced by meiosis
II are either spermatids or ova.

TOPIC 4: MOLECULAR EVENTS IN CELL GROWTH

(Robbins page 92-)
These events involve intercellular signalling pathways, including autocrine, paracrine and endocrine types.
4.1. Cell Surface Receptors
Cell growth occurs with binding of signals to specific receptors and these can be on the cell surface, in
the cytoplasm or in the nucleus. There are 3 main groups of cell surface receptors: receptors with
intrinsic kinase activity, receptors without intrinsic catalytic activity and G protein-linked receptors.
There are also several pathways by which information from activated cell surface receptors can be
transmitted to the nucleus, and these pathways are called signal transduction pathways.
A. Receptors with intrinsic kinase activity
These include receptors for epidermal growth factor, fibroblast growth factor and platelet-derived
growth factor and these particular receptors also have intrinsic tyrosine kinase activity. Others in
this group of these receptors have serine/threonine kinase activity. Each of these receptors has
an extracellular domain for binding to their respective ligands or growth factors, a single
transmembrane region and a cytoplasmic domain. The cytoplasmic domain has the kinase
activity.
B. Receptors without intrinsic catalytic activity
These include receptors for many cytokines and so this group is also called the cytokine receptor
superfamily. These receptors have an extracellular domain for binding the ligand, a single
transmembrane region and a cytoplasmic domain. Rather than the cytoplasmic domain having its
own enzyme activity, it activates protein tyrosine kinases in the cytoplasm and these in turn,
phosphorylate the receptor.
C. G protein-linked receptors
These include receptors for inflammatory chemokines and for adrenalin and glucagon. These
receptors contain seven transmembrane loops and so are also called seven-spanning receptors.
Ligands to these receptors activate the whole complex which then activates an effector system
and that system activates further messengers within the cell.

4.2. Signal Transduction Systems
These convert the information in the extracellular signals to intracellular signals. The signal
transduction systems characteristically comprise networks of sequential protein kinases. The main
signal transduction systems for cell growth are: Mitogen-activated protein kinase, also called MAP
kinase, phosphoinositide-3-kinase, inositol-lipid pathway, cyclic adenosine monophosphate and
JAK/STAT systems.
A. MAP Kinase System
This system is especially important in growth factor signalling and when active it can stimulate
cells to enter the growth cycle. Binding to a receptor with tyrosine kinase activity can activate the
MAP-kinase pathway which can lead to activation of the Ras protein. (Ras is one of the GTPase
(guanosine triphosphatase) proteins.) These proteins switch between being in active and inactive
forms. Inactive Ras can bind GDP (guanosine diphosphate). This GDP is then converted to the
active GTP form. This active GTP form in turn activates a series of kinases. GAP proteins
(GTPase activating protein) opposes Ras activation by converting Ras to its inactive GDP-binding
form. Activated Ras also binds to Raf which in turn phosphorylates MEK, one of the MAP kinases.
One of the last MAP kinases in that pathway is ERK and it is translocated into the nucleus. In the
nucleus it phosphorylates transcription factors such as c-jun and c-fos.
B. Phosphoinositide-3-kinase (PI-3 kinase) pathway
This pathway may also be activated by growth factors binding to receptor tyrosine kinases. This
PI-3 kinase pathway can activate membrane-associated lipid mediators, which in turn act as
second messengers in the cell. Activation of this pathway can ultimately lead to increased
glycogen synthesis through phosphorylation of glycogen synthase kinase 3.
C. Inositol-lipid pathway
This pathway acts downstream from either tyrosine kinase or seven-spanning G protein-linked
receptors. This ultimately produces IP3 (inositol 1, 4, 5-triphosphate) and DAG (1, 3-diacyl
glycerol). IP3 leads to release of calcium from the endoplasmic reticulum. DAG and calcium
activate protein kinase C which phosphorylates various cell components.

D. Cyclin AMP pathway
Activation of seven-spanning receptors leads to activation of adenylate cyclase and production of

C-AMP, which in turn acts as a messenger, activating protein kinase A.
E. JAK/STAT pathway
Cytokines bind one the cytokinase receptor superfamily. These lack intrinsic kinase activity. After
the receptor is activated by its ligand, it activates Janus protein kinases (JAKs) that are present in
the cytoplasm. These phosphorylate other proteins – signal transducers and activators of
transcription – STATS. This system mediates functional responses rather than cell proliferative
responses.
4.3. Transcription Factors
These include c-myc, p53 and Rb. The cell surface receptors allow the cell to receive information from
the environment and to transmit it into the cell. The signal transduction systems in turn carry this
information to the nucleus. This in turn can lead to transcription of genes. Such gene transcription is
controlled by transcription factors and transcription factors are also important in cell cycle regulation.
Transcription factors are composed of domains for DNA binding and there are also regulatory domains
that regulate transcription, either increasing it or decreasing it. Transcription factors are
phosphorylated by kinases and once phosphorylated this may alter their location in the cell or their
affinity for DNA.

TOPIC 5: CELL CYCLE AND THE

REGULATION OF CELL DIVISION
There are two important mechanisms that regulate cell cycling:
• Cyclin proteins
• Cell cycle checkpoints
5.1. Cyclins and Cyclin-dependent Kinases
Cyclins control entry into and progression through the cell cycle. They form complexes with cyclin-
dependent kinases (CDKs) and the active CDK complexes are in turn regulated by the binding of CDK
inhibitors and other kinases and phosphatases. Different specific combinations of cyclins and CDKs
are important in controlling progression through the cell cycle. For example, cyclin B binds to CDK1
and this controls the transition from G2 to M. This is shown in Figure 4-7 on page 96 of Robbins. After
mitosis, this cyclin B is degraded through the ubiquitin-proteasome pathway. The interface between
the G1 (Gap 1) phase and the S (synthesis) phase is important as here the cells decide either to
replicate or to become quiescent. The state of phosphorylation of Rb, the retinoblastoma protein, is
important at this point. During G0 and G1, Rb is bound to E2F transcription factors and it is inactive.
With progression through G1, cyclin Ds accumulate and activate CDKs. These hyperphosphorylate Rb
and that disrupts the binding to E2F. E2F becomes activated and activates transcription of genes
required for the cell to enter into S phase.
5.2. Checkpoints
Cell cycle checkpoints are points in the cell cycle when the processes of DNA replication and repair
and chromosome segregation are checked for accuracy. With DNA damage, for example due to
radiation, cell cycle arrest is initiated. If the DNA is successfully repaired, the cell cycle can be
continued. Cell cycle arrest can occur through either promoting inhibitory pathways or blocking
activating ones. For example, after sublethal DNA damage, p53 is quickly activated and that
increases the expression of p21 which is a CDK inhibitor.

