Mandatory Course key areas and exemplification of key areas
1 Differentiation in human cells
 Cellular differentiation
- All cells contain the same genes. However, once the cell becomes differentiated, the only
genes expressed are the ones needed to code for proteins specific for that type of cell. This
includes essential genes (e.g. for respiration) as well as genes characteristic for that type of
cell. This is also known as selective gene expression.
- Cellular differentiation is the process by which a cell develops more specialised
functions by expressing the genes characteristic for that type of cell.
 Somatic and germline cells
Somatic cells
- All differentiated cells with the exception of reproductive cells are known as somatic cells.
- Somatic cells form the main body tissue types:
- Epithelial: epithelial cells cover the body surface and line body cavities
- Connective: includes blood, bone and cartilage cells
- Muscle: muscle cells form muscle tissue
- Nerve tissue: nerve cells form nervous tissue
- The body organs are formed from a variety of these tissues.
- Somatic cells divide by mitosis to form more somatic cells.
- During cell division the nucleus of a somatic cell divides by mitosis to maintain the diploid
chromosome number. Diploid cells have 23 pairs of homologous chromosomes.
- In somatic cells, any errors made during mitosis are not passed to offspring.
Germline cells
- Germline cells divide by mitosis to produce more germline cells or by meiosis to produce
haploid gametes. (i.e. the only differentiation taking place in germline cells is towards the
production of gametes)
- Mutations in germline cells are passed to offspring.
 Stem cells
- Stem cells are unspecialised cells that can divide by mitosis to make copies of themselves
(self-renew) and/or differentiate into specialised cells.
Types of stem cells
Embryonic stem cells.
-The cells of the early embryo can make all of the differentiated cell types of the body.
The inner cell mass cells of an early embryo (blastocyst stage) are pluripotent (they have
the most potential) as they can make nearly all of the cell types in the body.
- These cells can self-renew, under the right conditions, in the lab. When grown in the lab
scientists call these embryonic stem cells.
Tissue (adult) stem cells
- Tissue (adult) stem cells are involved in the growth, repair and renewal of the cells found
in that tissue.
-They are multipotent. as they can make all of the cell types found in a particular tissue
type, e.g. red bone marrow and skin.
- For example, development of tissue (adult) stem cells in bone marrow (haematopoietic
stem cells) into red blood cells, platelets and the various forms of phagocytes and
lymphocytes.
- They have a much more limited differentiated potential as many of their genes are
already switched off thus adult stem cells can give rise to a limited range of cell types.
Therapeutic uses of stem cells
- Examples: the repair of diseased or damaged organs, eg corneal transplants, bone marrow
transplants and skin grafts for burns.
Corneal repair: Stem cells found at the edge of the cornea are used to repair a
damaged cornea from the same patient, there is no risk of rejection
Bone marrow transplant: To treat leukemia, stem cells are used to replace cancerous
bone marrow stem cells after they have been destroyed to stop the cance
Skin graft for burns: skin stem cells from the patient are isolated, grown and sprayed
on the wound. The burn heals faster, it reduces risks of infection and pain. There is no risk
of rejection.
Research uses of stem cells
- Stem cells can also be used as model cells to study how diseases develop or for drug
testing.
- Stem cell research provides information on molecular changes to explain how cell processes
such as cell growth, differentiation and gene regulation work, thus providing a much deeper
understanding on how gene regulation works and how drugs can interfere with these
processes.
Sources of stem cells
- Sources of stem cells include embryonic stem cells (from the inner cell mass of the
blastocyst (5 days old)), tissue stem cells and attempts to reprogram specialised cells to an
embryonic state (induced pluripotent stem cells [iPS]).
Ethical issues with stem cells and the need for regulations of their use
- Embryos used for research must not be allowed to develop beyond 14 days, around the
time a blastocyst would be implanted in a uterus. People are against it as they see it as
destroying a life.
- Regulation for the use of iPS cells. The techniques used to produce iPS cells use virus
vector which have been associated with cancer.
 Cancer
- Cancer cells divide excessively to produce a mass of abnormal cells (called a tumour).
- These cells do not respond to regulatory signals and may fail to attach to each other.
