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DNA contains the instructions that are translated from the nucleotide language of DNA into the amino acid language of proteins via an intermediary – RNA. The central dogma: DNA -> RNA -> protein. DNA vs. RNA –

  • Sugar phosphate backbones are different. Deoxyribose has an – H instead of the –OH group that ribose has. As a result, DNA is chemically far more stable (necessary because of its function)
  • DNA is double-stranded (protective)
  • In RNA, Thymine (has CH3 methyl group) is replace by Uracil (H)

DNA is transcribed into messenger RNA (mRNA) that is then translated into a protein. The code is redundant, with more coding power than is needed. AUG is the start codon; UAA, UAG and UGA are the three stop codons.

Initiation – the enzyme RNA polymerase binds to the promoter region upstream of the gene (not part of the coding sequence). DNA strands unwind and RNA synthesis is initiated by the RNA polymerase

Elongation – the RNA polymerase makes a complementary mRNA copy of the DNA sequence as it moves downstream unwinding the DNA and elongating the RNA transcript. As soon as it is transcribed, the DNA reforms a double helix

Termination – the polymerase transcribes the terminator sequence signaling the end of the gene. The RNA transcript is released and polymerase detaches from the DNA

As an additional control mechanism for gene expression, proteins called transcription factors mediate the initiation of transcription. Regions distant from the actual start of the gene called enhancer sequences. Proteins (TF's) bind to these before the gene can be transcribed. TF's can act as activators (increase) or repressors (prevent) to the presence of RNA polymerase.

In eukaryotic cells, the mRNA formed as pre-mRNA in the nucleus that is then extensively modified. It is then exported to the cytoplasm where protein synthesis occurs. Before export from the nucleus, mRNAs are protected by the addition of a 5' cap and a poly(A) 3' tail. This tags the RNA so it is recognized as self, and enhances its export out of the nucleus.

Eukaryotic genes contain intervening non-coding sequences (introns) in the middle of the coding and expressed sequences (exons). The introns are removed ('spliced out') before the mRNA leaves the nucleus. Exon splicing can occur in different ways to produce different proteins from the same gene sequence. For example, muscle α-tropomyosin has 12 exons which are spliced to produce different mRNAs for different tissues (striated muscle, smooth muscle etc)


tRNA has an amino acid attched to one end (3') and anticodon on the other. There is a separate tRNA for every amino acid. It has a folded structure, and so the two ends are quite close to eachother. An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA.

tRNAs carry their amino acids to the ribosome (protein synthesis factory) and base-pair with the mRNA. The ribosome is composed of two subunits – small and large, with two binding sites for the tRNAs:

  • A site for aminoacyl tRNAs –incoming tRNAs, carrying new amino acid
  • P site for peptidyl tRNAs – contains the growing peptide chain


The start codon (AUG) is the start of protein synthesis, not of the chain; it is not right at the 5' end of the mRNA. Methionine codes for AUG and is first amino acid of every polypeptide chain
  • The small subunit binds the mRNA and the initiator tRNA
  • The large subunit then binds with the initiator tRNA in the P site


  • A peptide bond forms between the amino acids; growing peptide chain is transferred on to the new amino acid
  • Translocation – everything moves along the mRNA; peptide chain in the A site moves into the P site, empty tRNA gets ejected


  • When a stop codon occurs in the mRNA, this is recognized by a release factor (protein), no tRNAs will recognize it
  • This causes the last tRNA to leave the ribosome and the newly synthesized protein is released. Ribosome subunits separate and mRNA is released; all are reused

Eukaryotic gene expression: Control of gene expression in prokaryotes is at the level of transcription; once the mRNA is formed, it is immediately translated. In eukaryotes, control occurs at many levels, but the most important is also at the transcription level:

Many antibiotics target bacterial transcription and translation. These processes are sufficiently distinct to prokaryotes vs. eukaryotes that it is possible to specifically inhibit them

  • RNA polymerase – rifampin
  • Protein synthesis – tetracycline, streptomycin, erythromycin, chloramphenicol

DNA damage, repair and mutation

Source Type of damage Repair mechanism
Replication Mismatched bases Proof-reading, mismatch repair
Spontaneous Base damage (makes code for wrong base) Base excision repair
Chemical Induced Base-adduct (adds extra chemicals onto bases) Base of nucleotide excision repair
Radiation Induced Double strand breaks