TOPIC 6: APOPTOSIS
(Robbins page 18-)
Apoptosis is a controlled, active form of cell death affecting specific single cells, quite unlike
necrosis which is a passive degenerative phenomenon generally affecting groups of cells. Apoptosis
has a characteristic morphological appearance, both with light and electron microscopy. It occurs under
wide ranging circumstances including: during embryogenesis, during hormone-regulated involution of
tissues, in mild tissue injury and in continuously proliferating cell populations, such as intestinal epithelium to
control cell numbers. It also occurs in the immune system, with the death of unwanted B and T cells and
with cell death induced by cytotoxic T cells, such as in viral hepatitis or in immune rejection. Apoptosis is
also critically important in removing cells that have sustained DNA damage and also in regulating normal cell
numbers. Suppression of apoptosis is significant in the growth of many cancers. Apoptosis is thus a
physiological response mechanism and too little or too much apoptosis underlies a variety of disease states.
With too little apoptosis, abnormal cells can persist, for example in carcinomas, particularly those with p53
mutations, or hormone-dependent tumours. Some autoimmune disorders are thought to arise when
autoreactive lymphocytes are not deleted by apoptosis and this has been demonstrated in a number of
animal models. Excessive apoptosis is thought to underlie some neurodegenerative disorders characterised
by neuronal loss, and also the lymphocyte depletion of AIDS. Ischaemia produces infarction and death by
necrosis, but mild ischaemia, such as at the periphery of an infarct, can cause cell death by apoptosis.
There has been considerable interest in controlling such mild ischaemia-induced apoptosis to reduce the
extent of damage that occurs with a myocardial infarct or a stroke. As cells undergo apoptosis they shrink
and the nuclear chromatin condenses forming compact masses, often round or crescent-shaped. An
apoptotic cell fragments into smaller apoptotic bodies and these are quickly ingested by surrounding cells,
both by usual phagocytic cells, such as macrophages and by surrounding parenchymal cells. No
inflammation occurs.

6.1. Biochemical Features
Various biochemical features characterise apoptotic cells and these correlate with the morphological
changes that occur. In apoptosis, the DNA undergoes a characteristic double stranded cleavage and
this occurs at internucleosomal sites, producing fragments of DNA 180-200 base pairs long. This is
due to the action of an endonuclease and results in a ladder pattern of DNA fragments demonstrated
with agarose gel electrophoresis.
Caspases, a family of cysteine proteases, are activated and cleave nuclear scaffold and cytoskeletal
proteins allowing many of the morphological changes of apoptosis to occur. Also, protein cross-linking
due to the action of tissue transglutaminase helps produce the shrunken, spherical apoptotic bodies.
The increased expression of phosphatidyl serine and thrombospondin on the surface of apoptotic cells
allows the recognition (and subsequent ingestion) of apoptotic bodies by neighbouring cells. No
inflammation occurs.
6.2. Molecular Regulation
The molecular regulation of apoptosis can be divided into 4 headings:

• Signalling pathways that initiate apoptosis
• Regulation, including its control and integration
• Execution or effector mechanisms
• Removal of the apoptotic bodies by phagocytosis
A. Initiation of apoptosis
Initiating stimuli can be divided into cases where a defined receptor is known, and cases where
the initiation pathway is poorly understood, such as mild ischaemia and mild hyperthermia and
non-genotoxic toxins. Cases with a known receptor include addition or withdrawal of trophic
hormones acting via receptors, activation of tumour necrosis (TNF)-alpha receptor and
activation of the Fas receptor. Activation of the TNF receptor on the plasma membrane is
known to lead to activation of specific death programmes.
B. Regulation of apoptosis
This includes the signalling pathways initiated in target cell killing by cytotoxic T lymphocytes
and also by Fas-Fas ligand interaction, for example. The Bcl-2 family of proteins play a major

role in the regulation of apoptosis via regulating mitochondrial function. With apoptotic signals,
pores form in the inner mitochondrial membrane: this reduces membrane potential and
produces mitochondrial swelling. Also, the outer mitochondrial membrane swells and
cytochrome c is released from the mitochondria into the cytoplasm. Cytochrome c release from
mitochondria is an early and important part of the apoptotic process. Bcl-2 family members play
a significant role in regulating mitochondrial permeability. Bcl-2 acts on mitochondria to block
the increase in permeability that occurs and it also acts by regulating other Bcl-2 family
members. Bcl-2 may also act as a docking protein, binding and sequestering other proteins
(such as Apaf-1) from the cytoplasm. After cytochrome c is released from the mitochondria, it
binds and activates Apaf-1. This in turn triggers an initiator caspase, the first step in the
proteolytic pathway that brings about cell death. Bcl-2 may also act to inhibit apoptosis by
binding Apa7-1. Activation of p53 and also Granzyme B also activate the pathway of execution
caspases. The role of p53 in apoptosis will be described later.
C. The execution or effector phase
These features are all essentially stereotyped occurring irrespective of the cell type or the cause
of apoptosis. The caspase family of protease enzymes are critical in this execution phase. The
‘c’ means they have cysteine protease activity and ‘aspase’ means they cleave after aspartic
acid residues. Caspases include initiator caspases, such as caspase 9 which binds to Apa7-1
and caspase 8 which is triggered by Fas-Fas ligand interactions. Execution caspases act on
the cytoskeleton, breaking it down in a controlled fashion and also act on endonucleases,
leading to the characteristic DNA fragmentation.
D. Recognition and removal of apoptotic bodies
Apoptotic bodies acquire molecular markers on their surfaces and that facilitates their
recognition by neighbouring cells. Exposure of phosphatidyl serine on the external leaflet of the
plasma membrane lipid bilayer is one important mechanism of recognition. The apoptotic
bodies disappear very quickly, generally without leaving a trace, though remnants of apoptotic
bodies can be seen within macrophages in the germinal centres of lymph nodes, a site where
there is obviously high cell turnover.

6.3. Specific Examples of the Occurrence of Apoptosis
This includes: signalling by Tumour Necrosis Factor (TNF) family of receptors, cell death induced by
cytotoxic T-lymphocytes, apoptosis after growth factor deprivation, and apoptosis induced by DNA
damage.
A. Signalling by TNF family of receptors
This includes binding of the Fas ligand (Fas L or CD95L) to the cell surface receptor Fas (also
called CD95). Fas ligand is produced by cells of the immune system and when it binds to Fas
on T cells, it activates apoptosis of these cells. This is important in limiting the duration of a T
cell response. Activation of one of the TNF receptors by its TNF cytokine also leads to
apoptosis. Activation of either of these receptors leads to their binding to adapter proteins in the
cytoplasm. These adapter proteins also contain so-called death domains which in turn bind to
initiate caspases, thus activating the execution phase of apoptosis.
B. Cytotoxic T-lymphocyte (CTL)-mediated apoptosis
On recognising foreign cells, CTLs express Fas ligand on their surface and so kill the target
cells by activating the Fas receptors on them. CTLs may also kill target cells by secreting
perforin, together with granzyme B. Perforin produces holes or pores in the cell membrane of
target cells allowing granzyme B into the cytoplasm. Granzyme B in turn activates many
caspases and thus the execution pathway. This rapid way of inducing apoptosis bypasses
many of the upstream signalling pathways.
C. Apoptosis after growth factor deprivation
Upon reduction in growth factors or cytokines, pro-apoptotic members of the Bcl-2 family move
from the cytoplasm to the outer mitochondrial membrane, altering the ratio of the various Bcl-2
proteins here. This change increases mitochondrial membrane permeability, releasing
cytochrome c, also activating the execution phase.
D. DNA damage-mediated apoptotis
Damage to the DNA of the cell that cannot be repaired induces apoptosis and this process is
critically mediated by p53. It is a very important process that ensures that cells that have
acquired mutations do not proliferate and pass on these mutations to daughter cells. When