- If the cancer cells fail to attach to each other they can spread through the body to form
secondary tumours.
2 Structure and replication of DNA
- All cells store their genetic information in the base sequence of DNA. The genotype is
determined by the sequence of DNA bases.
- The genotype is determined by the sequence of DNA bases.
Structure of DNA
- nucleotides contain deoxyribose sugar, phosphate and base.
- DNA has a sugar–phosphate backbone, complementary base pairing — adenine with thymine
and guanine with cytosine.
-The two DNA strands are held together by hydrogen bonds and have an antiparallel
structure, with deoxyribose and phosphate at 3' and 5' ends of each strand.
- The arrangement of the two strands with their sugar-phosphate backbones running in
opposite directions is known as antiparallel.
- Chromosomes consist of tightly coiled DNA and are packaged with associated proteins.
Replication of DNA
- DNA is the molecule of inheritance and can direct its own replication
- Prior to cell division, DNA is replicated by DNA polymerase
- DNA replication occurs at several locations on a DNA molecule.
- The requirements for DNA replication need to be explored.
- Replication of DNA by DNA polymerase and primer.
- DNA is unwound and unzipped to form two template strands.
- DNA polymerase can only add nucleotides to a pre-existing chain, so to begin to function a
primer must be present.
- A primer is a short sequence of nucleotides formed at the 3’ end of the DNA strand about
to replicate.
- The DNA chain is only able to grow by adding nucleotides to its 3’ end and that the reverse
is true of its complementary strand on the right.
- DNA polymerase can only add complementary DNA nucleotides to the deoxyribose (3') end
of a DNA strand. This results in one strand being replicated continuously (the leading
strand) and the other strand replicated in fragments (the lagging strand) which are joined
together by ligase.
- Requirements for DNA replication include:
- DNA template
- Primers
- DNA polymerase
- Ligase
- Free nucleotides
- Source of energy: ATP
3 Gene expression
- Only a fraction of the genes in a cell are expressed.
- Phenotype is determined by the proteins produced as the result of gene expression, i.e. it
is determined by the proteins made by the genes which are “switched on” (expressed).
- Gene expression is influenced by intra- and extra-cellular environmental factors.
- Gene expression is controlled by the regulation of both transcription and translation.
Structure and functions of RNA.
- RNA is also a type of nucleic acid but differs from DNA in a variety of ways.
- RNA is single stranded, contains uracil instead of thymine and ribose instead of
deoxyribose sugar.
- There are three different types of RNA molecules.
- mRNA involved in transcription carries a copy of the DNA code from the nucleus to
the ribosome.
- tRNA involved in translation: Each transfer RNA (tRNA) carries a specific amino acid.
tRNA folds due to base pairing to form a triplet anticodon site and an attachment site for a
specific amino acid.
- rRNA associates with certain proteins to form ribosomes.
Transcription
- Transcription of DNA into primary and mature mRNA transcripts in the nucleus.
- RNA polymerase moves along DNA unwinding and unzipping the double helix and
synthesising a primary transcript of RNA by complementary base pairing. Genes have introns
(non-coding regions of genes) and exons (coding regions of genes).
- The introns of the primary transcript of mRNA are removed in RNA splicing. The exons are
joined together to form mature transcript.
- Splicing refers to the process of removing any introns before the transcribed molecule is
translated.
Translation
- Translation is the synthesis of a protein under the direction of mRNA.
- mRNA is translated into proteins by tRNA at the ribosomes in the cytoplasm.
- mRNA consists of a series of base triplets called codons.
- Each anticodon carried by tRNA corresponds to a specific amino acid
- Each codon must be matched with its own anti-codon in the ribosomes.
- Triplet codons on mRNA and anticodons translate the genetic code into a sequence of amino
acids. - Start and stop codons exist.
- Before translation occurs, the ribosome must bind to the 5’ end of the mRNA template so
that the start codon can initiate the translation.
- The sequence of event after the binding of the ribosome is:
- Codon recognition of incoming tRNA
- Peptide bond formation
- exit of tRNA from the ribosome as the polypeptide is formed.
- Stop codons trigger the release of the polypeptide chain from the ribosomes.
