21- Gene expression

What is transcription?

Takes place in the nucleus of the cell and involves the formation of pre-mRNA which has a complementary sequence of bases to the DNA

A gene is made up of 756 base pairs, the mRNA transcribed from this gene is only 524 nucleotides long. Explain the difference.

Pre-mRNA is spliced to remove introns and leave only the coding exons which are used for translation

Compare DNA and RNA

DNA:

• Double polynucleotide chain

• Largest molecule of the 3

• Double helix shape

• Pentose sugar is deoxyribose

• Bases found are A,T,C +G

• Found in nucleus of cell

• Chemically stable

RNA:

• Single polynucleotide chain

• Smaller than DNA, bigger than tRNA

• Single linear strand

• Ribose pentose sugar

• Bases found are A,U,C +G

• Found in nucleus + cytoplasm

• Unstable

What are the 2 stages where gene expression can be controlled?

1. Transcription

2. Translation

Explain the regulation of transcription in eukaryotes

The general process involves in stimulation og gene transcription is as followed:

1. Transcriptional factors are found in cytoplasm of a cell

2. They move into nucleus

3. Each transcriptional factor has a site that binds to a specific base sequence of the DNA- the promotor region

4. Binding of the transcriptional factor to the promotor region allows attachment of RNA polymerase to the DNA and transcription is stimulated

5. mRNA is produced and subsequently translated into a polypeptide

NOTE: Transcriptional factors can also inhibit transcription

What is oestrogen?

A lipid- soluble, steroid hormone released into the bloodstream

How does oestrogen enter a cell?

Simple diffusion

How does oestrogen active a gene and inhibit transcription?

1. Oestrogen enters cytoplasm by simple diffusion

2. Binds to a specific transcriptional factor (sometimes called a receptor) in the cytoplasm, causing the transcriptional factor to change shape

3. This causes an inhibitor molecule to be released which exposes DNA binding site on the transcriptional factor

4. The transcriptional factor now moves into nucleus and binds to a specific sequence of bases on the promotor region, enabling the RNA polymerase to bind and initiate transcription

What is the function of the inhibitor molecule for oestrogens affect on gene transcription?

Prevents the transcriptional factor binding to DNA protomor region and initiating transcription and therefore synthesis of protein when a protein is not required

Explain regulation of translation (effect of RNA interference- RNAi- on gene expression)

In cytoplasm there are small double stranded sections of RNA called interfereing RNA (RNAi). These inhibit gene expression at the translation stage in a process called RNA interference

1. Double stranded RNAi is coded for by special regulatory genes

2. It moves into cytoplasm where it becomes single stranded and associates with nuclease enzyme

3. Binds to specific mRNA molecules with a complementary base sequence by complementary base pairing

4. The enzyme cuts mRNA in 2

5. mRNA can no longer be translatedso protein synthesis stops

Suggest when regulation of translation might be important in a cell

• If mRNA concentration is regulated it will limit the amount of protein synthesised- save energy/ be more efficient

• Stops the translation of viral RNA in a cell infected by a virus

What are some applications of RNAi in scientific research and medicine?

Used to identify role of genes in a biological pathway

• A specific seequence of RNAi which binds to mRNA transcribed from a particular gene is added to cells

• This prevents translation of that gene so protein is not produced. By observing the effect of lack of protein, the role of the blocked gene can be determined

Prevention of a genetic condition

• May be possible to use RNAi to prevent faulty protein from being produced (stops translation) and so prevent a condition which is caused by faulty genes

Why are all cells in an organism genetically identical?

All produced by mitosis following fertilisation of an egg cell

Tell me about cel differentiation and specialisation

During development, cells differentiate to become specialised cells because expression of genes is regulated

• Although all cells contain all genes, only specific genes are expressed in specific cells at any one time

Some genes whicih code for enzymes are expressed in all cells. Suggest which processes these enzymes might be involved in

• Respiration

• Transcription

• Translation

What are the 2 main featuress of stem cells?