DNA is damaged, p53 quickly accumulates on the cell and this blocks cell cycle progression at
G1, allowing time for DNA repair. If the damage is too great and repair not possible, p53
induces apoptosis of that cell. If p53 is absent or mutated, as in many cancers, this critical
check on DNA integrity is lost.
6.4. Genes that Regulate Apoptosis (Robbins p. 294-)
Many genes that limit or enhance the occurrence of apoptosis are also significant in cancer biology.
These include bcl-2, which (along with bcl-XL) inhibits apoptosis, while other members of that family
such as bax, bad and bcl-xs favour apoptosis. Overexpression of the bcl-2 protein occurs in 85% of
follicular B-cell lymphomas. These have a characteristic t(14;18) (q32;121) translocation that makes
the 14q 32 locus (the site of immunoglobulin heavy -chain genes) abut the bcl-2 gene, present on 18q
21. This produces overexpression of the bcl-2 protein, limiting apoptotic cell death of the B cells and
so allowing other mutations to accumulate.
The ratio of apoptosis-inhibiting (bcl-2, bcl-xl) to apoptosis-favouring (bax, bcl-xs, bad and bid) bcl2
family members is important in determining whether a cell will respond to an apoptotic stimulus. Bcl-2
homodimers favour cell survival; bax homodimers favour cell death. p53 activation increases bax
synthesis, favouring apoptosis and c-myc can also induce apoptosis if there is a limited supply of
growth factors in cells that are driven to proliferate by c-myc activation.

TOPIC 7: MOLECULAR BASIS OF CANCER

(Robbins page 276-295)
Damage to DNA that is insufficient to kill the cell is the basis of carcinoma. Key groups of genes
whose damage leads to cancer are: proto-oncogenes, tumour suppressor genes, apoptosis-related
genes and DNA repair genes.
7.1. Oncogenes
Proto-oncogenes are normal genes in the cell that promote normal cell growth and differentiation.
Examples of proto-oncogenes include growth factors and growth factor receptors, signalling proteins
and transcription factors. When these proto-oncogenes are abnormally activated (and this can occur
in a variety of ways), they are transformed into oncogenes. The structure of these genes may be
altered, they may be inappropriately expressed or they may be overexpressed.
Protein products of oncogenes:
A. Growth factors
Many growth factors are involved in normal cell proliferation and mutations of their coding regions
can make them oncogenic. Examples include: c-sis. This codes for the beta chain of platelet-
derived growth factor. In tumours, rather than this gene being mutated, it is overexpressed. That
also occurs with the genes hst-1 and int-2, which code for fibroblast growth factors.
B. Growth factor receptors
Many of the (normal) growth factor receptors are transmembrane proteins with an external binding
site and a tyrosine kinase domain in the cytoplasm. When these are turned into oncogenes the
receptors are permanently switched on without any growth factor binding to them. Growth factor
receptors may be activated by mutations, gene rearrangements or overexpression. For example,
point mutations on the ret proto-oncogene are associated with the MEN2A and 2B syndromes,
while rearrangements of the ret gene occurs in sporadic papillary cancer of the thyroid. More
commonly, overexpression of proto-oncogenes causes their abnormal activation. For example,
the EGF receptor gene (the normal form of c-erbB 1) is overexpressed in the majority of squamous

cell carcinomas, while the c-e r b B2 gene is overexpressed in many adenocarcinomas, for
example, of the breast.
C. Signal-transducing proteins.
The main example is mutation of the ras gene. Normally, in the inactive state, ras binds GDP
(guanosine diphosphate), and when stimulated by growth factors, ras is activated by exchanging
GDP for GTP. Activated ras in turn activates the MAP kinase pathway, thus eventually promoting
cell proliferation. Normally, the intrinsic GTPase activity of ras hydrolyses GTP to GDP, returning
ras to its inactive state. This GTPase activity is augmented by GAPs, GTPase-activating proteins.
With mutant and thus oncogenic ras, they bind GAP, but their GTPase activity is not augmented.
So, mutant ras persists in its active GTP-bound form. Ras is mutated in many cancers, including
those of the lung, colon and pancreas.
D. Nuclear transcription proteins
These proteins affect the cell’s progression through the cell cycle and mutations of these (or their
conversion to oncogenes) is often associated with malignant transformation. C-myc is amplified in
many carcinomas, including breast, colon and lung and also its control is abnormally regulated in
Burkitt lymphoma. Here, the c-myc proto-oncogene on chromosome 8 is translocated to
chromosome 14 next to the immunoglobulin locus. Thus c-myc is adjacent to a different enhancer
sequence and is overexpressed.
E. Cell cycle regulators
Mutations of cyclins, cyclin-dependent kinases and their inhibitors are very important for cell-cycle
control and mutations of these genes occur in a number of cancers. For example, cyclin D is
amplified in carcinomas of the breast and oesophagus, while it is translocated in mantle cell
lymphoma. CDK4 is amplified in melanoma.
Activation of Oncogenes
As indicated in the previous section, there are various mechanisms by which proto-oncogenes can be
converted into oncogenes. This can be by point mutations, such as occurs with ras. Many ras
mutations occur and they all reduce its GTPase activity. Chromosomal rearrangements can also
convert proto-oncogenes into oncogenes. Proto-oncogenes can be translocated to a new site so that
they come under the regulatory elements of immunoglobulin or T-cell receptor loci. In other words,

they are inappropriately switched on, while their coding sequence remains intact. This occurs with c-
myc translocation in Burkitt lymphoma.
Another way chromosomal rearrangements may produce oncogenes is through fusion of unrelated
sequences that combine and form hybrid genes. In CML (chronic myeloid leukaemia), there is a
translocation between chromosomes 9 and 22. Part of the c-abl proto-oncogene from chromosome 9
is moved to the bcr (breakpoint cluster region) on chromosome 22. The new hybrid c-abl-bcr gene
codes for a new protein that has tyrosine kinase activity. Also, in Ewing’s sarcoma, the EWS gene
fuses with the Fl-1 gene. The new EWS-FL-1 protein transactivates the c-myc promoter and thus c-
myc is overexpressed.
Finally, proto-oncogenes may be activated by overexpression of their DNA sequences, sometimes