Base and dimers

Double strand break repair

Base of nucleotide excision repair


DNA polymerase has a proofreading capacity; it can recognise when it has incorporated the wrong base, go back and replace it. Mismatch repair comes in after DNA has been replicated – (in E.coli, but similar in humans, e.g. MLH)

  • MutS protein scans the DNA for mismatches looking for distortions in the helix (hydrogen bonding inaccurate). If encountered, it binds and recruits other proteins
  • MutH nicks the DNA near the mismatch; exonuclease enzyme removes bases, including those flanking the mismatch
  • DNA polymerase fills the correct bases

Triplet repeats – error-prone sequences in certain genes. They code for repeated amino acids in the protein; often undergoing expansion from one generation to the next and leading to earlier onset and severity. E.g. Fragile X Syndrome (CGG), Huntington's (CAG)

Spontaneous change can be due to methylation, hydrolytic attack and oxidative damage. Hydrolytic damage leading to depurination and deamination is the most common spontaneous DNA damage. The base excision repair pathway:

  • A glycosylase enzyme recognizes the damaged base and removes it by hydrolyzing the glycosidic bond between the base and the sugar phosphate backbone
  • AP nuclease gets rid of the backbone

Apurinic or apyrimidinic (have no base attached to the sugar phosphate backbone) sites can also be repaired this way.

Induced damage repaired by excision repair pathways

  • Base excision (single damaged base removed)
  • Nucleotide excision (damaged base/s and surrounding nucleotides removed). A considerable amount of DNA is removed, and filled in by DNA polymerase. The last nucleotide is joined by DNA ligase.

Double strand breaks are severe lesions that can lead to loss of genetic information, chromosomal rearrangements and translocations as there is no intact normal strand to act as a template. They are repaired by recombination (2 systems):

  • Homologous recombination – usually error-free. There is strand invasion of the homologous chromosome that will still have accurate genetic information
  • Non-homologous recombination – error prone; DNA ends are simply joined back together (last resort)


Analysis of specific DNA sequences is by PCR (Polymerase Chain Reaction). It relies on thermal cycling and the activity of DNA polymerase which requires the

  • DNA template (single-stranded)
  • Primer (short piece of DNA to add on to) and
  • Free nucleotides to add to the growing chain.

It is highly sensitive and specific, able to detect a single DNA molecule. The DNA template is exponentially multiplied.

  • Denature (92-95˚C) to separate the strands by disrupting hydrogen bonds between the complementary bases
  • Anneal (50-65˚C) – added primers bind to strands in correct position (for right gene)
  • Extend (72˚C) – DNA polymerase enzyme added and builds up. A heat-stable enzyme must be used, like the Taq polymerase (from the thermophile Thermus aquaticus)

Visualisation of PCR products is by electrophoresis The DNAs sugar phosphate backbone has a negative charge; this charge pulls it through a mesh to the positive electrode. Shorter molecules will migrate through the mesh faster than the longer ones. It is stained so it appears fluorescent under UV light when bound to DNA. It is possible to estimate the size of DNA fragments by observing the distance of migration relative to the migration of standard DNA molecules of known size

PCR can be used to amplify different variable regions in each individual's genome. Patterns are inherited from parents; only identical twins will have identical pattern. Uses include history, disaster identification, paternity testing and forensics. Multiple different loci are compared:

SSRs (Simple Sequence Repeats) vary from person to person; there are about 120,000 SSRs. The repeats are dispersed throughout the genome, and occur at many different loci on chromosomes. Thus, if using PCR the primer must be a specific repeat adjacent to the SSR.

Growth and differentiation of cells –

MPF (mitosis promoting factor) is required for mitosis. It has 2 key subunits: mitotic cyclin and cyclin-dependant kinase (phosphorylates molecules when bound to cyclin)

After mitosis, mitotic cyclins undergo polyubiquination. Ubiquitin is a regulatory molecule that binds to and labels proteins for proteasomal degradation. Once cyclin is removed CDK becomes inactive.

E2F is a group of genes involved in DNA synthesis; they are inactive when bound to the protein Rb (retinoblastoma). If Rb is removed (either by degradation or more commonly phosphorylation using a kinase) the E2F is released and active; it can now induce the expression of S-phase genes.