• Undifferentiated but can differentiate into specialised cells

• Can replace themselves (to form more stem cells)

> When a stem cell divides, each new cell has the potential wither to remain a stem cell or to differentiate to become a specialised cell

What are the 4 types of stem cell?

1. Totipotent cells

2. Pluripotent cells

3. Multipotent cells

4. Unipotent cells

What are totipotent cells?

• Occur in a limited time in early mammalian embryos

• Can differentiate to produce any type of body cell, including placental cells

What are pluripotent cells?

• Found in embryos

• Can differentiate into all tissue types, except placental cells

What are multipotent cells?

• Found in many tissues of mature mammals

• Can differentiate to form a limited number of different cell types

e.g. stem cells in bone marrow can produce any type of blood cell

What are unipotent cells?

• Also founf in mature mammals

• Can only differentiate into 1 type of cell/ tissue

e.g. unipotent cells in the heart produce new cardiomyocytes- specialised cardiac muscle cells

What are induced pluripotent stem cells (iPS)?

• Pluripotent stem cells produced from differentiated adult body cells

• Specific protein transcriptional factors are used to ‘reprogramme’ the body cells

• iPS cells are able to divide to produce new iPS cells or differentiate into any type of body cell

• They could replace the use of embryonic stem cells in medical research and treatments

What are the potential uses of stem cells in treating human disorders?

• Stem cells are grown in culture in a laboratory

• Certain chemical stimuli are added which trigger the cells to differentiate into specialised body cells of a particular type

• These can then be used to replace damaged cells or tissues in a patient

What type of cell will be needed to treat the following disorders:

• Leukaemia

• Burns and wounds

• Osteoporosis

• Type 1 diabetes

• Heart damage caused by a heart attack

• Macular degeneration

• Blood cells

• Skin cells

• Bone cells

• B cells of the pancreas

• Cardiac muscle cells

• Retinal cells of the eye

> These cells could be produced from pluripotent embryonic stem cells or multipotent adult stem cells

What are the advantages of using pluripotent stem cells for treating human disorders?

• Can differentiate into any type of body cell

What are the disadvantages of using pluripotent stem cells for treating human disorders?

• Risk of rejection because they are not the patient’s own cells

• Ethical issues because sourced from embryos

What are the advantages of using multipotent stem cells for treating human disorders?

• No transplant rejection if stem cell is obtained from the patient

What are the disadvantages of using multipotent stem cells for treating human disorders?

• Can only differentiate into a limited number of cell types

Why do people use embryonic stem cells?

Easier to manipulate and acquire however are controversial

What are some ethical arguments for the use of embryonic stem cells?

• Some people argue that an embryo at such an early stage of development is just a ball of identical, undifferentiated cells, bearing no resemblence to a human being

• It is wrong to allow human suffering to continue when there is a possibility of alleviating it

• It makes no sense to destroy spare embryos from IVF fertility treatment that are not implanted or for them to just be stored indefinitely. As long as the prospective parents give their consent, they could be used for medical research

What are some ethical arguments against the use of embryonic stem cells?

• Some people feel that using embryos in this way undermines our respect for human life and could progress to the use of foetuses, and even new-born babies for research or the treatment of disease

• It is a move towards reproductive cloning and, even if this remains illegal in the UK, information gained could be used to clone humans elsewhere

• Embryos have the potential for life, so it is unethical to manipulate or destroy them

• It is wrong to use humans, including human embryos, as a means to an end, even if that end is the commendable one of alleviating human suffering

• The embryos themselves are unable to give consent. If adult stem cells were used instead, willing donors can be found, or they could even be obtained from the patient’s own tissues

Tell me about plant stem cells

• Mature plants have many totipotent cells- under correct conditions, these can differentiate into any other cell

e.g. A leaf develop into a whole plant or a cell from a carrot root placed on nutrient medium and develop into a new carrot plant

> These new plants will be genetically identical to one they came from

Define epigenetics

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

What is epigenetics?

• Phenotype of an organism is primarily determined by its genotype, but the environment can also influence phenotype

• Changes in environment can directly affect gene expression in eukaryotes without altering base sequence. The changes in gene function affect the phenotype and can be inherited

What is the epigenome?