producing several hundred copies of their DNA. N-myc is amplified in neuroblastoma and c -erb B2 is
amplified in breast cancer.
7.2. Tumour-Suppressor Genes
The normal physiological role of these genes is to regulate cell growth. Loss of their function is
responsible for their role in neoplasia and tumours tend to develop from cells in which both normal
alleles have been lost. For example, in familial retinoblastoma, children are born with one defective
copy of the Rb gene. When there is a mutation of the second normal allele, through a loss of the
chromosome or a point mutation, then the cancer, or retinoblastoma may develop. In other
circumstances, both copies of the normal allele are lost somatically.
Protein products of tumour suppressor genes
The signals for growth inhibition are less well known than those for growth promotion.
A. Molecules that regulate nuclear transcription and the cell cycle
These include Rb, WT-1 and p53. The Rb gene was the first tumour suppressor gene identified.
The Rb protein is a nuclear phosphoprotein. It is underphosphorylated in its active state and
hyperphosphorylated in its inactive state. When active, Rb blocks progression from G1 to the S
phase of the cell cycle. Active Rb binds the E2F family of transcription factors. With loss of
functional Rb, this brake on cell cycle progression is lost.

Similarly, mutations in other genes that control phosphorylation of the Rb proteins may have a
similar effect to that of loss of the protein. These genes include p16, cyclin D and CDK4 and at
least one of these four genes (including Rb) is mutated in the majority of cancers.
In normal cells, p53 functions as a tumour suppressor gene but there are mutations in this gene
in more than 50% of tumours. Generally, loss of both alleles is acquired in somatic cells, though
in the Li-Fraumani syndrome, one mutant allele is inherited and these people have a greatly (25-
fold) increased risk of developing cancer.
When a cell is subject to DNA damage, p53 levels increase rapidly. This causes cell cycle arrest
in late G1 due to the p53-dependent transcription of p21 which is a CDK inhibitor. By inhibiting
cyclin/CDK complexes, this prevents the phosphorylation of Rb which is necessary to allow
progression into S phase. DNA repair can then occur and if that is successful, mdm is activated,
which in turn down-regulates p53. If the DNA repair is not successful, p53 activates bax and IGF-
BP3. Bax binds to Bcl-2 allowing apoptosis to occur. If p53 is lost, DNA damage goes
unchecked and so may be passed on to daughter cells.
BRCA 1 and BRCA 2 are also tumour suppressor genes and patients with an inherited mutation of
either gene are at increased risk of developing breast cancer, and with BRCA 1, also ovarian
cancer. Mutations in these 2 genes account for about 80% of familial breast cancers (see also
Breast / Endocrine Module). The functions of the protein product of these genes is not clear; they
may be involved in transcriptional regulation or in DNA repair.
B. Molecules that regulate signal transduction
This includes the products of the NF-1 and APC genes. These normally produce down-regulation
of growth-promoting signals. Loss of this down-regulation allows tumours to form. The APC
protein is present in the cytoplasm and here it interacts with many proteins, including beta-
catenin. Beta-catenin can move to the nucleus and activate transcription of growth promoting
genes. APC produces degradation of beta-catenin, and with APC loss, beta-catenin levels
become elevated, leading to increased cell proliferation. Beta-catenin mutations that make it
unable to respond to APC have a similar effect.
The protein product of the NF-1 gene is neurofibromin and its function is to regulate signal
transduction via ras protein. Neurofibromin facilitates conversion of ras to its inactive state and
so with NF-1 loss, ras remains in its active form. People with an inherited mutation of NF-1
develop neurofibromatosis type 1.

C. Cell surface receptors
When TGF-beta binds to its receptor, there is increased transcription of growth-inhibitory genes.
This pathway is inactivated in 15% of colon cancers.
Cadherins are a family of glycoproteins that act partly as adhesion molecules between cells and
reduced cell surface expression of E-cadherin occurs in a number of cancers.
D. Other tumour suppressor genes
These include NF-2 gene, von VHL (von Hippel Lindau) gene, phosphatase and tensin
homologue (PTEN) and WT-1. Inactivation of the WT-1 gene is associated with the
development of Wilms tumour.
7.3. Genes that Regulate DNA Repair (Robbins p. 295-)
As we are all frequently exposed to DNA damaging agents, such as ultraviolet light, ionising radiation
and various other carcinogens in food and in the environment, there are DNA repair genes that protect
us from developing cancer. Errors also can occur during DNA replication which if not repaired would
be passed on to daughter cells. People with inherited defects of DNA repair mechanism are at greatly
increased risk of developing cancer.
In HNPCC (hereditary nonpolyposis colon cancer) which accounts for about 4% of colon cancers,
carcinomas occur in families, affecting the caecum and proximal colon. HNPCC arise from defects in
genes involved in DNA mismatch repair. If DNA mismatches are not repaired (eg pairing C with T
rather than G), then mutations may occur in important cell proliferation genes.
Cells with defective DNA repair genes have a replication error (RER) phenotype and this is also seen
as microsatellite instability. Microsatellites are short DNA sequences, 1-6 nucleotides long, which are
tandemly repeated several hundred times and the most common is (CA)n. These are non-coding
sequences and are present through the genome, found at tens of thousands of loci, and are the same
for that individual in all tissues. If cells have a defect in DNA mismatch repair, they are said to have an
RER (replication error) phenotype. RER cells have expansions and contractions of these
microsatellite repeats in the tumour cells, giving so-called microsatellite instability, the microsatellite
sequences thus varying in length between the tumour and the normal tissue. The mismatch repair

genes may be inactivated by somatic mutation, by hypermethylation of the promoter region or by

inheritance of a germline mutation. There are 4 main DNA mismatch repair genes that are involved in
the development of HNPCC: hMSH2, hMLH1, hPMS1 and hPMS2. With defects in one of these
genes, mutations are allowed to develop in TGF-beta and bax and mutations in these genes are
important for the development of colon cancer.
Using DNA obtained from formalin-fixed or (preferably) fresh tissue, mutations can be demonstrated in
the microsatellite loci. Compared with that individual’s normal DNA, mutations in microsatellite loci
produce band shifts in the tumour DNA. Two mononucleotide markers (BAT25, BAT26) and three
dinucleotide markers (D5S346, D2S123 and D17250) are evaluated. Microsatellite instability high
(MSI-H) tumours show instability in at least two of these five loci.
Patients with xeroderma pigmentosum also have defective DNA repair. Here the defect is in any of
the genes involved in nucleotide excision repair. This system repairs defects due to UV light induced
cross-linking of pyrimidine bases, which prevents normal DNA replication. The affected patients
therefore develop many skin cancers with exposure to UV light.
Patients with the inherited condition ataxia telangiectasia have increased sensitivity to ionising
radiation. They have progressive ataxia, with telangiectasia of the ears, conjunctiva and other areas
of the face. They are particularly susceptible to lymphoid malignancies. Patients with Bloom
syndrome are also sensitive to ionising radiation and may also have developmental defects. Patients
with Fanconi anaemia are sensitive to DNA cross-linking agents, such as nitrogen mustard. They
have various congenital abnormalities and also develop pancytopenia from bone marrow hypoplasia
and can go on to develop leukaemia.