There are several CDK inhibitors important in cell regulation; if inactivated they can lead to cancer:

  • p15 and p16 (cdk4/6 inhibitors)
  • p21 and p27 (general cdk inhibitors)

Differentiation is the process by which a cell undergoes a change to be an overtly specialized cell type. Cells are differentiated because there is tissue-specific gene expression; cells within a tissue express the proteins required for the appropriate functioning of that tissue. There is often a master regulatory gene that expresses a transcription factor which regulates the expression of many other required genes. Once a cell becomes committed to differentiate into a specific cell type, this type is generally maintained through subsequent generations. This phenomenon of cell memory is a prerequisite for the creation of organized tissues and for the maintenance of stably differentiated cell types.

Development is the succession of changes that take place in an organism as a fertilized egg gives rise to an adult. It comprises cell division, differentiation and morphogenesis. Both internal and external signals are required to achieve proper development. Sources of developmental information in the early embryo include cytoplasmic determinants and cell-cell signaling.

Gradients of molecules control axis formation (anterior-posterior and dorsal-ventral) and polarity determines segmentation. After segments have been determined, limb development begins, regulated by homeotic/Hox genes that encode transcription factors (turning on whole set of genes). In the developing limb, the AER (apical epidermal ridge) and ZPA (zone of polarizing activity) secrete signals that indicate the correct orientation for the limb.

Turnover of differentiated cells:

  • Most differentiated cells are not permanent and are replaced (though some are permanent)
  • Most permanent cells renew their parts
  • Some differentiated cells are replaced by simple duplication, others are generated from stem cells

Gene Therapy

Gene therapy is the use of genetic material to correct diseases caused by gene defects. Therapy may be:

  • Germ-line– treats gametes so all cells of the individual and subsequent generations are altered (banned)
  • Somatic cell – treat individual only so alteration in a single organ or tissue (current therapy)

Monogenic disorders (single gene defects) are the easiest to treat using gene therapy. Pathologies are due to direct loss of protein function, and so treatment involves correcting the gene and therefore gene expression. Multifactorial disorders are much more complex; extensive knowledge of the pathophysiology must be obtained to develop a therapy strategy. Cancers are the most commonly addressed disorders (65%), followed by CVD (9%) then monogenic diseases (8%). Gene therapy targets are – antigens (20%), cytokines (19%), tumour suppressors (11%), growth factors (8%) etc.

There are two methods of gene transduction:

  • In vivo – requires a vector to carry the functional gene that is then delivered into the individual.
  • Ex vivo – (more common) cells are harvested from the individuals and the gene-carrying vector is added; cells that take up the gene are selected for and grow in large quantities. This guarantees that the target cells are receiving the therapy, and there is no rejection

Common vectors are adenovirus (24%), retrovirus (21%), naked/plasma DNA (18%) and liposome-DNA complexes:

  • Viruses are the most efficient system and can produce permanent corrections if inserted into appropriate spot in the stem cell population. However, the size of gene carried is limited, it can elicit an immune response, and it involves random insertion (may interrupt coding sequences)
  • Liposomes also have random insertion, but can deliver large amounts of DNA
  • Naked DNA is the easiest method, but only limited cell types will take up this DNA

Disadvantages of gene therapy:

  • Short lived nature – unless it becomes integrated and is also inserted into stem cells or those that have a very long life, it is not a long-term cure. Patients must undergo multiple rounds of gene therapy
  • Immune response – can reduce effectiveness and makes repeated applications very difficult
  • Viral vector issues – toxicity, immune and inflammatory responses, and gene control and targeting issues
  • Multigene disorders – most commonly occurring but especially difficult to treat effectively

Folate metabolism

Vitamins are chemical compounds that the human body requires in very small amounts, but cannot be made from other food components. The water-soluble vitamins (C, niacin, thiamine, folate, B12 and other B vitamins) are not stored in the body and are not toxic; excess excreted in urine. Folate requirements – normal diets comfortably exceed the RDI (green leafy veg), but folate is relatively easily destroyed by cooking, and requirements are much higher during pregnancy (supplements should be taken before they become pregnant). Deficiency is found in alcoholics and the elderly (poor diet), those with malabsorption diseases and those on oral contraceptives or anticonvulsants. Folic acid is a complex organic molecule that has three structural components. N5 and N10 are the functional parts of the molecule. The glutamate can be extended to form a chain or 2 to 7 glutamates, forming polyglutamyl folic acid. It is the polyglutamyl forms that are functional in the body.

Sulphonamide antibiotics inhibit bacterial folic acid synthesis and so are toxic to them. All of the drugs in this group have a structural group analogous to PABA. This binds to the enzymes, inhibiting the bacteria from synthesizing folic acid.

