3.8 The control of gene expression

What are the types of gene mutations?

frameshift: addition, deletion, non-frameshift: substitution, inversion,

duplication and translocation of bases

How do gene mutations occur?

spontanteously

Define gene mutation

an alteration in the base sequence of DNA

Describe an addition mutation.

One additional nucleotide is inserted so this alters the genetic code.

What do framshift mutations lead to?

nonsense polypeptide chains as all the base triplets downstream from the mutation are changed

What is a substitution mutation?

when one nucleotide is replaced by another nucleotide with a different base

What is an inversion mutation?

where one nucleotide swaps with another

What is a duplication mutation?

Some sections of nucleotides are duplicated and so additional amino acids will be inserted into the polypeptide chain.

What is a translocation mutation?

This occurs when part of a chromosome breaks off and adds onto another chromosome.

What can increase the rate of mutations?

mutagenic agents

What is the impact of non-frameshift mutations?

this changes only one base. Only one triplet code is changed. Due to the degenerate nature of the triplet code, not all mutations result in a change to the encoded amino acid.

Give an example where more than one codon codes for the same amino acid.

codon on RNA:

ACU, ACA, ACC, ACG all code for Threonine

What is a totipotent cell?

cell that can divide and produce any type of cell.

Where are stem cells found?

- embyronic

- umbilibal cord blood cells (similar to adult stem cells)

- placental stem cells (develop into specific types of cell)

- adult stem cells (specific to a tissue or organ)

What are the two main characterisatics of stem cells?

potency

self renewal

What is self renewal?

the ability to maintain an unspecialised state

How is stem cell differentiation controlled?

expression a specific pattern of DNA, controlled by transcription factors

What are the types of stem cell?

totipotent, pluripotent, multipotent, unipotent

What are pluripotent stem cells?

- found in embryos and can differentiate into almost any type of cell.

What are multipotent stem cells?

- found in adults and can develop into a limited number of specialised cells (adult stem cells, umbilical cord blood stem cells)

What are unipotent stem cells?

can only differentiate into a single type of cell. They are derived from multipotent stem cekks and are made in adult tissue.

Give an example of unipotent stem cells.

cardiomyocytes

What are induced pluripotent stem cells (iPS cells)?

- a type of pluripotent cell that is produced from unipotent stem cells, which can be any body cell.

- These body cells are genetically altered to behave like embryonic stem cells, which are a type of pluripotent cell.

- This involves inducing genes and transcriptional factors within the cell to express themselves, ie to turn genes on that were otherwise turned off.

- As these genes are capable of being reactvitated, it shows that adult cells retain the same genetic information that was present in the embryo.

How are iPS cells similar to embryonic stem cells?

they can divide indefinitely to make an infinite supply

they are self-regenerative

What can pluripotent cells be used for?

- using cells to regrow damaged tissues - skin for serious burn damage

- treating neuro-generative diseases such as Parkinson's disease

What is potency?

the ability of stem cells to differentiate into specialised cells

Describe regulation of transcription in eukaryotes.

Transcription of target genes can be stimulated or inhibited when specific transcriptional factors move from the cytoplasm into the nucleus.

What is epigenetics?

It involves inheritable changes in gene function without changes to their DNA base sequences.

These inheritable changes are caused by changes in the gene's environment that inhibit transcription.

How do changes in the gene's environment inhibit transcription? (epigenetics)

- increased methylation of DNA

- decreased acetylation of histones.

Describe the nature of genes.

in year 1, we defined a gene as a base sequence of DNA that codes for the amino acid sequence of a polypeptide and a functional RNA. We also learnt that a gene must be transcribed to produce mRNA in order for a polypeptide to be made.

Describe the general theory of gene expression control in transcription.

only some genes are transcribed at a given time

Describe the general theory of gene expression control in translation.

mRNA might be destroyed or its translation by a ribosome blocked.

What are transcription factors?

regulatory proteins that bind to specific DNA sequences to control transcription

Describe the control of transciption by specific transcription factors.

Every gene has one or more DNA base sequences that control its expression called a promoter region. These regions are found near the gene it controls and is usually about 100 base pairs before the start of the gene. A protein, called a transcription factor, binds to the gene's promotor region and enable RNA polymerase to attach to the start of the gene and begin its transcription.