TOPIC 8: GENETIC DISORDERS

(Robbins Chapter 6)
A brief outline and commentary of this chapter will be given.
8.1. Mutations
A mutation is a permanent change in the DNA. If present in germ cells the mutation is transmitted to
offspring and may produce an inherited disease; if it arises in a somatic cell, it will not produce an
inherited disease, but may cause congenital malformations, cancer or other diseases. Mutations have
been divided into genome mutations (ie affecting the whole chromosome), chromosome mutations (ie
producing structural changes in the chromosome), and gene mutations, which are the most common.
These includes point mutations, with one base affected, and frameshift mutations, where one or two
bases are added or deleted, as well as other types.
A point mutation within a coding sequence may alter the code of a triplet and lead to substitution with
a different amino acid; this is also called a missense mutation. For example, sickle cell anaemia,
where valine is substituted for glutamic acid.
A nonsense mutation is when a point mutation changes an amino acid codon to a stop codon and thus
only a short peptide is produced. This occurs in beta-thalassaemia, where there is premature
termination of beta-globin gene translation.
Mutations within noncoding sequences also occur. A point mutation in a promoter or enhancer
sequence of a gene may affect binding of transcription factors, possibly stopping transcription of that
gene. Also, point mutations within (non-coding) introns can produce defective splicing of intervening
sequences and so abnormal mRNA transcripts that cannot be translated. Deletions and insertions into
the coding sequence can alter the DNA reading frame and so are called frameshift mutations.
Trinucleotide repeat mutations means there is amplification of a sequence of three nucleotides, and
this often includes guanine and cytosine. In Fragile X syndrome, there are up to 4000 repeats of the
CGG trinucleotide within a gene called FMR-I, and this is compared with up to 29 repeats in the
normal population. The trinucleotide expansion disrupts FMR-1 expression, producing the phenotype
of mental retardation.

Next, we look at Mendelian disorders, diseases with multifactorial inheritance and chromosomal
disorders.
8.2. Mendelian Disorders
These all result from expressed mutations in single genes of large effect, and this probably includes
over 5,000 disorders.
Single gene disorders:
A. Autosomal dominant
Here autosomal refers to the autosomes, or non-sex chromosomes. A dominant condition is

seen in either the heterozygote or the homozygote and it implies that a single copy of the
mutant allele is sufficient for the condition to be expressed. (Homozygous means that the alleles
at a single locus are identical; heterozygous means that they are different, usually one normal
and one abnormal.)
With autosomal dominant disorders, at least one parent of an index case is affected, both males
and females can be affected and either can transmit the condition. Some autosomal dominant
disorders arise from new mutations and therefore neither parent of the person with the condition
would be affected. Reduced penetrance occurs when a person inherits a mutant gene but is
phenotypically normal. This is different from variable expressivity, which is when the condition
is present in all those with the mutant gene, but is expressed differently. These variations are
probably due to modifying effects of other genes. Also, some conditions may not become
manifest until adult life, for example Huntington disease.
Most autosomal dominant disorders are ‘loss of function mutations’. If that involves an enzyme,
the person may be phenotypically normal. Effects are generally more pronounced with defects
of non-enzyme proteins, such as those that affect regulatory proteins and some key structural
proteins, such as collagen or cytoskeletal elements of the red cell membrane.
Gain of function mutations also occur, for example, the abnormal protein in Huntington disease.
This protein is toxic to neurons, so even heterozygotes develop a neurological deficit. Some
autosomal dominant disorders include familial adenomatous polyposis, polycystic kidney
disease, Huntington disease, neurofibromatosis, Marfan syndrome and achondroplasia.

B. Autosomal recessive disorders
Recessive means that the condition is seen only in the homozygote, so the mutant allele must
be present on both chromosomes. (Dominant and recessive refer to the expression of the
clinical conditions, not to the genes themselves.) As both alleles need to be mutant, the
condition does not usually manifest in the parents, but siblings may have it and siblings have a
one in four chance of being affected. Also, autosomal recessive disorders tend to show more
uniform expression than do autosomal dominant disorders, and complete penetrance is
common. Autosomal recessive disorders include almost all of the inborn errors of metabolism.
Common autosomal recessive disorders include cystic fibrosis, A1-antitrypsin deficiency, sickle
cell anaemia and alkaptonuria.
C. X-linked disorders
All sex-linked disorders are X-linked and almost all are recessive. A male, having only one X
chromosome, would be called hemizygous for an X-linked mutant gene and these disorders are
therefore expressed in the male. With X-linked disorders, the male does not transmit the
disorder to his sons, but all his daughters would be carriers. A female heterozygous for the
condition does not express the full phenotypic change, but because of random X chromosome
inactivation, she would usually express the disorder only partially. X-linked recessive disorders
include haemophilia A and B and Duchenne muscular dystrophy.
X-linked dominant conditions are unusual. These are transmitted by an affected heterozygous
female to half of her sons and half of her daughters. One example is Vitamin D-resistant
rickets.
Specific details about a number of individual genetic disorders are given in Robbins Chapter 6.
8.3. Disorders with multifactorial inheritance (Robbins page 165-)
This refers to the combined actions of environmental influences and two or more mutant genes. The
risk of expressing a multifactorial disorder is greater in siblings showing severe expression of the
disorder, and also, the greater the number of affected relatives, the higher the risk for other relatives.
The rate of occurrence of the disorder is the same for all first-degree relatives of the affected person.
The risk of both identical twins being affected is much less than 100% (often it is around 20-40%), but
it is greater than the chance that both non-identical twins will be affected. Expression of a