Different proteins can be expressed from one gene
- This happens as a result of alternative RNA splicing and post-translational modification.
- Different mRNA molecules are produced from the same primary transcript depending on
which RNA segments are treated as exons and introns.
- Post-translation protein structure modification: once translation is completed further
modifications to the protein may be required. These are cleavage and molecular addition, e.g.
cutting and combining polypeptide chains or by adding phosphate or carbohydrate groups to
the protein.
4 Genes and proteins in health and disease
Structures and shapes of proteins
- Proteins have a large variety of structures and shapes resulting in a wide range of
functions.
- Amino acids are linked by peptide bonds to form polypeptides.
- Hydrogen bonds are formed during the folding process and cause the chain to become
coiled or folded to form the three dimensional shape of the protein.
- Proteins are held in a three dimensional shape by peptide bonds, hydrogen bonds,
interactions between individual amino acids.
- Separation and identification of proteins can be done by agarose gel electrophoresis.
Mutations and genetic disorders
- Mutations result in no protein or a faulty protein being expressed.
- Genetic disorders are caused by changes to genes or chromosomes that result in the
proteins not being expressed or the proteins expressed not functioning correctly.
Single gene mutations
- Single gene mutations involve the alteration of a DNA nucleotide sequence as a result of
the substitution, insertion or deletion of nucleotides.
- Nucleotide substitution, can result in missense, nonsense and splice-site mutations.
- missense (replacing one amino acid codon with another)
- nonsense (replacing an amino acid codon with a premature stop codon — no
amino acid is made and the process stops)
- splice-site mutations (creating or destroying the codons for exon-intron
splicing).
- Nucleotide insertions or deletions result in frame-shift mutations or an expansion of
a nucleotide sequence repeat.
- These mutations have an effect on the structure and function of the protein synthesised
and the resulting effects on health.
Chromosome structure mutations
- The structure of a chromosome can be altered by deletion (loss of a segment of a
chromosome); duplication (repeat of a segment of a chromosome) and translocation (the
rearrangement of chromosomal material involving two or more chromosomes).
- The substantial changes in chromosome mutations often make them lethal.
5 Human Genomics
Bioinformatics
- Bioinformatics is the use of computer technology to identify DNA sequences.
- The enormous amount of data produced by DNA and protein sequencing can be managed and
analysed using computer technology and shared over the internet.
- Computer programs can be used to identify gene sequences by looking for coding sequences
similar to known genes, start sequences or sequences lacking stop codons.
- Computer programs can be used to identify base sequences that correspond to the amino acid
sequence of a protein.
(a)Sequencing DNA and its use
- The sequence of bases can be determined for individual genes and entire genomes. (A genome is
the complete DNA sequence of an organism).
(i) Comparative genomics
- Genome determination case studies including the human genome project and the comparison of
individual genomes using single nucleotide polymorphisms (SNPs) or comparing region with
repetitive sequences.
- Systematics compares human genome sequence data and genomes of other species to provide
information on evolutionary relationships and origins.
(iii) Genomics and medicine
- The information gained from DNA studies can provide information on the structure of the genes
and proteins involved in disease.
- It is important of distinguishing between neutral and harmful mutations as only harmful mutations
will cause disease.
This information can be used for:
- Rational drug design, i.e. the synthesis of specific drugs that will bind to proteins involved
in disease or prevent their synthesis by binding to a specific region of DNA preventing
transcription or by binding to mRNA preventing translation.
- Personalized medicine. The aim of personalized medicine is to improve the understanding
of the genetic component of risk of disease and the likelihood of success of a particular
treatment. Personal medicine can be extremely complex as many disease are caused by changes in
more than one gene. The study of inherited differences in responding to drugs is called
pharmacogenetics. It will improve the choice of effective drugs.
(b)Amplification and detection of DNA sequences.
(1) The polymerase chain reaction (PCR) is a technique for the amplification of DNA in vitro
(i.e. obtaining many copies of a small section of DNA (200-1500bp))
- PCR is done by amplification of DNA using complementary primers for specific target sequences
at the two ends of the region to be amplified.
- PCR depends on a process of thermal cycling. A cycle consists of three steps each carried
out at a different temperature.