• All the chemical tags which have been added to a person’s genome

> DNA is wrapped around histone proteins in nucleus to produce chromatin

>DNA and histones have chemical tags attached to them

> Chemical tags are either an acetyl group or a methyl group

> Environmental factors such as diet, stress and toxins can affect number of these groups attached to the genome

Describe acetylation of histones

• Acetyl groups bind to histone proteins

• This causes DNA to be less tightly wrapped around the histones- reduces condensation of chromatin

• This makes promotor regions accessible

• Transcriptional factors can bind to promotor regions and initiate transcription

• Genes are switched on

Histone proteins are positively charged. Does the addition of an acetyl group increase or decrease the positive charge?

Decrease- DNA is negatively charger due to phosphate groups so is attracted to positive histones. Addition of an acetyl group decreases positive charge so DNA is less attracted

What might be a potential source of acetyl groups?

Transferred from acetylcoenzyme A

Describe decreased acetylation

• Acetyl groups are removed from histone proteins

• This causes DNA to be more tightly wrapped around the histones which increases condensation of chromatin

• This makes promotor regions inaccessible

• Transcriptional factors cannot bind to the promotor regions to initiate transcription

• Genes are switched off

Describe methylation of DNA

• Methyl groups added to cytosine bases in DNA

• This results in decreased acetylation of histones

• DNA wraps more tightly around histones- chromatin becomes more condensed

• Promotor regions become inaccessible to transcriptional factors

• Transcription inhibited

• Genes are switched off

Explain how epigenetic changes caused by the environmental influences can be inherited using an example (Starvation experiments)

• If pregnant mice are starved, the resulting offspring (F1) have a higher risk of developing metabolic diseases like diabetes due to epigenetic changes in the offspring during gestation

• This effect could still be seen in the second generation (F2), even though both F1 and F2 offspring were well-fed

• The epigenetic changes acquired by F1 generation during gestation were passed on to its offspring

What is cancer?

Results from mutations in genes that control cell division, causing rapid. uncontrolled growth and division of cells by mitosis. This results in the formation of a mass of abnormal cells (tumour)- not all tumours are cancerous

What is a benign tumour?

• A mass of cells that has not spread into neighbouring tissues

• Location of tumour is restricted to one part of the body and in one type of tissue

• Not cancerous

• However, have the potential to develop into cancers

• Most common method of removal is surgery to cut away affected tissue

What are malignant tumours?

• Uncontrollably dividing cell groups that may spread or metastasise to other parts of the body

• Surgery is often combined with other treatments like:

> Chemotherapy= drugs that kill the fast-growing cancer cells

> Radiation therapy= uses ionising radiation to damage the DNA of the cancer tissue to kill the abnormal cells

What 2 types of genes play major roles in controlling rate of cell division by mitosis?

• Proto-oncogenes

• Tumour suppressor genes

What is the normal role of proto-oncogenes and the role when mutated?

NORMAL:

• Stimulates cell division

MUTATED:

• Becomes an oncogene

• Gene is permanently active

• Cells divide continuously by mitosis

What is the normal role of tumour suppressor genes and the role when mutated?

NORMAL:

• Inhibits cell division

• Repairs faulty DNA

• Instructs cell death of faulty cells (apoptosis)

MUTATED:

• Inactivated

• Cells divide uncontrollably by mitosis

Give an example of gene mutation in a tumour suppressor gene

• p53= protein that stops the cell cycle moving from G1 to S phase in mitosis

• p53 encoded by the p53 gene

• If gene mutates the p53 protein is unable to halt the cell cycle and cells divide uncontrollably (cancer)

• Mutated p53 genes found in 70% of colon cancers, 30-50% of breast cancers and 50% of lung cancers

• Abnormalities of p53 gene can also be inherited

> Some cancers are caused by inherited mutations, however most are acquired during an individual’s lifetime

BRCA1 and BRCA2 are tumour suppressor genes that are normally expressed in breast tissue, producing proteins that help to repair damaged DNA. The graph shows the incidence of women developing breast cancer below the age of 70 years, both in the general population, and in populations of women with mutations in BCRA1 or 2.