multifactorial characteristic may lack a distinct phenotype (eg height), or it may have a distinct
phenotype, such as diabetes mellitus. Many of the multifactorial diseases are very common, for
example, hypertension, ischaemic heart disease, gout and diabetes mellitus.
8.4. Cytogenetic disorders (Robbins page 168-)
This refers to chromosomal mutations and this may be an abnormal number of chromosomes, or an
abnormality of the structure of one or more chromosomes.
A female has a normal chromosome count of 46,XX and a male 46,XY. An exact multiple of the
haploid number is called euploid, and if it is not an exact multiple of 23, it is called aneuploid. The
main causes of aneuploidy are nondisjunction and anaphase lag.
Nondisjunction is a failure of segregation of chromosomes or chromatids at cell division and that can
occur during meiosis or mitosis. When nondisjunction occurs during the production of gametes, the
gametes formed have either one extra chromosome (trisomy) or one less chromosome (monosomy).
In anaphase lag, as the name suggests, one homologous chromosome in meiosis or one chromatid in
mitosis lags behind and is left out of the cell nucleus. This gives one normal cell and one with a
monosomy. Monosomies or trisomies involving the sex chromosomes are usually compatible with life,
while monosomies of autosomes usually do not allow a live birth. Some autosomal trisomies are
compatible with survival.
Mosaicism is when mitotic errors occur early in development and produce two or more populations of
cells in the one person. This is more common in the sex chromosomes than in the autosomes.
Another group of chromosomal abnormalities relates to the structure of chromosomes. These occur
after chromosome breakages, which are followed by loss or rearrangement of the genetic material.
Deletion means loss of a part of a chromosome. A ring chromosome occurs when there is loss at
each end of a chromosome and fusion of the damaged ends. They usually do not divide properly in
meiosis or mitosis. Inversion is a rearrangement that involves two breaks within one chromosome and
the segment in the middle is reincorporated in an inverted manner. Depending on where the breaks
occur, this may be compatible with normal function. Isochromosomes occur when one arm of a
chromosome is lost and the remaining arm is duplicated, so the chromosome consists of two short
arms only or two long arms.

In translocation, a segment of one chromosome is transferred to another. With balanced reciprocal

translocations, there are single breaks in each of two chromosomes, with exchange of material.
These persons may be phenotypically normal, though they are at increased risk of producing
abnormal gametes. Robertsonian translocation occurs between 2 acrocentric chromosomes and the
breaks usually occur close to the centromeres. With transfer of the segments, one very large and one
very small chromosome is produced and usually the small one is lost. While that person is usually
phenotypically normal the offspring may be abnormal.
Cytogenetic abnormalities involving autosomes include trisomy 21 (Down syndrome), trisomy 18

(Edwards syndrome) and trisomy 13 (Patau syndrome). Cytogenetic disorders involving sex
chromosomes include Klinefelter syndrome, XYY syndrome and Turner syndrome.
8.5. Single Gene Disorders With Nonclassic Inheritance (Robbins page 176-)
This includes disorders characterised by triplet -repeat mutations, disorders characterised by mutations
in mitochondrial genes, disorders associated with genomic imprinting and disorders associated with
gonadal mosaicism.
A. Triplet repeat mutations
Fragile X syndrome is the best understood of this type of disorder and it is the second most
common cause of mental retardation. There are long repeating sequences of three nucleotides
and usually that includes guanine and cytosine. The affected gene is the FMR-1 gene and
within this is a region in which there are multiple tandem repeats of the nucleotide sequence
CGG in the 5´ untranslated region. In normal people, the average number of repeats is 29,
while affected males have between 230 and 4000 repeats, or full mutations. Normal males and
females transmitting the abnormality have 50-230 repeats. While Fragile X syndrome affects
males, approximately 50% of carrier females may be affected, manifest as mental retardation.
Other diseases associated with unstable repeat expansions include Friedrich ataxia, myotonic
dystrophy and spinocerebellar ataxia type 1.

B. Mutations in mitochondrial genes
The unique feature of mitochondrial DNA is maternal inheritance. Ova contain mitochondria,
while sperm contain few, if any. So, the mitochondrial DNA in the zygote derives from the
mother and so mothers pass on mitochondrial DNA to all of their offspring; and then the
daughters, but not the sons, will continue to pass it on. Mitochondrial DNA particularly codes for
enzymes involved in oxidative phosphorylation and hence the defects particularly affect organs
dependent on oxidative phosphorylation. Also, during cell division, mitochondria (and thus
mitochondrial DNA) is randomly distributed to daughter cells. So when a cell with mutant and
normal mitochondrial DNA divides, the proportion of normal and mutant DNA in that cell is very
variable, and therefore so is the expression of the disorders.
C. Genomic imprinting
This is the differential expression of a gene depending on whether it was inherited from the
mother or the father. The DNA sequence is not altered, but the expression of the gene is
modified. For most genes, the effect of imprinting does not apply. The molecular basis of
imprinting is not clear but it is thought to be probably associated with different patterns of DNA
methylation. Two syndromes associated with imprinting are Prader-Willi syndrome and
Angelman syndrome. When inherited from the father, a deletion on the proximal arm of
chromosome 15 produces Prader-Willi syndrome, and when inherited from the mother produces
Angelman syndrome. The lack of expression of the maternally imprinted genes in Prader-Willi
syndrome and the lack of the paternally imprinted genes in Angelman syndrome underlie this
abnormality. In each case, the deletion affects the chromosome that normally expresses these
genes and therefore there is no active copy in the cell.
D. Gonadal mosaicism
This occurs when a mutation occurs early in embryonic development. The mutation may only
affect cells that become gametes and not somatic cells. Thus that individual may be
phenotypically normal, but they may pass the abnormal gene to their offspring and more than
one child may be affected. Osteogenesis imperfecta may be transmitted in this manner.

TOPIC 9: LABORATORY TESTS
Basic molecular tests to be aware of are given below. The underlying principles are important, not the
specific technical details.
9.1. Gel electrophoresis and blotting including Southern blotting, Northern blotting and Western
blotting.
9.2. Polymerase chain reaction (PCR).
9.3. DNA sequencing.
9.4. Direct gene diagnosis
9.5. F.I.S.H. (fluorescent in-situ hybridisation).
9.6. Basic cytogenetics, including karyotyping.
9.1. Gel Electrophoresis and Blotting
After DNA is isolated from tissues or blood or cell cultures it is often necessary to separate the various
fractions that have been isolated and this is usually done with electrophoresis. The samples are
separated in a gel (agarose or polyacrylamide) in the presence of an electric field. The gel contains a
network of small pores and as DNA is negatively charged it moves through the gel according to its
size. The smaller fragments move faster than the larger ones and so separation of the fragments
according to their size can be achieved. An intercalating dye, which binds to the DNA between the
bases, is included, so that the DNA can be visualised on the gel. Gel electrophoresis is also used for
separation of RNA and protein according to the size of the fragments.
Further analysis of the DNA fragments can then be performed with Southern blotting. This technique
was described in 1975 by a Professor E.M. Southern. Later, this technique was adapted for RNA
(Northern blotting) and for protein (Western blotting). In Southern blotting, the DNA fragments are
separated by gel electrophoresis and then transferred or blotted to a sheet of nitrocellulose or a nylon
membrane. The arrangement of DNA on the membrane exactly matches how the DNA was arranged
on the gel. The DNA is fixed on to the membrane by UV light and the membrane can be used for later
analysis. Further analysis is based on the principle that complementary strands of DNA will hybridise
or bind together, should the conditions be right. A probe (made of DNA) will only hybridise to DNA
with the correct matching sequence. Probes can be synthesised chemically, created using PCR, or
cloned. Cloning generally is able to produce the largest probes. The probe is radioactively labelled
and is hybridised with the membrane. If the correct target sequence is present, the probe will

