TOPIC 8: The control of gene expression

Gene mutations arise during

DNA replication

Gene mutations occur

spontaneously

The mutation rate is increased by

mutagenic agents

what are stem cells?

undifferentiated/ unspecialised cells capable of:

• dividing by mitosis to replace themselves

• differentiating into other types of specialised cells

Totipotent cells

divide and produce any type of body cell

Pluripotent cells are found

in embryos

multipotent and unipotent cells are found in

mature animals

Epigenetics

heritable changes in gene function, without causing changes to the bases of DNA

gene expression at a transcriptional level involve

methylation

acetylation

changes in the environment that inhibit transcription by

increasing methylation of DNA

decreasing acetylation of associated histones

methylation

addition of methyl groups on cytosine of DNA

makes the DNA less accessible to transcriptional factors

acetylation

addition of acetyl groups

makes DNA more negatively charged

causing more repulsion

so the DNA-histone complex is less condensed

making it easier for transcription factors to bind onto the DNA

Gene mutations might arise during DNA replication. They include

addition, deletion, substitution, inversion, duplication and translocation of bases.

translocation of bases

a group of dna bases become separated from the DNA sequence on one chromosome and are inserted into the DNA sequence on another chromosome.

Induced pluripotent stem cells (iPS cells) can be produced from

adult somatic cells

siRNA - small interfering RNA

1. double stranded rna is broken up by an enzyme into siRNA

2. one of the two strands of siRNA combines with an enzyme

3. the siRNA strand pairs with complementary bases on a mRNA strand

4. the enzyme cuts the mRNA into smaller sections

iPS induced pluripotent stem cells

unipotent stem cells genetically altered to bring a fully differentiated cell back to being pluripotent

PCR

method used to amplify DNA

involves a mixture of dna nucleotides, dna polymerase, primers

95°C - breaking of hydrogen bonds using heat

65°C - annealing of primers, primers bind to DNA

75°C - dna polymerase (taq polymerase) joins nucleotides

role of primers

stops DNA strands from rejoining back together

binds to DNA

oestrogen

steroid hormone

can initiate transcription

binds to the receptor site of the transcription factor

transcription factor changes shape slightly to become complementary (induced fit)

transcription factor with oestrogen attached to it binds on to dna

initiates transcription

lipid soluble nature of oestrogen means that is can freely diffuse across the cell membrane

genome project

(all) the total DNA in an organism/cell

maps out all genes

Recombinant DNA technology

involves the transfer of fragments of DNA from one organism, or species, to another.

Fragments of DNA can be produced by several methods, including:

• conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase

• using restriction enzymes (restriction endonuclease) to cut a fragment containing the desired gene from DNA

• creating the gene in a ‘gene machine’ - artificially synthesise genes

VNTRs

variable number tandem repeats

regions found in the non-coding part of DNA.

Suggest two features of the structure of different proteins that enable them to be separated by gel electrophoresis

mass OR sequence of amino acids

charge

R groups differ

What are the 3 methods for making dna fragments ?

1. Using reverse transcriptase
2. Using restriction endonuclease enzymes
3. Using gene machine

What are palindromic sequences?

Sequences that consist of base pairs that read the same in the opposite direction

What is in vivo cloning?

Where gene copies are made within a living organism- as the organism grows and divides it replicates the dna creating multiple copies of the gene.

What is in vitro cloning?

Where gene copies are made outside of living organism using the polymerase chain reaction

What method is used in in-vitro cloning?

Polymerase chain reaction (PCR)

Explain the 4 steps in the polymerase chain reaction

1. Reaction mixture is set up that contains the dna sample, free nucleotides, primers and DNA polymerase
2. Dna mixture is heated to 95degrees to break hydrogen bonds and then cooled to between 50-65 so primers can bind to strands
3. Reaction mixture is heated to 72 so DNA polymerase can work which lines up free nucleotides along template and specific base pairing allows complementary new strands to form
4. Two new copies of fragment of dna are formed and one cycle complete- cycle starts again this time all 4strands are used (each cycle doubles dna)

What is a DNA microarray?

A glass slide with microscopic spots of different dna probes attached to it in rows

Explain the process of using dna probes to locate certain alleles (3 steps)

1. Sample of dna is digested into fragments using restriction enzymes and separated using electrophoresis
2. Fragments are then transferred to nylon membrane and incubated with a fluorescently labelled dna probe - if allele is present the probe will bind to it
3. Membrane is exposed to uv light so if the gene is present there will be a fluorescent band

What are the 2 most common labels used on dna probes?

Fluorescent label- detected using uv light
Radioactive label- detected using x Ray Film

Explain dna probes

Short single strands of dna that have specific base sequence that is complementary to the base sequence of part of the target allele so it will bind to the target allele if it's present in the dna sample Probe has a label attached so that is can be detected

What is gene therapy?