The main dietary form is polyglutamyl-FH2; this must be converted (oxidized and intestinal enzyme remove the polyglutamyl chain) into folic acid to be absorbed in the GIT. Within cells (mainly liver) folic acid is converted to its active form – tetrahydrofolate (FH4). It is transported in the blood as monoglutayl-FH4 derivatives (mainly methyl-FH4) and stored and utilized in cells as polyglutamyl-FH4derivatives. These are required for 1-C metabolism; many biosynthetic reactions require donation of chemical groups containing single carbon atoms:

  • Polyglutamyl-FH4 derivatives (and vitamin B12) act as coenzymes, supplying these chemical groups
  • Biosynthetic pathways include those for making purines and pyrmidines, as well as choline and creatine synthesis
  • Defects in 1-C metabolism most affect rapidly dividing cells, due to impaired DNA/RNA synthesis (haemopoiesis)

Synthesis of tetrahydrofolate involves the conversion of what is absorbed in GIT into the fully reduced form. The same enzyme – dihydrofolate reductase – is involved in two successive reductions, with NADPH as the reducing agent. We cannot make use of dietary folate without this enzyme. Because FH2 is a much better substrate, it is immediately reduced so significant concentrations are not found in the body. Some pharmacological compounds inhibit this enzyme:

  • Methotrexate: used in various forms of cancer, especially leukaemia. It is structurally very similar to folic acid, except it has an amino acid instead of hydroxyl group, as well as an extra methyl group. Methotrexate is a potent competitive inhibitor of the enzyme – it binds 100x more tightly than folate. By binding to the enzyme it inhibits folate metabolism; it is less toxic to cells that have lower requirements of 1-C metabolism and DNA/RNA synthesis. A potentially lethal dose is given, followed by a large 'rescue' dose of the readily-utilized form (5-formyl-FH4)
  • Trimethoprim is used as an antibiotic as it too is an analogue of folic acid (so inhibits dihydrofolate reductase). It is much more effective at inhibiting bacterial enzymes than the mammalian one so has minimal effects on humans

Tetrahydrofolate (FH4) derivatives (coenzymes) – Donors of 1-C fragments (below) are amino acids – glycine, tryptophan, histidine (formate) and most importantly serine. A range of different 1-C groups can be attached to N5 and/or N10 by enzyme reactions. These can then be used in biosynthetic reactions.

There are various interconversions between FH4 derivatives; all are reversible except for the irreversible formation of 5-methyl-FH4. As a result, this derivative predominates in cells and the blood. There is only one reaction that can take the methyl group off 5-methyl-FH4, requiring Vitamin B12 as a coenzyme (is B12 is deficient, methyl-FH4cannot be utilized). The methyl-tetrahydrofolate 'trap' – in B12 ¬¬deficiency (dietary deficiency or lack of intrinsic factor), essentially all FH4 is converted into and trapped as unusable methyl-FH4, leading to deficiency of usable FH4 derivatives and anaemia similar to that arising from a folate deficiency.


Separation techniques

  • Gel filtration (permeation chromatography, or size exclusion) based on size
    • Smaller molecules move through the beads and so travel slower
    • Larger molecules push past the beads and so travel faster
  • Ion-exchange chromatography based on charge. The beads are negatively charged:
    • Neutral amino acids (e.g. phenylalanine) will move faster and elute earlier
    • Basic/positively-charged amino acids (e.g. arginine) move slower as they are attracted to the beads

PKU is an autosomal recessive disease usually involving a mutation in the gene coding for the PAH enzyme (phenylalanine hydroxylase). This enzyme usually catalyses the conversion of phenylalanine to tyrosine; inactivation causes an accumulation of phenylalanine in body fluids. Symptoms (neurological problems, delayed psychomotor maturation, tremors, hyperactivity, eczema) are a result of accumulation of substrate, rather than lack of product, as tyrosine can be derived from other sources.

Restricting the intake of phenylalanine (in almost all protein-containing foods) can reduce the effects of PKU. This diet is commenced immediately upon diagnosis; adherence to the diet is crucial early in life and many recommend that it be followed for life. The artificial sweetener aspartame (in diet coke) is converted into phenylalanine, so must be avoided. Pregnant woman with PKU must initiated the diet prior to conception and maintain it throughout pregnancy as excess phenylalanine will cross the placenta and induce developmental defects in the foetus.