(i)DNA heated at 95°C to separate strands (Denaturation)
(ii) the reaction is then cooled to 60°C for primer binding to target sequences.
(iii) The reaction is heated to 72°C so that the heat-tolerant DNA polymerase replicates
the region of DNA. (72°C is an optimum temperature for heat-tolerant DNA polymerase).
- Repeated cycles of heating and cooling amplify this region of DNA.
(2) Detections of DNA sequences
(1) DNA sequences can be detected by DNA Probes and arrays
- The probes are short single stranded fragments of DNA that are complementary to a specific
sequence. Fluorescent labeling of probes allows detection.
- The probes are arranged in arrays (i.e. many probes orderly arranged on a small slide). Arrays of
DNA probes are used to detect the presence of specific sequences in samples of DNA (The sample
DNA contains nucleotides with fluorescent labels.)
DNA probes can be used for detecting single gene mutations and comparing gene expression in a
diseased and healthy cells to understand the genetic basis of the disease.
(2) PCR can be used to detect DNA sequences:
- Applications for DNA profiling: it allows the identification of individuals
through comparison of regions of the genome with highly variable numbers of
repetitive sequences.
- By screening a cell sample from a patient for the presence or absence of a
particular sequence, a diagnosis of disease status or risk of disease onset can be
made.
6- Metabolic pathways
- Metabolism encompasses the integrated and controlled pathways of enzyme catalysed
reactions within a cell.
-These pathways can have reversible and irreversible steps
- Metabolic pathways may exist that can bypass steps in a pathway thereby providing
alternative routes.
- Enzymes often act in groups or as multi-enzyme complexes. DNA and RNA polymerases are
part of multi-enzyme complexes.
(i)Types of metabolic pathways:
- Anabolic pathways require energy and involve biosynthetic processes (e.g. protein synthesis)
- Catabolic pathways release energy and involve the breakdown of molecules (e.g. respiration)
- ATP plays a key role in the transfer of energy between catabolic and anabolic pathways.
(ii) Activation energy, active sites and induced fit
- Activation energy is the energy needed to contort the molecule into a highly unstable state
(Transition state) so that chemical bonds can be broken and a chemical reaction can take place.
Out with cells, this energy is provided in the form of heat energy. In cells, enzymes lower the
activation energy needed for a chemical reaction so that they can take place at lower
temperatures.
- The active site of an enzyme is a groove or a hollow created by the chemical structure of and
the bonding between the enzyme’s amino-acids. The substrate shows affinity (chemical
attraction) to the active site. The specificity of an enzyme is attributed to a compatible fit
between the shape of its active site and that of its substrate.
- When the substrate enters the active site, the shape of the active site and that of the
enzyme changes due to chemical interactions between the substrate and the active site. This
change in shape is called the induced fit and it makes the active site fit even more snuggly
around the substrate so that the conditions for the chemical reaction to take place are
optimized.
- When the reaction involves 2 or more reactants, the active site has a role in orientating
reactants.
- Finally, after the reaction has taken place, the products have low affinity for the active
site and they are released from the enzyme-substrate complex.
iii)Control of metabolic pathways
- Regulation can be controlled by intracellular and extracellular signal molecules.
- 1-Control by selective gene expression
Genes coding for specific enzymes can be switched on/off. This results in the control of metabolic
pathways by the presence or absence of particular enzymes.
- 2- Control through the regulation of the rate of reaction:
Genes for some enzymes are continuously expressed. These enzymes are always present in the
cell and their control involves regulation of their rate of reaction.
-a- Control by concentration of substrate/product
Most metabolic reactions are reversible. The presence of a substrate or the removal of a
product will have an effect on both the rate of reaction and drive a sequence of reactions in a
particular direction, i.e. towards equilibrium.
- b- Control through competitive and non-competitive inhibition
Competitive inhibition: inhibitor molecule binds to active site. Competitive inhibition can be
reversed by increasing substrate concentration.
Non- competitive inhibition: Binding by the non-competitive inhibitor changes shape of active site
so that it is no longer affinity to the substrate
- c- Control through feedback inhibition: the end product binds to an enzyme that catalyses
a reaction early in the pathway and inhibits its function.