Topic 8: Control of gene expression

• An organism develops from a single fertilised egg, this fertilised egg has the ability to mature into any body cell - it is totipotent
• They later differentiate and become specialised for a particular function
• The specialised cells only make proteins that are required for it to carry out its function
• They are still capable of making other proteins but this would be wasteful
• Genes are prevented from expressing themselves by: preventing transcription, or preventing translation

• Only a few cells retain the ability to differentiate into other types of cells, these are stem cells
• Stem cells are undifferentiated dividing cells that occur in adult animal tissues and need to be constantly replaced
• They have the ability to divide into an identical copy of themselves - self-renewal
• They originate from: embryos, umbilical cord, placenta, adult bone marrow

Types of stem cell include:

• Totipotent: found in early embryo, differentiate into ANY type of cell
• Pluripotent: differentiate into ALMOST ANY type of cell
• Multipotent: differentiate into a LIMITED number of specialised cells
• Unipotent: differentiate into only ONE type of cell

• For transcription to begin, the gene is switched on by transcriptional factors, each has a site that binds to a specific base sequence of DNA
• When the transcriptional factor binds it causes that region of DNA to begin transcription
• When a gene is not being expressed, the site on the transcriptional factor that binds to DNA is inactive

Hormones like oestrogen can switch on a gene and start transcription by combining with a transcriptional factor receptor site:

• Oestrogen is lipid soluble, it passes through the cell membrane
• Once inside the cytoplasm, oestrogen binds with a complementary receptor site on the transcriptional factor
• Oestrogen changes the shape of the DNA binding site on the transcriptional factor, it is now active and can bind to start transcription

Epigenetics - the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself - environmental factors can cause heritable change in gene functions without changing the base sequence of DNA

The epigenome

• DNA is wrapped around proteins called histones, both are covered in chemicals called tags
• Chemical tags form a second layer - the epigenome
• This determines the shape of the DNA-histone complex, the DNA code is fixed, the epigenome is flexible
• Genes that are inactive are tightly packed, ensuring they cannot be read - epigenetic silencing
• Active genes are unwrapped so the DNA is exposed and can be easily transcribed
• Chemical tags respond to changes in environmental factors - diet, stress, etc.
• The epigenome of a cell is the accumulation of all signals that it has received - cellular memory

• Where the association of histones with DNA is weak - it is less condensed, so the DNA is accessible by transcriptional factors - it is switched on
• Where the association of histones with DNA is strong - it is more condensed, the DNA inaccessible by transcriptional factors - it is switched off

Decreased Acetylation

• An acetyl group is transferred to a molecule
• In this case, the group donating is acetylcoenzyme A
• Decreased acetylation increases the positive charges on histones - increases their attraction to the phosphate groups on DNA
• Association between DNA and histones is stronger - gene inaccessible/switched off

Increased Methylation

• Addition of methyl group to the cytosine bases
• Inhibits transcription by preventing the binding of transcriptional factors and attracting proteins that condense the DNA-histone complex - making genes inaccessible

• In eukaryotic and some prokaryotic cells, the translation of mRNA produced by a gene can be inhibited by breaking mRNA down before it can be translated

Small interfering RNA (siRNA)

• An enzyme cuts large double-stranded molecules of RNA into smaller sections - siRNA
• One of the siRNA strands combines with an enzyme
• The siRNA molecule guides the enzyme to mRNA and pairs up bases
• The mRNA is cut into smaller sections by the enzyme
• mRNA can no longer be translated
• Genes are not expressed

BENIGN TUMOUR

• Large, grows slowly
• Cells well differentiated
• Cells produce adhesion molecules that make them stick together and they remain within the tissue - primary tumours
• Surrounded by a capsule of dense tissue, remains compact
• Less likely to be life-threatening, can disrupt organ function
• Localised effect, rarely reoccur after treatment

MALIGNANT TUMOUR

• Large, grows rapidly
• Cells undifferentiated
• Cells do not produce adhesion molecules and spread to other regions of the body, a process called mestasis - secondary tumours
• No capsule, finger-like projections penetrate surrounding tissue
• Life-threatening, abnormal tumour tissue replaces normal tissue
• Systematic (whole body) effect, more frequently reoccur after treatment

• Cancer cells derive from a single mutant cell
• Intial mutation caused uncontrolled mitosis

ONCOGENES

• Mutations of proto-oncogenes
• Proto-oncogenes stimulate a cell to divide when growth factors attach to a protein receptor on its surface
• If a proto-oncogene mutates into an oncogene it can become permanently activated
• Therefore cells divide too rapidly and out of control