Involves altering the defective genes inside cells to treat genetic disorders and cancer

What are the two types of gene therapy?

Somatic therapy
Germ line therapy

What does germ line therapy involve?

Involves altering alleles in sex cells so every cell of offspring won't suffer from disease

What does somatic therapy involve?

Involves altering the alleles in body cells particularly the cells that are most affected by the disorder, does not affect sex cells so offspring could still inherit disorder

Explain oestrogen's role in initiating transcription

1. oestrogen is lipid soluble so diffuses across cell membrane
2. binds to the receptor on a transcription factor (complementary)
3. Changes the shape of transcription factor's binding site so it can bind to DNA

How can epigenetics impact tumour suppressor genes

If the gene is hypermethylated, transcription of the protein is inhibited so the gene is turned off meaning it no longer slows down cell division

how can epigenetics impact oncogenes

If the gene is hypomethylated (reduced methylation) the gene would be permanently switched on, leading to uncontrolled cell division

what are oncogenes

Mutated version of proto-oncogene. Genes coding for proteins that stimulate mitosis (potentially causing cancer)

how is translation regulated

RNA interference (RNAi)

how does RNAi work

1. Enzyme cuts the mRNA into siRNA
2. One strand of the siRNA combines with another enzyme
3. siRNA-enzyme complex binds via complementary base pairings to another mRNA molecule
4. Once bound the enzyme with cut up mRNA can't be translated

features of benign tumours

- grow at a slow rate
- produce adhesion molecules sticking them together and to a particular tissue
- often surrounded by a capsule to they remain compact (and can be removed in surgery)
- impact is localised

features of malignant tumours

- grow very rapidly
- cell nucleus becomes large and cell can become unspecialised again
- do not produce adhesion molecules so metastasis occurs
- tumour isn't encapsulated so can grow projections into surrounding tissues and develop its own blood supply
- can be life-threatening and recurrence is more likely

what is metastasis

cancer cells leaving a tumor and invading other parts of the body

what does sequencing a genome mean

working out the DNA base sequence for all the DNA in a cell

what can the genome be used for?

directly sequence proteins that derive from the genetic code which is useful for example to identify antigens in a vaccine

what is taq polymerase

DNA polymerase from bacteria in hot springs that has a high optimum temperature so it doesn't denature at high temperatures of PCR

How are DNA fragments amplified in vivo method (4 points)

- Addition of promotor and terminator regions to the fragments of DNA
- Use of restriction endonucleases and ligases to insert fragments of DNA into vectors
- Transformation of host cell using these vectors
- Use of marker genes to detect genetically modified cells or organisms

different type of marker genes

fluorescent, radioactive or antibiotic resistance

how is a vector inserted into a host cell

the host cell is mixed with Calcium ions and heat shocked to make the membrane more permeable

what is a dna probe

short, single-stranded pieces of DNA that are radioactively or fluorescently labelled so they can be identified

how do DNA probes work

they have complementary base sequences to the allele that is being screened for, so if the patient has the allele, the DNA probe will hybridise and the label indicates the presence

explain the process of DNA hybridisation

- DNA is heated to break hydrogen bonds and make it single stranded
- DNA is mixed with probes then cooled so any complementary sequences can align and bond

what determines a unique fingerprint

variable number tandem repeats (VNTRs) where the probability of two individuals having the same VNTRs is very low

process of genetic fingerprinting

- Collection and extraction (using PCR)
- Restriction endonucleases added to cut DNA into smaller fragments close to the VNTRs
- DNA samples put into small wells in agar gel with a voltage applied
- DNA is negatively charged so DNA samples move through gel towards positive end of gel
- Different lengths of DNA (VNTRs) are separated
- Alkaline added to separate double strands of DNA
- Different DNA probes bind to VNTRs
- VNTRs and DNA probes transferred to nylon sheet
- Sheet exposed to x-rays or UV light and will fluoresce if probes were used

why do different length VNTRs separate in gel electrophoresis

agar gel creates resistance so smaller pieces of DNA can move faster and further along the gel

explain how induced pluripotent cells (iPS) are produced

• obtain adult somatic cells from patient

• add specific transcription factors associated with pluripotency to cells so they express genes

• transcription factors bind to promoter regions of DNA, stimulating or inhibiting transcription

• culture cells to allow them to divide by mitosis

evaluate the use of stem cells in treating human disorders

for:

• can divide and differentiate into required healthy cells, so could relieve human suffering by saving lives and improving quality of life

• embyros are often left over from ivf and so would otherwise be destoryed

• iPS cells unlikely to be rejected by patient’s immune system as made with patient’s own cells

• iPS cells can be made without destruction of embryo

against:

• ethical issues with embryonic stem cells as obtaining them requires destruction of an embryo and potential life

• immune system could reject cells and immunosupprsessant drugs are required

• cells could divide out of control, could cause cancer/tumours