Mutation and Gene Expression

What is a mutation?

A random change in DNA base sequence

Explain how the structure of DNA is related to it’s functions

• Weak hydrogen bonds between nitrogenous bases so easily broken for DNA replication

• Double helix structure wrapped around histones

  • Makes DNA compact so it fits inside of the nucleus

• Stable due to phosphodiester bonds

• Long to store lots of information

• Base sequence allows information to be stored

• Double stranded so replication occurs semi-conservatively

What is a mutagenic agent?

Any factor that causes an increase in the rate of mutations

What is cancer?

Uncontrolled, rapid cell division that results in the formation of a malignant tumour

What is a chromosome mutation?

• Mutations/changes in the number of chromosomes in daughter cells

• Arises spontaneously due to chromosomal non-disjunction

How does the structure of DNA allow for accurate replication?

• Two polynucleotide strands therefore semiconservative replication is possible

• Hydrogen bonds bonds strand together

• Hydrogen bonds are weak so allows strands to separate

• Base sequence exposed so the strands can act as a template

• Adenine pairs with thymine and cytosine with guanine

• New DNA contains one parent strand and one new strand

Describe the behaviour of chromosomes during mitosis and explain how this results in the production of genetically identical daughter cells

• The nuclear envelope breaks down

• Chromosomes condense, become visible and shorten

• Chromosomes are made up of two identical chromatids

• Chromosomes move to the equator of the cell and attach to spindle fibres

• Spindle fibres contract and centromeres divide

• Chromatids move to opposite poles of the cell

• Each pole receives all of the genetic information

• Nuclear envelope forms around each group of chromosomes at each pole

What is a duplication mutation?

• DNA bases are duplicated and repeated

• This increases the number of bases within a gene

What is an inversion mutation?

A section of DNA bases are inverted and read in the opposite direction

What is a translocation mutation?

• Sections of DNA base sequence are removed and added elsewhere

• Often occurs between non-homologous chromosomes but can also happen on the same chromosome

When do mutations normally occur?

During semi-conservative replication

What is a missense mutation?

A mutation where a change in one base causes a change in an amino acid in a polypeptide

What is a nonsense mutation?

A mutation that changes an amino acid-coding codon into a premature stop codon

What is a zygote?

The cell formed when two gametes fuse together

What are stem cells?

Undifferentiated cells that can differentiate into any other type of cell

What does genome mean?

The the complete set of genes within a cell

What does proteome mean?

The full range of proteins a cell can produce

How does differentiation allow cells to become specialised?

• Some genes are ‘switched on’- these genes are transcribed to produce mRNA and then the mRNA is translated to produce specific proteins

• Other genes are switched off- there is no transcription and translation so no proteins are produced

Where do we get totipotent stem cells and what type of cells can they differentiate into?

• Found in embryos/zygotes within the first few cell divisions

• They can differentiate into any other type of cell

Where do we get pluripotent stem cells and what type of cells can they differentiate into?

• Found in the blastocyst (8/16 cell stage of embryos)

• Can differentiate into any cell except for placenta cells

Where do we get multipotent stem cells and what type of cells can they differentiate into?

• Found in the bone marrow of adults and somatic cells of mammals

• Can produce a limited number of different cell types

Where do we get unipotent stem cells and what type of cells can they differentiate into?

• Found in mature mammals inside organs

• Can only differentiate into one type of cell- an example of this is cardiomyocytes (you need to know this example)

What are cardiomyocytes and how are they formed?

• They are specialised heart muscle cells

• They are formed from unipotent stem cells in the heart that have specialised

What are the two fates of stem cells?

• Self-renewal

• Specialisation

What is the use of unipotent stem cells in the heart?

• We can treat diseases or damage to the heart such as heart failure, cardiomyopathy, heart attacks

• These unipotent stem cells found in heart tissue can differentiate into cardiomyocytes to allow heart muscle cells/tissue to regenerate

What are iPS cells?

Induced pluripotent stem cells

How are iPS cells formed?

• Multipotent stem cells are extracted from adults

• Cells are reprogrammed to become pluripotent

• The multipotent stem cells are infected with a virus that introduces genes into the cell

• When the genes are integrated into the cell’s DNA, the proteome of the cell is altered

• The cells begins to transcribe and translate different sets of genes

  • Regulatory genes coding for transcription factors are transcribed and the mRNA translated, which then means we can induce a cells to differentiate into any other type of cells by controlling what genes are expressed and which ones are not

What are some ethical issues about using embryonic totipotent stem cells?