hybridise to it. Unbound probe is washed away using salt solutions of varying concentrations and
varying temperatures. The membrane, with the bound radioactive probe is then placed next to a
photographic film for a few hours or days and then developed, producing an autoradiograph. A black
band indicates where the probe had hybridised and the size of the target fragment can be worked out
according to how far it had migrated in the gel. Northern analysis uses similar principles, except for
RNA being used rather than DNA. Northern blotting is useful to see if a cell is actually expressing a
gene and comparing the different intensities of the bands gives an indication of the relative amounts
that are expressed. Then to know that the mRNA in a cell is actually being translated, techniques
such as Western blotting which measures protein expression, are also used. Here, antibodies are
used as probes, rather than DNA or RNA.
9.2. PCR
The polymerase chain reaction or PCR gives selective and rapid amplification of specific DNA or RNA
sequences. Enzymatic amplification of the target DNA fragment occurs and that fragment is bounded
or flanked by two stretches of nucleotide primers often about 18-20 base pairs long. These primers
hybridise to the opposite strands of the sequence that is being targeted.
Target DNA for amplification
~~~~~~~~~~~~~~~~~~~~~~~
________________________________
∪ ∪
Nucleotide primers
The pair of primers are used to start the new DNA synthesis.
A very small amount of DNA containing the target, the two primers, a solution of the four
deoxynucleotides (dNTPs) and a Taq polymerase are mixed together in a small tube. Then a
repeated sequence of heating and cooling steps occur using a thermal cycler or PCR machine. There
is initial heating to 95°C which is a denaturation step, which separates the 2 DNA strands. Then the
mixture is rapidly cooled to approximately 54°C and the primers hybridise to their complementary DNA
stretches. Then reheating to 72°C occurs and this is the best temperature for DNA synthesis. The
Taq polymerase uses the dNTPs to produce new DNA strands. This cycling reaction then continues,

often for about 25-35 cycles. This produces a great amplification of the amount of target DNA. The
end product of the PCR is then analysed using gel electrophoresis.
PCR has a great range of applications. It is used to produce short (often around 200-600 base pairs
long) fragments of DNA required for further uses in molecular biology, for example, producing a probe
for Northern blots, for identification of suspected mutations within genes or for identification of viruses
or microorganisms, such as atypical mycobacterium. Reverse transcriptase-polymerase chain
reaction (RT-PCR) looks for the presence of RNA in a sample. To do this, the RNA has to be copied
into a DNA molecule, using a reverse transcriptase enzyme.
9.3. DNA Sequencing
DNA sequencing can be performed biochemically, using radioactive labels, but more commonly now
automated sequencing equipment is employed and these use fluorescently labelled probes. These
techniques are based on gel electrophoresis procedures and use high resolution polyacrylamide gels.
Simply, using a single strand DNA template, radiolabelled oligonucleotides are generated in four
separate reactions, one for A, one for T, one for G and one for C. Each of these oligonucleotides have
one fixed end and one that ends sequentially at each A, T, G or C. Thus there are many fragments,
each of varying length, ending with A, and so on with T, with G and with C. Four lanes are run on a
sequencing gel, one for each nucleotide and they are fractionated according to size on the
polyacrylamide gel. Autoradiography is performed and the DNA sequence can be read from the film.
Automated sequencers are now more commonly used and they also have data analysis capabilities.
The sequence obtained can be analysed and compared with known sequences used web-based data
bases and a variety of computer software.
9.4. Direct Gene Diagnosis (Robbins page 183-)
These include techniques that demonstrate loss of a restriction enzyme site. Restriction
endonucleases cut DNA at very specific sequences and if there is a mutation, altering the DNA
sequence, a restriction site may be lost. This can be detected using a gel, when different sized DNA
fragments will be seen.
Allele-specific oligonucleotide probes can also be used. Radioactively labelled oligonucleotide probes
specific for the sequence being investigated are applied to target DNA that has been bound to a filter.
If there is a mutation in the sequence, the probe will bind less strongly and if the filter is washed under

conditions of high stringency (low salt concentration and high temperatures), the loosely bound probe
will be washed off and no signal detected. With a perfect match between the probe and the target
DNA, the probe should remain bound, and so a signal will be detected with autoradiography.
Another technique uses the fact that some mutations affect the size of the DNA fragment produced
and that size difference can be detected using PCR. For example, with Fragile X syndrome, variation
in the length of DNA is produc ed by variation in the number of trinucleotide repeats and this size
difference can be detected with gel electrophoresis.
9.5. Fluorescence in situ Hybridisation (F.I.S.H.)
This is also based on the principle of detecting a labelled probe hybridising with target DNA. Here the
probe is labelled with a fluorescent dye and applied to either a metaphase spread or to an interphase
nucleus and the resultant reaction can be visualised with a fluorescent microscope. F.I.S.H. probes
can be for centromeric regions of chromosomes and so can be useful for chromosome counting, for
example in the detection of trisomy 21. They can be made to detect smaller specific sequences on a
chromosome. So-called chromosome painting is also used where whole chromosomes can be
labelled with fluorescent probes, allowing multiple chromosomes to be detected simultaneously.
9.6. Karyotyping
This is the study of chromosomes. Target cells, commonly lymphocytes from peripheral blood, are
cultured and stimulated to proliferate using phytohaemagglutinin. Colchicine is added to arrest the
dividing cells in metaphase. This is when the chromosomes are maximally contracted and most easily
seen and analysed. Hypotonic saline is added, causing the cells to swell. They are then dropped
onto a slide and the cell membrane shatters and the groups of metaphase chromosomes are spread
on the slide. The metaphase chromosomes are then stained by various means to highlight the
different banding patterns. They are then photographed, or an image is captured, and the karyotype is
then analysed.

ENABLING OBJECTIVES AND SELF-TEST TASKS
Question 1-10 take you through some of the background knowledge expounded in this module. Now that
you have been through the module, can you answer these questions?
SELF-TEST TASK
1. What is DNA and how is it constructed?

2. How is the information in DNA converted into protein?
3. What are the key features that distinguish mitosis from meiosis?
4. How may a cell receive a growth signal and by what pathways does that signal ultimately
cause the cell to proliferate?
5. Why do defects in the Rb or p53 genes so commonly underlie the development of many
cancers?
6. How does overexpression of the Bcl-2 gene contribute to the development of cancer?
7. What is the difference between an oncogene and a tumour suppressor gene?
8. How does the proto-oncogene ras become oncogenic?