Describe and explain the data in the graph

• Populations which possess the mutated BRCA1 or BCRA2 gene have an increased incidence of breast cancer compared to the total population

• BCRA1 and 2 are tumour suppressor genes. Mutation inactivates these genes. DNA damage is not repaired and the faulty cells divide, passing on the damaged DNA to the daughter cells. Uncontrolled cell division can then occur, resulting in cancer

Describe hypermethylation of a tumour suppressor gene

• Gene is inactivates

• No transcription of gene

• No translation so no protein produced

• Protein no longer inhibits cell division

• Cell divides uncontrollably by mitosis

Describe hypermethylation of a proto-oncogene

• Permanent activation of the gene- becomes an oncogene

• Continuous transcription and translation produce protein continuously

• Protein stimulates cell to divide too quickly by mitosis

Explain how increased methylation of DNA could lead to a tumour suppressor gene being inactivated

Transcription is inhibited- condensation of the chromatin increases which means transcriptional factors are unable to bind so the tumour suppressor gene is not expressed and protein is not produced

Explain how decreased methylation of a proto-oncogene could lead to the formation of cancer

DNA is less tightly coiled around histones so transcriptional factors can bind to the promotor region- the gene is switched on permanently so protein is produced which stimulates cells to divide too quickly

Increased concentrations of oestrogen can result in development of breast cancer

Suggest how increased levels of oestrogen could cause cells to divide too rapidly

• Breast cells contain transcriptional factors which act as oestrogen receptors

• When oestrogen binds to these, the inhibitor molecule is released and the DNA binding sites are exposed. The transcriptional factors move into the nucleus and bind to the promotor region of the proto-oncogene

• Proto-oncogene is switched on and stimulates cell division

Explain how acquired mutations (caused by mutagenic agents) are prevented, diagnosed, and treated

PREVENTION:

• Avoid the mutagen e.g. sunscreen to protect against UV, vaccination to protect against virus which can cause cervical cancer

DIAGNOSIS:

• Normally happens after symptoms appear

• Regular screening of high risk individuals

TREATMENT:

• Depends on type of cancer- radiotherapy, chemotherapy

Explain how hereditary mutations (caused by mutagenic agents) are prevented, diagnosed, and treated

PREVENTION:

• Maybe pre-emptive surgery e.g. mastectomies to prevent breast cancer

DIAGNOSIS:

• Can genetically screen for particular genes

• More frequent screening

TREATMENT:

• Early diagnosis due to screening may result in lower doses of radiotherapy or chemotherapy

Describe how altered DNA may lead to cancer (5 marks)

• DNA altered by mutation

• Mutation changes base sequence of gene controlling cell growth of tumour supressor gene

• Change protein structure

• Tumour suppressor genes produce proteins that inhibit cell division by mitosis

• Uncontrolled and rapid cell division= malignant tumour

Define genome

the complete set of genes in a cell/ organism

Define proteome

the full range of proteins produced by a given cell

What is DNA sequencing?

An automated, laboratory-based procedure used to determine the order of nucleotides and their bases in the DNA of different organisms

>This enables sequence of amino acids in proteins, coded for by the genome to be determined

Why is determining the genome (and proteome) of organisms such as bacteria relatively easy?

• Most prokaryotes have a single piece of circular DNA that is not associated with histones

• DNA does not contain non-coding regions

What applications could determining genome be?

• Identification of potential antigens for use in vaccine production against pathogenic bacteria

• Understanding of the proteins involved in the metabolic pathways of the organism- aid design of drugs to treat infections e.g. malaria

What is the human genome project?

Underway to identify all the proteins produced in humans

• Has produced a complete DNA sequence and mapped approximately 20,000 genes in the human genome, providing a map of gene loci on individual chromosomes

• Took 13 years to complete

• Non-coding DNA and presence of regulatory genes means that knowledge of the genome cannot easily be translated into the proteome

• Currently thought that only approx 1.5% of human genome consists of coding DNA