TUMOUR SUPRESSOR GENES

• Slow down cell division, repair mistakes in DNA and tell cells when to die - apoptosis
• Prevent tumours by maintaining normal cell division rate
• If a TSG mutates, it becomes inactivated - it stops inhibiting cell division

Abnormal methylation of tumour supressor genes

• Hypermethylation occurs in a specific region of TSG
• This leads to the TSG being inactivated
• Transcription of the promoter regions of TSG is inhibited
• TSG silenced
• Inactivation = increased cell division

Oestrogen concentration and cancer

• Oestrogen plays a central role in the menstrual cycle
• Women’s chances of cancer often increase after menopause due to increased oestrogen concentration
• Fat cells of the breast produce more oestrogen
• Tumour produces even more oestrogen when present

GENOME - the complete set of genes or genetic material present in a cell or organism

DNA sequencing

• Determining the complete DNA base sequence of an organism uses the technique of whole-genome shotgun (WGS) sequencing
• Researchers cut DNA into many small, easily sequenced, sections and use computer algorithms to align overlapping segments to create the genome

PROTEOME - All the proteins produced in a given type of cell, at a given time, under specified conditions

Producing DNA fragments

• Isolate, clone and transfer genes into microorganisms
• Microorganisms grown to provide a ‘factory’ for the production of the desired gene
• The DNA of two different organisms that have been combined in this way is recombinant DNA
• The resulting organism is transgenic, a GMO - Genetically Modified Organism

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

USING REVERSE TRANSCRIPTASE

• Cell that readily produces the protein is selected - eg. beta cells
• Reverse transcriptase is then used to make DNA from RNA, this DNA is known as complementary DNA - cDNA
• DNA polymerase is used to build up the complementary nucleotides on the cDNA
• The double strand of DNA is the required gene

USING RESTRICTION ENDONUCLEASES

• Restriction endonucleases - produced by bacteria to defend themselves by cutting up DNA
• Some cut between 2 opposite base pairs leaving 2 straight blunt ends
• Others cut unevenly, each strand of the DNA has unpaired exposed bases - STICKY ENDS
• Restriction Endonucleases make staggered cuts, recognise 6 base pairs
• 4 bases exposed - mirror images
• This a 6 base pair palindrome

GENE MACHINE

• Desired sequence worked out, fed into computer
• Checked for biosafety and biosecurity
• Computer designs a series of oglionucleotides - assembled into the desired gene
• No introns or non-coding DNA
• Gene replicated using polymerase chain reaction (PCR)
• Also constructs a complemtary strand
• Using sticky ends the gene can be inserted into a bacterial plasmid

IN VIVO - within a living organism, as the organism grows and divides it replicates the DNA making multiple copies of the gene

Part 1 - MAKING RECOMBINANT DNA

• The vector (plasmid) is isolated
• Vector cut open using the same restriction endonuclease used to isolate the DNA fragment containing the target gene, the sticky ends of the vector and DNA fragment will be complementary
• Vector DNA and DNA fragment are mixed together with DNA ligase (enzyme), DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector - LIGATION
• The new combination of bases is called recombinant DNA

Part 2 - Transforming cells

• Vectors (plasmids) and host cells (bacterial cells) mixed together in a heated medium containing calcium ions, these conditions make the bacterial membrane permeable - allowing the plasmids to pass through into the cytoplasm
• Only as few as 1% take up the plasmid, sometimes plasmids will have closed up again without incorporating the DNA fragment

Bacterial cells that have taken up the plasmid will have antibiotic resistance

• All bacterial cells grown on a medium that contains the antibiotic ampicillin
• Bacterial cells that have taken up the plasmid will be ampicillin resistant
• These cells will survive
• Bacterial cells without the plasmid will die

However, some cells will have taken up the plasmid and closed without incorporating the gene, to eliminate these we use marker genes

ANTIBIOTIC RESISTANT

• Bacterial cells that survived the first antibiotic are cultured in an agar
• A tiny sample of each colony is transferred onto a replica plate in the same position
• The replica plate contains an antibiotic that cells have resistance to if they took up the plasmid
• Colonies not killed were the successful ones, these are grown from the first plate

FLUORESCENT

• Jellyfish gene for fluorescence (GFP) is inserted into a plasmid
• The gene to be cloned is transferred to the middle of the GFP gene
• Any Bacterial cell that has taken up the plasmid with the desired gene to be cloned will not glow

ENZYME

• Required gene inserted into gene that makes lactase
• Lactase makes solutions turn blue
• Cells won’t turn blue if the desired gene was successfully incorportated

IN VITRO - cloning outside of a living organism using polymerase chain reaction

POLYMERASE CHAIN REACTION

• A method of copying fragments of DNA - automated, rapid, efficient

Requires the following:

