The control of gene expression

What is base addition?

One or more nucleotides is added

What is base deletion?

One or more nucleotides is deleted

What is base substitution?

A nucleotide is replaced with another base

What is duplication?

One or more bases is repeated

What is inversion?

A sequence of bases is reversed

What is translocation?

A sequence of bases is moved from one location in the genome to another. This could be movement within the same chromosome or movement to a different chromosome

What is a frameshift?

When the mutation affects the whole of the rest of the code

What are mutagenic agents?

Things that increase the rate of mutations

Describe acting as a base

Chemicals called base analogs can substitute for a base during DNA replication, changing the base sequence in the new DNA

Describe altering bases

Some chemicals can delete, or alter, bases

Describe changing the structure of DNA

Some types of radiation can change the structure of DNA, which causes problems during DNA replication

What are stem cells?

Unspecialised cells that can develop into other types of cell

Describe totipotent stem cells

Cells that can mature into any type of body cell, occur only for a limited time in early mammalian embryos

Describe pluripotent stem cells

Cells that can mature into any type of body cell (except placental cells), found in mammalian embryos

Describe multipotent stem cells

Can divide to form a limited number of different cell types, found in mature mammals, e.g. bone marrow

Describe unipotent stem cells

Can only differentiate into one type of cell, found in mature mammals, e.g. cardiomyocytes

How do stem cells become specialised?

- Stem cells all contain the same genes - but during development not all of them are transcribed and translated
- Under different conditions, certain genes are switched on or off
- Genes that are expressed get transcribed into mRNA, which is then translated into proteins
- These proteins modify the cell - they determine the cell structure and control cell processes
- Changes to the cell produced by these proteins cause the cell to become specialised

How do bone marrow transplants work?

- Bone marrow contains stem cells that can become specialised to form any type of blood cell
- These can replace faulty bone marrow in patients that produce abnormal blood clots
- Can be used to treat leukaemia and lymphoma

Describe stem cell therapies of the future

- Spinal cord injuries
- Heart disease and damage caused by heart attacks
- Bladder conditions
- Respiratory diseases
- Organ transplants

Describe adult stem cells

- Obtained from body tissues of an adult
- Can be obtained from bone marrow in a relatively easy operation
- Not as flexible as embryonic stem cells - can only specialise into a limited range of cells, not all body cell types (multipotent)

Describe embryonic stem cells

- Obtained from early stage embryos
- Embryos created in a lab using IVF
- At 4-5 days old, stem cells removed and the rest is destroyed
- Divide unlimited times and develop into all types of body cell (pluripotent)

Describe induced pluripotent stem cells (iPS cells)

- iPS cells are created by scientists in the lab
- Process involves 'reprogramming' specialised adult body cells so that they become pluripotent
- Adult cells are made to express a series of transcription factors that are normally associated with pluripotent, causing adult cells to express genes associated with pluripotency
- This can be done by injecting a virus

What are the ethical considerations of using stem cells?

- Obtaining stem cells from embryos by IVF raises issues as the embryos could develop into a foetus
- Some think they should only use adult cells
- Less objections to unfertilised cells - only survive a few days
- iPS cells are body's own, body would not reject

What are the benefits of stem cell therapy?

- Could save many lives - e.g. people waiting for transplants
- Might be possible to make stem cells identical to patient's own cells - body would not reject
- Could improve quality of life - e.g. replace damaged cells in eyes of blind people

What are transcription factors and what do they do?

Transcription factors bind to promoters near the start of target genes, they control expression by controlling rate of transcription

What do activators do? Give an example

Stimulate or increase rate of transcription, e.g. help RNA polymerase to bind to target gene

What do repressors do? Give an example

Inhibit or decrease rate of transcription, e.g. bind to target gene, preventing RNA polymerase from binding

What is oestrogen?

Steroid hormone

How does oestrogen act as an activator?

1. Oestrogen binds to transcription factor, forming oestrogen-oestrogen receptor complex
2. Complex moves from cytoplasm into nucleus where it binds to promoter near start of target gene

What is RNAi and what does it do?

RNA interference, small lengths of non-coding RNA, RNAi stops mRNA from target genes being translated into proteins

What is siRNA?

Short interfering RNA

How does siRNA interfere with gene expression?

1. Double-stranded siRNA associates with several proteins and unwinds

2. One of the single strands is selected and the other strand is degraded

3. Single strand binds to target mRNA, base sequence of siRNA is complementary to base sequence in sections of target mRNA

4. Proteins associated with siRNA cutes mRNA into fragments so it can no longer be translated

5. Fragments move into a processing body, which contains 'tools' to degrade them

What is miRNA?

MicroRNA

Why is miRNA less specific than siRNA?

miRNA isn't fully complementary to target mRNA, s o it may target more than one mRNA molecule

How does miRNA interfere with gene expression in mammals?

1. When miRNA is first transcribed, it is a long, folded strand processed into a double strand, then 2 single strands by enzymes in cytoplasm

2. One strand associates with proteins and binds to target mRNA

3. miRNA-protein complex physically blocks translation of target mRNA

4. mRNA is moved to a processing body, where it can be stored or degraded. When it's stored, it can be returned and translated at another time

What are acquired mutations?

