iModule 8 (Genes) Revision Notes

What is a Stem Cell?

 a unspecialised/undifferentiated cell
 potential to form different types of cells

How does a stem cell be come a specialised cell?

 differentiation
 3 changes: cell shape, number of organelles, new content
 occurs by controlling gene expression (some gene are activated, other genes are
inhibited)

Stem Cell in Animals/Mammals/Humans?

 Totipotent = Zygote
 Pluripotent = Embryonic Stem Cells
 Multipotent = Bone Marrow Stem Cell
 Unipotent = Tissues

What are Induced Pluripotent Stem Cells (iPS Cells)?

turning unipotent body cells into pluripotent cells (like embryonic stem cells), involves
activating certain deactivated genes using transcription factors

Stem Cell Therapy in Humans?

 2 uses,
 use stem cells to produce tissues/organs for transplant
 use stem cells to treat irreversible diseases e.g. heart disease, type 1 diabetes, paralysis
(inject stem cells at site of disorder – will differentiate to become local specialised cells
e.g. heart muscle cells, beta cells of pancreas, neurones)

Stem Cell in Plants?

 In embryo = Zygote/Embryonic Stem Cells

 In adult = Meristem Cells in Stem/Shoot/Root

Uses of Stem Cells from Plants?

 traditionally cuttings were taken from plants (stem/shoot/root) and used to grow
genetically identical plants – possible due to presence of meristem cells
 tissue culture (micro propagation) = large scale application of cuttings
 process,
 take cutting from shoot/stem/root (called explant)
 place explant in nutrient rich medium so meristem cells divide by mitosis
 produces a mass of meristem cells (called callus)
 take each meristem cell and grow in plant growth factor medium to promote
differentiation and formation of shoot/root
 transfer plant to soil and greenhouse
 then transfer to field
What is Controlling Gene Expression?

 either Activating or Inhibiting a Gene

 activating gene = protein made
 inhibiting gene = protein not made

Example of activating genes?

 using oestrogen
 oestrogen can enter a cell by simple diffusion and bind to receptors on the transcriptional
factor
 causes transcriptional factor to change shape
 so transcriptional factor can now enter nucleus and bind to promoters on the DNA to
activate transcription
= activated genes (protein to be made)

Example of inhibiting genes?

 using siRNA (small interfering RNA)

 making siRNA = double stranded RNA cut down into small sections, made single
stranded, then attaches to an enzyme
 siRNA will bind to complementary sections on mRNA = the enzyme will cut the mRNA
so translation cannot occur = gene inhibited (protein not made)

What is Epigenetics?

 Heritable changes in gene function without changes to base sequence of DNA

 Changes may due to lifestyle, stress, diet
 Chromatin (DNA-Histone Complex) is surrounded by an Epigenome (chemical layer)
 Epigenome can either cause the Chromatin to become more condensed or more loose
 Chromatin becoming more condensed means transcription factors cannot reach the DNA
and the gene will be inactivated
 Chromatin becoming more loose means transcription factors can reach the DNA and the
gene will be activated
 These changes may be brought about by Acetylation or Methylation

How does Methylation and Acetylation affect the Genome?

 Increased Methylation = adding methyl groups, this attracts proteins which condense the
DNA-Histone Complex so transciption factors cannot gain access (gene inhibited)
 Decreased Acetylation = removing acetyl groups, increases positive charges on the
Histone which increases the attraction to the phosphate groups on DNA which condense
the DNA-Histone Complex so transciption factors cannot gain access (gene inhibited)

What is a Gene Mutation?

  a change in the base sequence of DNA
 2 types = substitution and insertion/deletion
 substitution = replace one base for another, changes one triplet code
can be silent (new triplet code codes for same AA), mis-sense (codes for a different
AA, so protein shape changes slightly), non-sense (codes for a stop codon, so
polypeptide chain not produced)
 insertion = adding a base, deletion = removing a base
both insertion/deletion causes frameshift, all the triplet codes after the mutation
changes, so normal polypeptide chain/protein not produced

What is Cancer?

 formation of a malignant tumour

 due to uncontrolled cell division (mitosis)

Malignant vs Benign Tumour?

Malignant Tumours,

 Rapid Growth (rapidly dividing cells)

 Cells are unspeicialised
 Cells can spread (Metastasis)
 Systemic Effects
 Requires Surgery/Chemotherapy/Radiotherapy

What normally controls Cell Division (mitosis)?