9. What malignancies are associated with defective DNA repair enzymes?
10. Why are mitochondrial DNA abnormalities maternally inherited?

CASE SCENARIOS
Questions 11-14 are aimed to test how you can now apply the principles of molecular biology to
some clinical situations. The following case scenarios and their linkage with your enabling
objectives are illustrative of the knowledge you should have gained from study of this module.
11. A 25-year old woman presents to the Emergency Department on your receiving night with a sudden
onset of severe constant abdominal pain in the left upper quadrant. Examination reveals that her
blood pressure is 150/90, pulse 110, respiration 25 and the abnormalities on completing the physical
examination are confined to the abdomen. She has palpable bilateral lobulated renal masses, of
which the left is very tender to deep palpation. Her family history reveals that her father died of a
stroke consequent upon malignant hypertension and was found at autopsy to have some kind of
cystic renal disease. A paternal aunt is currently on haemodialysis for end-stage renal failure. With
reference to the family history and your knowledge of Mendelian genetics propose a provisional
diagnosis of her renal disease, and the likely cause of her acute presentation.
12. You are a paediatric plastic surgeon who has a particular interest in cleft lip and palate. A young
mother has presented with her first-born child, a son, who has bilateral cleft lip. There is no family
history of this disorder, however the mother wishes to know what the risk of cleft lip or palate will be
in any subsequent children. You are able to tell her that because the cleft lip in her son is bilateral
the risk to any subsequent child is about 6%, or 1 in 16. The mother then states that this must mean
that this disorder can be inherited and asks you to explain the genetic basis.
13. Haemochromatosis and haemophilia A are both recessive genetic disorders. The gene that is
mutated in the causation of haemachromomatosis is on an autosome, and the gene that is mutated
in the causation of haemophilia A is on the X-chromosome. Using your knowledge of Mendelian
genetics calculate the risk of haemochromatosis and haemophilia A in the sons and daughters when
both parents carry the mutated gene for haemochromatosis, when both parents carry the mutated
gene for haemophilia A, and when only the mother carries either of these mutated genes.
14. Replication error repair (RER) testing is used to screen families where colorectal cancer has
clustered for HNPCC. Explain the basis for this test and the expected outcome on affected
individuals.

MULTIPLE CHOICE QUESTIONS
Now try these MCQs. The first 3 are Type A (1 from 5).
1. Which type of genetic disease occurs most frequently in the population?
A. Chromosome abnormality
B. Autosomal dominant
C. Autosomal recessive
D. Multifactorial
E. X-linked
2.1 to 2.5
What type of underlying genetic abnormality (listed A-E) is most commonly seen with each of the following
genetic diseases as listed from 2.1 to 2.5?
2.1 Osteogenesis imperfecta

2.2 Cystic fibrosis
2.3 Angelman syndrome
2.4 beta-thalassaemia
2.5 Fragile x syndrome
A. Point mutations
B. Gonadal mosaicism
C. Genomic imprinting
D. Deletions
E. Triplet repeat mutations

3. What is the most common genetic change underlying the development of tumours?
A. Somatic activation of the ras proto-oncogene

B. Activation of the c-myc gene by chromosomal rearrangement
C. Inherited inactivation of the p53 gene
D. Loss of the p53 gene by somatic mutation
E. Point mutation of the Rb gene
Type X (Multiple True-False)
4. The following characteristics suggest that the disease is inherited as a multifactorial trait:
1. The disease occurs more frequently in the children of an affected person than among the
grandchildren.
2. The incidence of the disease in the population is 3% and is 50% in the offspring of the
affected person.
3. The risk of developing the disease is greater if both parents are affected than if only one
parent is affected.
4. The disease occurs more frequently in women than in men.

Answers
1. Answer to come
2. 2.1 – B
2.2 – D
2.3 – C
2.4 – D
2.5 – E
3. D
4. 1–T
2–F
3–T
4–T

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STEM MODULE: CELLULAR & MOLECULAR BIOLOGY

GOALS AND OBJECTIVES......................................................................................................... 5

TOPIC 1: DNA AND RNA ........................................................................................................ 7

TOPIC SUB-HEADINGS INCLUDE:

MULTIPLE CHOICE QUESTIONS

 Royal Australasian College of Surgeons – April 2000 Page 1 of 45

Although not in your recommended reading list, access to a current pharmacology

 Royal Australasian College of Surgeons – April 2000 Page 2 of 45

The Structure and Function of Genes

• The structure of DNA, mitosis and meiosis.

The Structure and Function of the Cell

 Royal Australasian College of Surgeons – April 2000 Page 3 of 45

The Human Genome Project

 Royal Australasian College of Surgeons – April 2000 Page 4 of 45

GOALS AND OBJECTIVES

Suggested references include:

Werner M et al: Interphase cytogenetics in pathology: principles, methods, and applications of

For an overview of apoptosis:

 Royal Australasian College of Surgeons – April 2000 Page 5 of 45

 Royal Australasian College of Surgeons – April 2000 Page 6 of 45

TOPIC 1: DNA AND RNA

1.1. Structure of DNA

 Royal Australasian College of Surgeons – April 2000 Page 7 of 45

1.2. Types of DNA

1.3. Structure of RNA

 Royal Australasian College of Surgeons – April 2000 Page 8 of 45

1.4. Types of RNA

 Royal Australasian College of Surgeons – April 2000 Page 9 of 45

TOPIC 2: FROM DNA INTO PROTEIN

2.1. DNA Replication

 Royal Australasian College of Surgeons – April 2000 Page 10 of 45

 Royal Australasian College of Surgeons – April 2000 Page 11 of 45

2.4. The Genetic Code

Translation occurs on ribosomes in the cytoplasm. Information encoded by mRNA is translated in

 Royal Australasian College of Surgeons – April 2000 Page 12 of 45

 Royal Australasian College of Surgeons – April 2000 Page 13 of 45

TOPIC 3: CELL DIVISION

 Royal Australasian College of Surgeons – April 2000 Page 14 of 45

 Royal Australasian College of Surgeons – April 2000 Page 15 of 45

TOPIC 4: MOLECULAR EVENTS IN CELL GROWTH

4.1. Cell Surface Receptors

A. Receptors with intrinsic kinase activity

B. Receptors without intrinsic catalytic activity

 Royal Australasian College of Surgeons – April 2000 Page 16 of 45

4.2. Signal Transduction Systems

A. MAP Kinase System

B. Phosphoinositide-3-kinase (PI-3 kinase) pathway

 Royal Australasian College of Surgeons – April 2000 Page 17 of 45

D. Cyclin AMP pathway

Activation of seven-spanning receptors leads to activation of adenylate cyclase and production of

4.3. Transcription Factors

 Royal Australasian College of Surgeons – April 2000 Page 18 of 45

TOPIC 5: CELL CYCLE AND THE

There are two important mechanisms that regulate cell cycling:

5.1. Cyclins and Cyclin-dependent Kinases

 Royal Australasian College of Surgeons – April 2000 Page 19 of 45

 Royal Australasian College of Surgeons – April 2000 Page 20 of 45

6.1. Biochemical Features

6.2. Molecular Regulation

The molecular regulation of apoptosis can be divided into 4 headings:

 Royal Australasian College of Surgeons – April 2000 Page 21 of 45

C. The execution or effector phase

D. Recognition and removal of apoptotic bodies