The Control of Gene Expression

What are mutations?

Mutations are changes in the sequence of nucleotides in DNA molecules

What are the four types of mutations?

Insertion/deletion, duplication, inversion and translocation mutations

What is an insertions mutation?

Where one or more nucleotide pairs are inserted or deleted from the sequence. This type of mutation alters the sequence of nucleotides after the insertion/deletion point known as a frameshift

What is a duplication mutation?

One or more bases are repeated and therefore produced a frame shift.

What is an inversion mutation?

A group of bases becomes separated from the DNA sequence and then rejoin at the same position but in the reverse order. This therefore affects the amino acid that is produced

What is a translation mutation?

A group of bases become separated from the DNA on one chromosome and are inserted into the DNA sequence on another chromosome. This can often lead to significant effects on the phenotype

How do mutations happen?

Gene mutations can arise spontaneously during DNA replication and can be caused by mutagenic agents that affect DNA.

What are the 3 causes of gene mutations?

Chemical mutagens (for example alcohol, benzene and substances in asbestos and is tar in tobacco), ionising radiation (alpha and beta, but also UV and X-ray) and spontaneous errors in DNA replication

What effects can mutations cause?

Mutations can either have beneficial, neutral or harmful effects

Give an example of a neutral effect of a mutation

An example of where a mutation has a neutral effect is in cases where the mutation occurs in a non-coding region of DNA or is a silent mutation. A mutation can also be neutral when a change in tertiary structure of the protein has no effect on the organism

Give an example of a beneficial effect of a mutation

An example of a beneficial effect that a mutation has had is the development of trichromatic vision in humans

Give an example of a harmful effect of a mutation

An example of a harmful effect that mutations have caused is the mutation in the CFTR protein which causes cystic fibrosis

What are stem cells?

Stem cells are undifferentiated cells which can keep dividing to give rise to other cell types.

What are the different types of stem cells?

Totipotent, Pluripotent, Multipotent and Unipotent

What are totipotent stem cells?

Totipotent stem cells are stem cells that are able to differentiate into any type of cell found in the body and into extra embryonic cells such as those in the placenta.

Where are totipotent stem cells found?

Totipotent cells are found in the embryo at an early stage called the blastomere

What is another name for totipotent stem cells?

Embryonic stem cells

What are pluripotent stem cells?

Pluripotent stem cells are cells that can form any cell type in the body, however cannot form extra embryonic cells.

Where are pluripotent stem cells found?

Pluripotent stem cells are also found in the early stages of an embryo. These are often used in replacing damaged tissues in human disorders.

What are multipotent stem cells?

Multipotent stem cells are cells that can differentiate into other cell types but are more limited.

Give an example of a multipotent stem cell

The cells in the bone marrow and umbilical cord

What are unipotent stem cells?

Unipotent stem cells are cells that can only differentiate into one type of cell

How can pluripotent stem cells be created?

Pluripotent stem cells can also be created from unipotent stem cells and are therefore known as induced pluripotent stem cells (iPS)

How can oestrogen affect transcription?

Oestrogen has the ability to alter transcription through altering molecules called transcription factors

What are transcription factors?

Transcription factors are molecules that bind to a specific site on DNA to begin the process of transcription.

Describe the process in which oestrogen controls transcription

The lipid soluble nature of oestrogen means that it can freely diffuse across the cell membrane where it binds to a receptor on a transcription factor. The binding alters the shape of the DNA binding site on the transcription factor and makes it able to bind to the DNA. The transcription factor therefore enters the nucleus via the nuclear pore where it binds to DNA. This stimulates the transcription of the gene that makes up the DNA.

What is sRNA and what is it used for?

sRNA, also called silencing RNA, is used for short term switching off of genes

How does siRNA (small interfering RNA) switch genes off short term?

siRNA binds to a complementary sequence of mRNA. As mRNA is usually single stranded and the cell therefore detects the double stranded form on mRNA and views it as abnormal. Therefore, the mRNA is broken down by enzymes preventing translation

What is Epigenetics?

Epigenetics involves heritable changes in gene function, without changes to the base sequence of DNA. It shows that environmental factors can make changes to the function of genes which can be inherited

How does methylation work?

DNA methylation is a process by which methyl groups are added to DNA. Methylation modifies the function of the DNA, typically acting to suppress gene transcription. DNA methylation alters the expression of genes in cells as they divide and become specialised. The change is permanent and prevents the cell from converting back into a stem cell or a different cell type. The methylation is through the addition of a CH₃ chemical group to cytosine bases, which both prevents binding of transcriptional factors to DNA and stimulates decreased acetylation of histones

How does DNA acetylation work?

DNA acetylation also changes DNA structure. Histones are positively charged proteins closely associated with DNA, which is negatively charged. Decreased acetylation of histones increases their positive charge, so they bind DNA more tightly. When this happens, transcriptional factors can no longer access the DNA, so the gene is switched off.

What do proto-oncogenes do?

Proto-oncogenes cells to divide by producing proteins that stimulate cell division, allow the checkpoints of the cell cycle to be passed, and can cause cancer if mutated

What do oncogenes do?