 2 genes: proto-oncogene & tumour-supressor gene

 both produce proteins to control cell division
 proto-oncogene stimulates cell division
 tumour-suppressor gene inhibits cell division
 proto-oncogene produces growth factor and receptor protein, when the growth factor
binds to receptor protein on cells it stimulates DNA replication that leads to cell division
 tumour-suppressor gene produces a protein that inhibits cell division

Cancer?

 caused by mutation of genes that control cell division

 causes of mutation = random or mutagens (chemicals/radiation)
 mutation of proto-oncogene leads to formation of a oncogene = over production of
growth factor or receptor proteins permanently active = over stimulation of cell division
(uncontrolled cell division)
 mutation of tumour-suppressor gene = loss of protein to inhibit cell division
(uncontrolled cell division)

Oestrogen and Cancer?

 Oestrogen leads to activation of genes – high levels of oestrogen can lead to over activation
of Proto-Oncogen forming an Oncogene = Cancer (uncontrolled cell division)

Epigenetics and Cancer?

Main Example = increased methylation of tumour suppressor genes leads to inhibition of

tumour suppressor genes leading to cancer (uncontrolled cell division)

What is Genetic Engineering?

 changing the genetic make-up of an organism's DNA by adding or removing a gene

 the DNA becomes Recombinant
 the Organism becomes Genetically Modified (Transgenic)

Why do we Genetically Engineer Animals?

 to give them additional characteristics

 so they can make useful products (proteins)

Examples of genetic engineering in animals?

 additional characteristics,
 add gene for disease resistance
 add gene for growth hormone for growth
 making useful products,
 use to produce anti-thrombin = protein used to make blood clot (people with certain
genetic disease may not produce), use milk producing animal to produce, add gene for
anti-thrombin next to milk producing gene in animal, therefore anti-thrombin protein
will be made in the milk (easily extracted)

Why do we Genetically Engineer Plants?

 to give them additional characteristics

 so they can make useful products (proteins)

Examples of genetic engineering in plants?

 additional characteristics,
 add gene for disease resistance
 add gene for pest resistance
 add gene for pesticide resistance
 add gene to promote growth for high yield
 produce genetically modified tomatoes = prevented from softening therefore remain
hardened (easy for storage and transport), involves preventing formation of softening
enzyme, a gene is added that is complementary to the the softening enzyme gene, so its
mRNA will bind to the mRNA of the softening enzyme preventing translation of the
softening enzyme
 making useful products,
 use to make golden rice (rice that contains beta-carotene, a pre-cursor to vitamin A to
treat malnutrition deficiency)
 use to make protein raw material for polymers
Why do we Genetically Engineer Bacteria? so they can make useful products (proteins)

Genetically engineering bacteria?

 to make useful proteins e,g, Insulin

 normally used animal sources (problems = limited supply, infection risk,
immunorejection)
 involves adding human insulin gene to a plasmid, then inserting this into a bacteria = the
bacteria now has the gene/code to produce the human insulin protein

involves 5 steps =

1. Isolation, 2. Insertion, 3. Transformation, 4. Identification, 5. Growth/Cloning

1. Isolation

 either by Reverse Transcriptase or Restriction Enzyme or Gene Machine

 RT = enzyme found in virus, converts RNA into DNA, obtain mRNA for insulin, the RT
will convert it into cDNA (single stranded complementary DNA), DNA Nucleotides and
DNA Polymerase added to make it double stranded
 RE = enzyme found in bacteria, cuts DNA at certain base sequences (called recognition
sites) by breaking bond between sugar and phosphate, can cut straight or staggered,
staggered used in GE as it leaves exposed bases called 'sticky ends' [cuts staggered at 6
base pair palindromes, were the 6 bases read forward are identical to 6 bases read
backward on both strands]
 GM = build DNA base sequence from know Amino Acid Sequence of the Protein (uses
oligosacchairdes)

end result = Isolated Human Insulin Gene

2. Insertion

 cut plasmid using the same RE from isolation stage

 leaves complementary sticky ends
 join human insulin gene with plasmid via the sticky ends
 use DNA Ligase to join the sugar-phosphate backbone

= Recombinant plasmid (carrying human insulin gene)

3. Transformation

 mix recombinant plasmid with bacteria

 add Ca2+ ions and heat shock
 bacteria will become permeable and take up the recombinant plasmid

= Genetically Modified Bacteria (carrying recombinant plasmid with human insulin

gene)