• Obtaining stem cells from discarded embryos is seen as unethical by some as they believe an embryo to have the right to life at fertilisation

• Creating embryos by IVF just to destroy them for stem cells can be seen as unethical, immoral and is illegal in some countries

• Some people believe we should only be using adult stem cells as it avoids the destruction of an embryo

  • This is an issue though because adult stem cells can only differentiate into a smaller range of other cells

• There are fewer objections to stem cells being obtained from egg cells that haven’t been fertilised from sperm but have been artificially activated to start dividing

  • This is because the cells wouldn’t survive past a few days if implanted in the womb

  • Some people argue however that improving the quality of life for patients is more important than these factors

What are the advantages of iPS cells? (May need to add to this one)

• Ethical because it avoids destroying an embryo

• Less likely to be rejected and destroyed by the immune system

  • This is because the multipotent stem cells that iPS cells made from come from the person to be treated, so they’ll have the same antigens as any other body cell so an immune response isn’t triggered

Why is it important the agar growth medium in tissue culture is sterile?

• Prevents microbial growth

• No competition for resources like glucose

• Prevents disease which may kill the tissue being grown

What are the advantages of tissue culture in agriculture?

• All crops are genetically identical

• Same yield

• Same size for harvesting

• No sexual reproduction so can cultivate sterile crops

• All adapted to the same conditions

What are the disadvantages of tissue culture in agriculture?

• Reduced genetic diversity

• All susceptible to the same pathogens

What does a gene being expressed mean?

• A gene is ‘switched on’

• This means the gene can code for/transcribed into mRNA which then is translated to produce a protein

Essentially gene expression is the production of a protein coded for by a gene

What are the ways we can control transcription?

• Blocking the promoter region to RNA polymerase cannot bind to it to begin transcription

• Inhibiting RNA polymerase

• Inhibit enzymes involved in splicing

• Methylation and acetylation in epigentics

What are the ways we can control translation?

mRNA broken down by enzymes

How do transcription factors work in general?

• They are proteins found in the cytoplasm

• They move into the nucleus before transcription

• RNA polymerase and transcription factors both bind to the promoter region to form a translation initiation complex

• Transcription factors can either act as activators or repressors of transcription

What is the name of genes that are code for transcription factors?

Regulatory genes

How do activator transcription factors work?

• The stimulate and increase the rate of transcription

  • They can bind upstream of the gene (at the promoter region)

• They help RNA polymerase to bind to the start of the gene and activate transcription, increasing the rate of transcription

How do repressor transctiption factors work?

• Inhibits or decreases the rate of transcription

• They bind to the promoter region of a target gene and prevents RNA polymerase from binding (by blocking it)

• This prevents transcription

Why do transcription factors remain in the cytoplasm until they are needed for the regulation of transcription?

• They are kept in the cytoplasm to prevent over-transcription

• Uncontrolled transcription could be harmful if transcription factors stimulate an increased rate of transcription of proto-oncogenes which could lead to cancer

How can oestrogen regulate the rate of transciption?

• Oestrogen is a lipid soluble, steroid hormone

• It binds to a oestrogen receptor on a transcription factor

What are some circumstances that may lead to an organism being exposed to increased oestrogen levels?

• Female mammals are going to always have higher oestrogen levels than male mammals

• IVF

• Pregnancy

• Hormone replacement therapy (HRT)

• Contraception pills

How does oestrogen enter a cell and why?

• Oestrogen can enter a cell through the phospholipid bilayer by simple diffusion

• This is because oestrogen is a lipid soluble steroid hormone so it is not repelled by the hydrophobic fatty acid tails of the phospholipid bilayer

How can oestrogen regulated the rate of transcription of genes?

• Oestrogen enters a cell by simple diffusion due to being a lipid soluble steroid hormone

• Oestrogen then binds to a oestrogen receptor on a transcription factor forming an oestrogen-oestrogen complex

• The oestrogen-oestrogen complex then moves into the nucleus

• The complex will then bind to the promoter region of a gene and can act as an activator, helping RNA polymerase to bind or a repressor, blocking RNA polymerase from binding to the promoter region

Why does oestrogen act in some cells and not others?

• Some cells do not have transcription factors with oestrogen receptors

• Some cells contain enzymes that will hydrolyse oestrogen

How may oestrogen stimulate the growth of tumours/cancer when binding to transcription factors?

• Oestrogen can act as a repressor to block the promoter region of DNA from RNA polymerase, decreasing the rate of transcription

• If this occurs in a tumour suppressor gene, proteins that inhibit the cell cycles are not coded for so there is no inhibition of the cell cylce

• This can lead to uncontrolled cell division (cancer)

What is a nucleosome?