Mutations that occur in individual cells after fertilisation

What causes tumours?

When mutations occur in genes that control rate of mitosis, causing uncontrolled cell division

What are cancers?

Tumours that invade and destroy surrounding tissue

Name 2 genes that control cell division

Tumour suppressor genes and proto-oncogenes

What happens when tumour suppressor genes are functioning normally?

They slow cell division by producing proteins that stop cell division or cause apoptosis

What mutation occurs in tumour suppressor genes and what does it cause?

Hypermethylation means genes are not transcribed by proteins which slow cell division are not translated, so cells are able to divide uncontrollably

What happens when proton-oncogenes are functioning normally?

They stimulate cell division by producing proteins that make cells divide

What mutation occurs in proton-oncogenes and what does it cause?

Hypomethylation causes them to act as oncogenes, increasing production of proteins that encourage cell division, so cells are able to divide uncontrollably

What is hypermethylation?

When too many methyl groups added

What is hypomethylation?

When too few methyl groups are added

Describe the features of malignant tumours

- Cancerous
- Grow rapidly and invade and destroy surrounding tissues
- Cells can break off the tumours and spread to other parts of body in bloodstream or lymphatic system

Describe the features of benign tumours

- Not cancerous
- Grow slower and often covered in fibrous tissue that stops cells invading other tissues
- Often harmless, but can cause blockages and put pressure on organs
- Can become malignant

Describe the features of tumour cells

- Nucleus is larger than normal, may have more than one nucleus
- Have an irregular shape
- Don't produce all the proteins needed to function correctly
- Different antigens on surface
- Don't respond to growth regulating processes
- Divide by mitosis more frequently

Why does increased exposure to oestrogen over an extended period of time increase risk of breast cancer?

- Oestrogen stimulates certain breast cells to divide and replicate, increasing risk of mutations
- If cells become cancerous, oestrogen stimulates replication of these cells, causing tumours to develop quickly
- Research suggests that oestrogen is able to introduce mutations directly into DNA of certain breast cells, again increasing risk of cells becoming cancerous

What is epigenetics?

Involves heritable changes in gene function without changes to the base sequence of DNA

Name 2 epigenetic mechanisms

Methylation of DNA and acetylation of histones

How does methylation of DNA alter gene expression?

- Methyl group (CH3) is added to cytosine at a CpG site (where C and G are next to each other)
- Increased methylation changes DNA structure - blocking transcription if this happens in a promoter region
- Added by methyltransferases

How does acetylation of histones alter gene expression?

- When an acetyl group (COCH3) is added to histone
- Causes chromatin to be less condensed, allowing genes to be transcribed
- Added by acetyltransferases
- When acetyl groups are removed, chromatin becomes highly condensed and genes can't be transcribed
- Histone deacetylase enzymes remove acetyl groups

How can epigenetics be used to treat disease?

- Epigenetic changes are reversible
- HDAC inhibitor drugs (e.g. romidepsin) inhibit HDACs

What is the problem with using epigenetics to treat disease?

These changes take place normally in a lot of cells, so drugs have to be as specific as possible

What is a genome?

Complete set of DNA in an organism

What is a proteome?

Complete set of proteins made by organism

What must be done before genomes can be sequenced?

DNA must be split into smaller fragments as gene sequencing methods only work on fragments

Why is it relatively easy to determine the proteome of simple organisms?

They don't have much non-coding DNA

Give an example of when it is useful to know the proteome of simple organisms?

Identifying antigens on bacteria to develop vaccines

Why is it more difficult to determine the proteome of more complex organisms?

They contain large sections of non-coding DNA and complex regulatory genes, which makes it more difficult to translate genome into proteome

How have sequencing methods become more specialised?

The techniques are often automated, more cost-effective and can be done on a large-scale, pyrosequencing can sequence 400 million bases in 10 hours

What does recombinant DNA technology involve?

Transferring a fragment of DNA from one organism to another

Why can transferred DNA be used to produce a protein in cells of recipient organism?

Because genetic code is universal and transcription and translation mechanisms are similar

What are transgenic organisms?

Organisms that contain transferred DNA

Why is mRNA often easier to obtain than DNA?

Because the cells that produce the protein coded for by target gene will contain complementary mRNA

How is reverse transcriptase used to make DNA fragments?

1. mRNA is isolated from cells

2. mRNA is mixed with free DNA nucleotides and reverse transcriptase

3. Reverse transcriptase uses mRNA as a template to synthesise new strands of complementary DNA (cDNA)

What are palindromic sequences of nucleotides?

Antiparallel base pairs

What do restriction endonucleases do?

Recognise specific palindromic sequences (recognition sequences) and cut (digest) DNA at these places

Why do different restriction endonucleases cut at different specific recognition sequences?

The shape of recognition sequence is complementary to enzyme's active site

How are restriction endonucleases used to make DNA fragments?