4. Identification

 identify which of the bacteria have taken up the recombinant plasmid and of these which
ones have accepted the new gene (human insulin gene)
step 1 = choose a plasmid that carries an Ampicillin Resistance Gene, so when Ampicillin is added
only the bacteria that have taken up the recombinant plasmid will survive (as they will have
obtained the ampicillin resistance gene)

step 2 = use gene markers (antibiotic resistant, fluorescent, enzyme) to identify which of the
remaining bacteria have accepted the human insulin gene, the human insulin gene will be placed in
the middle of these gene markers, if the bacteria accepts the human insulin gene they will reject the
gene marker & if the bacteria rejects the human insulin gene they will accept the gene marker
 antibiotic resistant = tetracycline resistance gene lost if human insulin gene accepted,
so bacteria no longer resistant to tetracycline, add tetracycline by replica plating (on
another plate that carries a few of the bacteria from each colony in their same
position), the ones that die are the ones that we want, identify on original plate
 fluorescent = fluorescent gene lost if human insulin gene accepted, so identify
bacteria showing no fluorescence
 enzyme = enzyme gene lost if human insulin gene accepted, therefore add colourless
substrate, where there is no colour change select those bacteria (as enzyme not made
to breakdown colourless substrate for colour change)

end result = Genetically Modified Bacteria

5. Growth/Cloning

 grow genetically modified bacteria (carrying human insulin gene)

 they will produce the protein (human insulin)

What is PCR?

 polymerase chain reaction

 used to replicate DNA artificially
 step 1: heat to 95oC, hydrogen bonds break, double strand separates, left with 2 template
strands
 step 2: cool to 55oC, primers bind (short single stranded sections of DNA) to start of
each template strand, prevents the templates from rejoining and allows DNA Polymerase
to bind to build the new strand
 step 3: heat to 72oC, DNA nucleotides attach to complementary bases, DNA Polymerase
joins sugar-phosphate backbone of the new strands
= 2 copies of DNA (each made of 1 original strand, 1 new strand)

Polymerase Chain Reaction vs Semi-Conservative Replication?

 PCR can only replicate short DNA fragments, SCR can replicate whole DNA
 PCR use 95oC, SCR uses DNA Helicase
 PCR uses primers, SCR does not require primers

In-vitro vs In-vivo method of DNA Replication?

 In-vitro = PCR
 In-vivo = using bacteria to replicate DNA (add DNA fragment to the plasmid, then
replicate the bacteria to make many copies of DNA fragment)
  benefits of in-vitro = more rapid, less complex
 benefits of in-vivo = more accurate (less mutations), less chance of contamination

What is a DNA Probe?

 short single stranded section of DNA

 has a specific base sequence, so it binds to complementary genes
 is radioactively/fluorescently labelled
 if gene is present in DNA, DNA probe will bind to it and show up be
radioactivity/fluorescence

What is Genetic Screening?

 analyse an individual's DNA for the presence of a particular gene (e.g. mutated allele)
 use DNA Probes (single stranded section of DNA, complementary to a particular gene,
is radioactively labelled)
 obtain individuals DNA, make it single stranded, add the specific DNA Probe for the
gene to be screened for, if the gene is present the DNA Probe will bind, will show up as
radioactivity on an X-ray film

What is Genetic Fingerprinting?

 used to produce a unique 'fingerprint' of an individual's DNA (produces a specific

banding pattern)
 used in forensics and paternity testing
 involves analysing the individual's introns (non-coding DNA)
 introns contain repetitive sequences called variable number tandem repeats (VNTR)
 the number and length of the VNTR are unique for each individual organism

involves 5 steps:
1. Extraction, 2. Digestion, 3. Separation, 4. Hybridisation, 5. Development

1. Extraction
 extracting the individual's DNA
2. Digestion
 cutting the DNA down into fragments
 use Restriction Enzymes that cut just outside the VNTR (leaves the VNTR of the
introns)
3. Separation
 separate out the DNA fragments by gel electrophoresis
 add alkali to make the separated fragments single stranded
 transfer the fragments to a nylon membrane by Southern Blotting
 add UV light so the DNA fragments set
4. Hybridisation
 add radioactively labelled DNA Probes complementary to the DNA fragments
5. Development
 add photographic film and take an x-ray to produce the banding pattern picture

What is Genome Sequencing?

 determining base sequence of a genome (full set of DNA)

 uses Whole-Genome Shotgun (WGS) to cut DNA into smaller sections to be sequenced
  Bioinformatics is the science by which the information is collected and analysed
 uses = supports phylogenetic classification, identify genes related to diseases

What is a Proteome?

 full set of proteins produced by a certain genome