This consists of DNA wrapped around 8 histone molecules

What is epigenetics?

• Heritable changes in gene function without changes to the base sequence of DNA caused by the environment

• These changes are a result of environmental factors, not mutations

• This occurs by the methylation of DNA or the acetylation of histones

How does methylation affect the transcription of DNA?

• Methyl groups bind to a CpG site of the DNA base sequence

  • This is where a cytosine and guanine liked by a phosphate bond are adjacent in the DNA

• Increased methylation changes the DNA structure to make it more compact so RNA polymerase and transcription factors cannot bind to the promoter region as it is less accessible

• Transcription of the gene cannot take place, so the gene is not expressed

• The enzyme methyl transferase attaches the methyl group to the cytosine

How does acetylation affect the transcription of DNA?

• Addition of acetyl groups to histones causes the histones to pack less tightly together

• RNA polymerase and transcription factors are then able to access the promoter region and a gene is transcribed

• Histone acetyl transferase adds acetyl groups to histones

How does acetylation reduce the tight packing of DNA around histones?

• Histone tails have a positive charge

• DNA nucleotides are negatively charged

• Acetyl groups are negatively charged

• They neutralise the histone charge so DNA is no longer strongly attracted to the histone

What affect does demethylation of DNA have on transcription?

• When methyl groups are removed, the structure of DNA changes to be less compact so that RNA polymerase and transcription factors are more able to bind to the promoter region of DNA

• This increases the rate of transcription

What effect does deacetylation of histones have on transcription?

• When acetyl groups are removed from histone molecules, DNA is made more compact as the positive charge on the histone is now no longer neutralised meaning it is more attracted to the negatively charged DNA bases

• RNA polymerase and transcription factors are less able to bind to the promoter region of DNA as it is less accessible meaning the rate of transcription is reduced

What is RNAi?

• In eukaryotes, gene expression is also affected by RNA interference (RNAi)

• This is where small, double stranded RNA molecules stop mRNA from target genes being translated into proteins

  • A similar process to RNAi can also occur in prokaryotes

• The molecules involved are called siRNA (small interfering RNA) and miRNA (micro-interfering RNA)

Outline how siRNA regulates transcription

• mRNA is made double stranded by the enzyme RNA-dependent RNA polymerase by joining nucleotides together to make a new complementary strand with the original strand

  • The double stranded/dsRNA is made from the mRNA fragment to be hydrolysed

• The double stranded RNA is cleaved by the enzyme dicer, forming siRNA that is complimentary to the mRNA that is to be destroyed

• siRNA associates with RISC (the RNA-induced silencing complex)

• The original strand is degraded/broken down/hydrolysed to expose the bases on the RISC-siRNA strand to allow for complimentary base pairing with the mRNA to be destroyed

• The RISC-siRNA strands bind to the complimentary strand of mRNA and the RISC protein hydrolyses mRNA into fragments so it can no longer be translated

• The fragments then move into a processing body which contains tools to degrade them

How does miRNA work and how does it differ from siRNA?

• miRNA is shorter than siRNA

• miRNA is not fully complimentary to the target mRNA

• miRNA is less specific than miRNA and therefore may target more than one mRNA molecule

  • This is because the miRNA has a shorter base sequence meaning that it is more likely that you will have a molecules of miRNA that are complimentary to multiple strands than a longer strand of siRNA which, because its longer, is specific to one strand

• Its produced in the same way and associates with proteins

• Blocks the translation of mRNA rather than hydrolysing it

• mRNA is degraded in the processing body or stored for transcription later

What is a proto-oncogene and how do they work?

• A gene that codes for proteins that increase the rate of cell division

  • These proteins are growth factors

How can RNAi lead to prevent cancer?

• si/miRNA can destroy mRNA fragments that could produce proteins from proto-oncogenes

• If no proteins that increase the rate of cell division are produced then cell division is not increased

What is a malignant tumour?

• Less compact tumours that grow rapidly and metastasize to other parts of the body by travelling through the bloodstream of lymphatic system

• They are more likely to be life threatening

What is a benign tumour?

• More compact tumours that grow very slowly

• Not cancerous and less likely to be life threatening

• Usually enclosed in fibrous tissue that stops the invasion of other tissues

• These are often harmless but can cause blockages or put pressure on organs

• Some benign tumours can become malignant

When is a tumour classed as cancerous?

When it invades other tissues (metastasizes)

What is a tumour?

An abnormal mass of cells

How do tumour suppressor genes work?

• These genes produce a protein that slows cell division or causes a cell to self-destruct (apoptosis)

How can base sequence alteration in a tumour suppressor gene lead to cancer?