1. DNA sample is incubated with specific restriction endonuclease, which cuts DNA fragment out via hydrolysis

2. Sometimes the cut leaves sticky ends - small tails of unpaired bases at each end of fragment

3. Sticky ends can be used to bind (anneal) DNA fragment to another pieces of DNA that has sticky ends with complementary sequences

How is a 'gene machine' used to make DNA fragments?

1. Sequence that is required is designed (if one doesn't already exist)

2. The first nucleotide in the sequence is fixed to some sort of support, e.g. a bead

3. Nucleotides are added step by step in the correct order, in a cycle of processes that includes adding protecting groups

4. Short sections of DNA called oligonucleotides (roughly 20 nucleotides long) are produced

5. Once these are complete, they are broken off from support and protecting groups removed

6. Oligonucleotides can then be joined together to make longer DNA fragments

What are protecting groups?

The make sure that the nucleotides are joined at the right points, to prevent unwanted branching

Define gene cloning

Making lots of identical copies of a gene

Define in vivo cloning

Where gene copies are made within a living organism

Define in vitro cloning

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

Describe the process of in vivo cloning

1. DNA fragment of interest from gene using same restriction endonuclease

2. Same enzyme used to cut vector DNA (plasmid), to ensure they are complementary

3. Fragment DNA inserted into vector DNA, aided by ligase (ligation)

4. Marker gene also added to vector DNA, to allow identification of transformed cells, e.g. antibiotic resistance or fluorescence

5. Vector inserted into host cell, e.g. bacteria

6. Host multiplies, producing many copies of cloned gene

7. To produce proteins, they must contain specific promoter and terminator regions

Name 2 ways of using marker genes

Antibiotic resistance marker genes or fluorescent marker genes

Describe the process of in vitro cloning (PCR)

1. Reaction mixture containing DNA sample, free nucleotides, primers and DNA polymerase is set up

2. Mixture heated to 95c to break H-bonds between strands and cooled to 50-65c so primers can bind (anneal) to strand

3. Mixture heated to 72c so DNA polymerase can line up free nucleotides alongside each template strand, new complementary strands formed

4. Two new copies of fragment of DNA are formed and one cycle of PCR is complete, cycle starts again

What does PCR stand for?

Polymerase chain reaction

What are primers?

Short pieces of DNA that are complementary to bases at start of fragment you want

What is the equation for finding number of double strands?

2^n (n \= number of cycles)

What is the equation for finding number of single strands?

2 x 2^n (n \= number of cycles)

How does genetic engineering work?

Using recombinant DNA technology

How are transformed plants produced?

1. Gene that codes for desirable protein is inserted into plasmid

2. Plasmid is added to bacterium (vector) and inserted into plant cells

3. If right promoter region has been added, transformed cells will produce desired protein

How are transformed animals produced?

1. Gene that codes for desirable protein inserted into an early embryo or into egg cells of female

2. Promoter regions that are only activated in specific cell types can be used to control exactly which cells the protein is produced in

3. If protein is only produced in certain cells, it can be harvested more easily, producing protein in wrong cells can damage the organism

What are the benefits of transformed organisms?

- Agricultural crops can be transformed so they give higher yields or are more nutritious - reduces famine and malnutrition

- Crops can be transformed to have resistance to pests or droughts

- Enzymes can be produced from transformed organisms, so can be produced in large quantities for less money

- Drugs, e.g. insulin, can be produced quickly, cheaply and in large quantities

What are the concerns about transformed organisms?

- Monoculture makes whole crop vulnerable to disease, and reduces diversity

- 'Superweeds' that are resistant to herbicides

- Contamination f crops from GM crops

- Introduction of toxins into food industry

- Forcing smaller companies out of business

- 'Unethical' designer babies

What are promoter regions?

Tell RNA polymerase where to start

What are terminator regions?

Tell RNA polymerase where to stop

How are transformed microorganisms produced?

In vivo cloning

How can gene therapy be used to treat a disease caused by 2 recessive alleles?

Add a dominant allele

How can gene therapy be used to treat a disease caused by a dominant allele?

You can 'silence' an allele by inserting a fragment of DNA into the middle of the allele

What are liposomes?

Spheres made of lipid

Describe somatic gene therapy

- Altering alleles in body cells, particularly cells most affected
- Does not affect sex cells, so offspring could still inherit disease

Describe germ line gene therapy

- Altering alleles in sex cells
- Illegal
- Every cell of any offspring will be affected so won't suffer from disease

What are the ethical issues surrounding gene therapy?

- Some people are worried that the technology could be used in other ways, e.g. treating cosmetic effects of ageing
- Potential to do more harm than good (e.g. risk of over expression of genes)

What are DNA probes used for?

To locate specific alleles or to see if a person's DNA contains a mutated allele

What are DNA probes?

Short strands of DNA with complementary base sequence to target allele so it will hybridise to target allele, label attached so it can be detected

Describe the process used when DNA probes are used to detect genes

1. Sample of DNA digested into fragments using restriction enzymes and separated using electrophoresis

2. Separated DNA fragments transferred to nylon membrane and incubated with fluorescently labelled DNA probe, if allele is present, DNA probe will hybridise with it

3. Membrane exposed to UV light and if gene is present, there will be a fluorescent band