The PAH gene is 79,277 nucleotides (1,356 nucleotides in the expressed sequence) and 13 exons. Multiple mutations have been found in the exons of the enzyme:

  • Glycine to alanine – silent, only adding a methyl group
  • Cysteine to alanine – effect, taking away sulfur
  • Phenylalanine to alanine – effect, removing large aromatic group
  • Aspartic acid to alanine – effect, extra carboxyl group
  • Leucine to isoleucine – silent, same molecular formula (isomers)

It is not possible to screen using genetic testing, due to cost and the fact that most of the mutations are point mutations and therefore difficult to identify. Tandem mass spectrometry (MS/MS) can be used to screen very large sample numbers quickly for increased levels of circulating phenylalanine; this is diagnostic for all mutations. The technique separates molecules based on mass and structure. Advantages

  • Many disorders can be screened on the same sample at the same time. As well as PKU, it can detect 20 other amino acid metabolism disorders and 10 fatty acid metabolism disorders
  • Can specify analytes to be detected
  • Simple sample preparation
  • Fast analysis time (2 min/sample)
  • Small sample volume (3µL blood)
  • Low running costs
  • Has other non-clinical uses, including detection of illegal drug use in athletes

Genetics in congenital disease

Autosomal (all chromosomes except sex chromosomes) recessive – when both parents are heterozygotes (carriers): ¼ affected, ½ carriers, ¼ normal in each pregnancy. 2/3 of unaffected individuals will be carriers. AR diseases are often enzyme defects, resulting in too little protein to carry out normal function. Only 10-20% of normal protein levels are required for normal function, therefore AR do not occur in heterozygotes.

Autosomal dominant – male-to-male transmission may occur. Some AD aspects of phenotypic expression:

  • Reduced penentrance – individuals with appropriate genotype fail to express phenotype (characteristic)
  • Variable expressivity – severity of phenotype varies in individuals with same genotype
  • New mutations – sporadic phenotype without affected parents
  • Delayed onset – death may be due to unrelated causes prior to phenotype expression
  • Anticipation – increase in severity with successive generations; associated with triplet repeat disorders (Huntington)
  • Germline mosaicism – recurrence in a clinically unaffected parent
  • Imprinting disorders – every autosomal gene has a maternal and paternal copy; generally both copies are functional. In a small subset, one copy is turned off (imprinted) in a parent-of-origin dependent manner. The allelic expression of an imprinted gene depends upon whether it resided in a male or female the previous generation (phenotype depends on sex of transmitting parent)
  • Sex-limited phenotype – expressed in only one sex
  • Sex influenced phenotype – expressed in both sexes but to differing degree

AD diseases are generally either –

  • Loss of function (reduced expression) – a non-active protein from the mutant allele will bind with the normal protein product of the remaining normal alleles, resulting in inactivation of the complex, and reduced gene product activity
  • Gain of function – due to:
    • Increased gene dosage
    • Ectopic or temporally altered mRNA expression – mutation affects time or place of gene expression, often in the gene's regulatory domain (e.g. hereditary persistence of fetal Hb)
    • Increased protein activity – mutation alters recognition site for protein degradation; protein remains active
    • Dominant negative effects – antagonize the activity of the remaining wild type
    • Altered structural protein
    • Toxic protein alterations new protein functions

X-linked disorders (recessive, girls have two x chromosomes so only good one expressed, boys have only one so always expressed) – mother may be an obligate, probable or occasionally manifesting carrier. Impact:

  • Males who inherit the mutated gene on their X chromosome will usually show the effects of the disorder
  • Females will usually be unaffected because of the random 'switching off' of one member of the X chromosome pair (becomes a Barr body). They may be mildly affected if the X chromosome inactivation is skewed

Gene mapping

Finding where specific genes are in relation to their spot on chromosome Linkage produces gametes with non-parental (recombinant) genotypes. In crossing over (meiosis prophase 1), homologous chromosomes physically exchange segments, contributing to diversity of offspring. The closer together two loci are the less likely they are to be separated.

The frequency of combination between markers indicates their relative locations and the distances between them. Using linkage and probability, we can obtain gene order and relative distance between them (measured in centiMorgans). Linkage analysis requires individuals that are heterozygous for at least two loci (sites of specific gene). Various DNA markers are used:

  • Microsatellites – repetitive DNA segments, typically 2-8 base pairs long; measure different numbers of repeats
  • Single nucleotide polymorphisms (SNPs) – occur at very high frequency in the genome; only useful in large numbers
  • Restriction fragment length polymorphisms (RFLPs)

Linkage analysis is by LOD (log of odds of linkage scores); this changes according to frequency