• If a mutation occurs in a tumour suppressor gene, it can affect the sequence and order of amino acids, changing the tertiary structure of the protein, making it non-functional

• Cells then divide uncontrollably, resulting in a tumour

How can abnormal deacetylation of histones with tumour suppressor genes wrapped around then lead to cancer?

• Removal of acetyl groups from histones means that the DNA is more tightly packed around the histone so the promoter region of the tumour suppressor gene is less accessible to RNA polymerase and transcription factors

• This decreases the rate of transcription of tumour suppressor genes so cells divide uncontrollably, resulting in a tumour

How can abnormal methylation of tumour suppressor genes lead to cancer?

• Methyl groups bind to the CpG sites of DNA making it more compact

• This makes the promoter region of the tumour suppressor gene less accessible so RNA polymerase and transcription factors are less able to bind

• The rate of transcription decreases

How may a base sequence mutation of proto-oncogenes lead to cancer?

• A base mutation in a proto-oncogene may cause it to become overactive (too much transcription of the gene)- the mutated gene is called an oncogene

• Or lots of protein is produced causing the cells to divide uncontrollably by mitosis and form a tumour

• This is due to the alteration of the primary and therefore tertiary structure of the protein

How may abnormal acetylation of proto-oncogenes lead to cancer?

• More acetyl groups bind to the histones with proto-oncogenes

• The gene is less tightly wound around histones meaning the promoter region of the gene is more accessible

• RNA polymerase and transcription factors bind more easily to the promoter region so the rate of transcription increases

• More growth factor proteins are produced which leads to uncontrolled cell division

How may abnormal demethylation of proto-oncogenes lead to cancer?

• Methyl groups are removed from CpG sites

• The DNA becomes less compact so the promoter region is made more accessible to RNA polymerase and transcription factors

• This increases the rate of transcription

• More growth factor proteins are produced which leads to uncontrolled cell division

How may oestrogen cause breast cancers to grow?

• Oestrogen may stimulate cells to replicate

  • As more cells divide there is a greater chance of mutations occurring meaning there is an increased chance of a mutation occurring in a tumour suppressor or proto-oncogene (to form an oncogene) that can lead to cells becoming cancerous

• Stimulate rapid replication if the cells do become cancerous

• Introduce mutations directly into the DNA of certain breast cells

How oestrogen and alc can lead to cancer- extra research, do at a later date

What are BRCA genes and how would a mutation in these genes lead to breast cancer?

• BRCA genes are breast cancer genes- everyone has these genes

  • They are cancer suppressor genes

• BRCA 1 and 2 mutations can cause a tumour suppressor gene to be altered if a mutation occurs within them, coding for a non-functional protein

• Certain alleles can increase the risk of breast cancer

BRACA genes are a genetic factor that increase the risk of breast cancer. What preventative measure can be taken to prevent breast cancer?

• People can be scanned for mutations in their BRCA genes

• If there are mutations, people may have their breasts removed (mastectomy) to prevent the potential metastasization of a breast tumour

Why is developing a drug against mutated tumour suppressor genes difficult?

• Mutated tumour suppressor genes are transcribed

• The protein produced in translation doesn’t function

• A drug which targets a mutated tumour suppressor gene would have to restore the function of a tumour suppressor protein which is difficult

What is the one-hit hypothesis of cancer?

• If one allele of a tumour suppressor gene is mutated, the cell cannot produce enough functional protein in order to regulate cell division

• As a result, the cell begins to divide uncontrollably even though there is some inhibition by the suppressor proteins, leading to cancer

• In proto-oncogenes, when they are mutated to form oncogenes, only one allele needs to be mutated in order for cell division to begin to be uncontrolled

What is the two hit hypothesis of cancer?

• If a mutation occurs in just one allele of a tumour suppressor gene, the cell can still make enough functional protein in order to regulate cell division

• However, if both become mutated, the cell does not produce any functional protein and therefore the cell divides uncontrollably

• In proto-oncogenes, only one mutation is needed to produce an oncogene that codes for a protein that causes uncontrolled cell division

According to the two-hit hypothesis, why is cancer more likely to develop with age?

• Natural mutation rates are slow so it takes considerable time for both tumour suppressor genes to mutate so people are diagnosed with cancer later in life

• Its though that some people are born with one mutated allele of a tumour suppressor gene, so only one more mutation is needed for them to develop cancer and they are therefore at greater risk and may develop it earlier so some cancers carry an inherited increased risk

What is a mutated proto-oncogene called?

An oncogene