Oncogenes are formed from mutated proto-oncogenes and by result are permanently switched on resulting in cell division that is uncontrolled. It does this by permanently activating a cell surface receptor or coding for a growth factor.

What do tumour suppressor genes do?

Tumour suppressor genes - control cell division, cause the cell cycle to stop when damage is detected. They also play a role in the programming of apoptosis (cell death). When these are switched off the cell cycle becomes unregulated.

Draw a table to show the differences between benign and malignant tumours

What does abnormal methylation of tumour suppressor genes and oncogenes do?

Increased methylation also called hyper-methylation plays an important role in controlling tumour suppressor genes and oncogenes. The hyper-methylation of a tumour suppressor gene called BRAC1 can lead to breast cancer.

How are increased oestrogen concentrations linked to breast cancer development?

These elevated levels of oestrogen are found in fatty tissues called adipose tissue in the breast of postmenopausal women. Oestrogen binds to the transcription factor which activates the genes promoting cell division, leading to tumour formation.

Give one example of an application of sequencing the genome of a simpler organism?

The identification of potential antigens for use in vaccine production

What is the human genome project?

The Human Genome Project is an international scientific project which has successfully determined the sequence of bases of a human genome.

What are the potential applications of the human genome project?

Screening for mutated sequences, carriers and preimplantation screening as well as screening for disorders such as Huntington's disease before the symptoms appear.

What is the significance of comparing genomes between species?

It allows evolutionary relationships between species to be determined

Other than genome wide comparisons, what has gene sequencing been used for?

Gene sequencing has allowed for the sequences of amino acids in polypeptides to be predicted and has allowed for the development of synthetic biology

What is reverse transcriptase?

An enzyme that is found in only some viruses and bacteria and catalyses the formation of a double strand of DNA from a single strand of RNA.

What are restriction endonucleases?

Restriction endonucleases are enzymes, extracted from bacteria, that cut DNA at specific sequences, usually six base pairs in length

What are the most useful endonucleases?

The most useful restriction endonucleases are those that make staggered cuts, as they leave sticky ends on the DNA

Why are sticky ends important?

Sticky ends are important because if the same restriction endonuclease is used to cut two DNA fragments, then the ends will be complementary. This allows then to attach together before stronger covalent bonds form.

Why are vectors used to place DNA fragments?

This is because a DNA fragment would be digested by enzymes if it was placed in a cell, therefore a vector is used to insert DNA into cells

How can DNA fragments be placed in plasmids?

Plasmid and gene are cut with the same restriction enzyme to create complementary sticky ends. The fragments are incubated with the plasmids. If a plasmid takes up the insert, base pairing takes place between the complementary ends which are then sealed with the use of DNA ligase which forms phosphodiester linkages. A recombinant DNA molecule is created

What can be done to stimulate bacterial cells to take up plasmids?

Electroporation

How does electroporation help bacteria take up plasmids?

It increases the permeability of bacterial membranes thus increasing the chance of success.

How can we check to see if the DNA has been taken up by the bacteria?

Using gene markers

What is DNA profiling?

DNA profiling is a technique used to identify individuals by characteristics of the DNA.

Give one example of DNA profiling?

PCR

What is the full name of PCR?

Polymerase chain reaction

What is PCR used for?

It is used to amplify DNA by making millions of copies of a given DNA sample

How does PCR occur?

A reaction mixture is set up by mixing the DNA sample, primers, free nucleotides and DNA polymerase. The mixture is then heated to 95 degrees to break the hydrogen bond and separate the two strands. The mixture is then cooled to a temperature between 50-65 degrees, depending on the primers used, so that they can bind the strands. The temperature is then increased to about 70 degrees as this is the temperature DNA polymerase works at. The DNA polymerase is call Taq polymerase and is from bacteria that live in hot springs. DNA polymerase creates a copy of the sample by complementary base pairing using the free nucleotides. This cycle is repeated around 30 times and gives rise to an amount of DNA sufficient to create a DNA profile.

What is In-vitro cloning?

Gene cloning that can be done with PCR. It is fast, automated and reliable once conditions are established. It does not require living cells and can have problems such as contamination and errors

What is In-vivo cloning?

Gene cloning that can be done using recombinant plasmids in bacteria. It is accurate and useful as the gene is placed in cells where it can be expressed, however it is very time consuming and requires monitoring of cell growth.

What is a DNA probe?

A short, single stranded DNA molecule that is designed to be complementary to a sequence to be detected.

How can DNA probes be seen?

The labelling of fragments either uses radioactive isotopes or a fluorescent dye which glows under certain wavelengths of light

What is genetic fingerprinting?

It is a technique that can detect differences in people's DNA.

How does genetic fingerprinting work?

It uses variable number tandem repeats (VNTRs) which are short repeating sequences or bases. The probability of two individuals having identical VNTRs is extremely low, therefore VNTR analysis can be used in genetic fingerprinting

What is gel electrophoresis?

A process used to separate the DNA fragments and proteins according to their size using an electric current

For what can genetic fingerprinting be used?

Genetic fingerprinting can be used in the fields of forensic science, medical diagnosis as well as animal and plant breeding