• DNA fragment to copy
• DNA taq polymerase - thermostable, can withstand high temperature
• Oglionucleotide primers
• Nucleotides
• Thermocycler - a computer-controlled machine that varies temperature precisely over a period of time

1. SEPARATION

• DNA fragments, primers and DNA polymerase placed in thermocycler
• Temperature is set to 95 causing the two strands of DNA fragment to separate due to the breaking of hydrogen bonds

2. ADDITION

• Temperature 55, causes primers to join (anneal) to complementary bases on the end of the DNA strands
• Primers prevent the two strands from rejoining

3. SYNTHESIS

• Temperature 72, optimum temp for DNA taq polymerase to add complementary nucleotides along the DNA strands
• 2 new copies of DNA fragments are formed

DNA PROBES

• A short single stranded length of DNA that has some sort of label attached to make it easily identifiable
• Radioactively labelled probes - made up of nucleotides with the isotope 32P, identified using x-ray film
• Fluorescently labelled probes - emit fluorescent light under certain conditions

Used to identify particular alleles as follows:

• DNA probe made that has complementary bases to part of the DNA sequence with the gene you want to find
• Double-stranded DNA that is being tested treated to separate the strands
• Separated DNA strands are mixed with the probe which binds to the complementary base sequence on one of the strands - DNA HYBRIDISATION
• The site at which the probe binds can be indentified by the fluorescence or radioactivity

• A section of DNA or RNA is combined with a single-stranded section of DNA
• The 2 strands of DNA are separated by heating - denaturation
• When cooled the complementary bases recombine - anneal
• Given sufficient time all DNA partners will pair up

• Determine the desired allele - genetic libraries
• Fragment of DNA produced complementary to allele
• Multiple probes formed using PCR
• Marker attached
• DNA heated to separate 2 strands
• Separated strands cooled in a mixture with many probes
• If the DNA contains the allele one of the probes is likely to attach
• DNA washed clean of an unattached probes
• Remaining hybridised DNA will now be fluorescent
• Dye detected by shining light on the fragments
• Genetic screening
• Personalised medicine
• Genetic counselling

• Analysis of fragments that have been cloned using PCR
• 95% of human DNA made up of non-coding DNA known as Variable Number Tandem Repeats (VNTR)
• Some is between genes - some is within genes (introns)
• Some of the non-coding DNA is repetitive - hypervariable region

GEL ELECTROPHORESIS

• Used to separate DNA fragments according to size
• DNA fragments placed on agar gel and voltage is applied across it
• The resistance of the gel means that the larger the fragments are the more slowly they move
• Over a period of time, the small fragments move further
• DNA fragments of different lengths separated
• Only DNA around 500 bases long can be sequenced so larger genes must be cut into smaller fragments using restriction endonucleases

EXTRACTION

• Obtain a small sample of blood, saliva, hair, etc.
• Separate the DNA from the rest of the cell using a solvent
• If the sample is very small many copies are made using PCR

DIGESTION

• Restriction endonucleases are used to hydrolyse the DNA into fragments
• The DNA fragments will be of varying sizes, some contain hypervariable regions
• They will cut close to, but not within, core sequenced

SEPARATION

• DNA fragments separated according to size using gel electrophoresis
• DNA fragments placed on gel at pH7, voltage applied
• DNA fragments move towards positive electrode as they are negatively charged
• Gel immersed into an alkali to seperate the double strands
• The single strands are transferred into a nylon membrane using a technique called Southern blotting
• Membrane covered with several layers of absorbent paper which draws the liquid containing DNA

HYBRIDISATION

• Separated DNA single strand mixed with radioactive or fluorescent DNA probes
• Probes bind with known core sequences at complementary bases
• Specific temperatures and pH are needed
• Complementary base pairings occur between DNA and probes

DEVELOPMENT

• A phosphate containing substrate is then added and placed on an X-Ray film
• The alkaline phosphatase removes the phosphate causing the substrate to fluoresce - fogging the X-Ray film
• The developed film shows dark bands where the probe has bound to the hypervariable region

GENETIC RELATIONSHIPS & VARIABILITY

• To determine genetic variability
• Resolve questions of paternity
• More closely related individuals have closer genetic fingerprints

FORENSIC SCIENCE

• Blood, hair, saliva, etc. left at the scene of a crime
• Can determine whether a suspect was present at a crime scene

PLANT & ANIMAL BREEDING

• Prevent undesirable interbreeding in farms and zoos
• Can identity plants or animals with a desirable gene

MEDICAL DIAGNOSIS

• Diagnose diseases like Huntington’s disease
• Compare genetic fingerprints to determine the probability of a person developing symptoms and when