What proportion of cytosine bases are methylated in human DNA?

3%

What does a DNA nucleotide comprise?

deoxyribose, a purine or pyramidine base, a phosphate group.

What is methylated DNA?

DNA that is not transcribed

Describe methylation of cytosine.

during DNA methylation, a methyl group (CH3) is added to carbon atom 5 of a cytosine residue.

When does methylation of cytosine most often occur?

where cytosine is linked by a phosphodiester bond to a guanine base, represented by CpG.

What do methylated CpG repeats do?

Repeated CpG sequences are common in the DNA near gene promoters. The presence of repeated CpG sequences near a gene promoter inhibits the activity of the enzyme RNA polymerase. As a result, the affected gene cannot be transcribed; it is effectively silenced.

What is a nucleosome?

A region of DNA wound around histone proteins

When is a gene accessible to RNA polymerase?

when the winding of DNA around a histone protein is loose.

What are 'tails' on histone proteins?

histone molecules have side branches, or 'tails'

What is acetylation?

addition of an acetyl (COCH3) group to a molecule

How does acetylation help control transciption?

- histone tails contain the amino acid leucine

- leucine can be acetylated

- when acetylated, the histones become more loosely packed so that RNA polymerase can bind.

Describe epigenetic inheritance in rats.

females that recieve good care when they were young responded better to stress later and nurtured their offspring more effectively than those who recieved lower quality care

this. epigenetic information was passed onto the offspring's DNA without going through the gametes

Describe how gestational diabetes is an example of epigenetic inheritance.

- when the mother experiences gestational diabetes, the unborn foetus is exposed to higher levels of glucose which causes epigenetic changes in the daughter's DNA. This increases her chances of experiencing gestational diabetes if she becomes pregnant

Describe evidence for epigenetics.

during the earliest stages of development, there is a specialised cellular mechanism that searches the genome and erases the epigenetic stages. Unfortunately, a few of these tags remain and are passed onto the offspring.

Describe how epigenetic changes causing disease can be combatted.

Epigenetic changes are reversible, which make them good targets to combat the diseases tehy create. Incresaed methylation can lead to a gene being switched off, sod rugs that stop methylation can be used to treat diseases.

E.g. azacytidine is a drug used in chemotherapy for types of cancer that are caused in increased methylation of tumour suppressor genes.

Describe how drugs can be used to deal with decreased acetylation of histones.

Decreased acetylation of histones can lead to genes being swithced off. HDAC inhibitor drugs can be used to treat cancers that are caused in this way. The drugs inhibit the activity of histone deacetylase enzymes which remove the acetyl groups from the histones. Without the activity of HDAC enzymes, the genes remain acetylated and the protein can be transcribed.

What is the problem with developing drugs to counteract epigenetic changes?

these changes take place in many cells, so the drugs need to be as specific as possible.

e.g. cancer drugs need to be aimed at targetting dividing cells and avoid damaging normal body cells

Describe how epigenetics is used in the development of diagnostic tests.

Epigenetics has been used to develop diagnostic test that can detect the early stages of diseases such as cancer, brain disorders and arthritis. They can identify the level of DNA methylation and histone acetylation at an early stage so individuals can seek early treatment and have a higher chance of survival.

What is Acetylation?

an acetyl group is transferred to a molecule from acetyle CoA

What is deacetylation? What are its impacts?

The removal of an acetyl group which increases the positive charges on histones and increases the attraction to the phosphate groups of DNA. So, there is a stronger association between DNA and histone proteins and the DNA is not accessible to transcription factors. These transcription factors cannot initiate mRNA production from DNA. i.e. the gene is switched off

What is methylation? What are its impacts?

This is the addition of a methyl group CH3, which is added to the cytosine bases of DNA. It inhibits the transciption of genes in two ways.

How does methylation inhibit transcription of genes?

1. prevents the binding of transcription factors to DNA

2. attracts proteins that condense the DNA; histone complex (by inducing deacetylation of the histones). This makes the DNA inaccessible to transcription factors

What is siRNA?

small interfering RNA

it is usually 21 base pairs long, with two bases overaging at each end.

How is siRNA created? What does it bind with, and what happens when it does?

The mRNA is used as a template to produce a complimentary mRNA strand using the enxyme RNA-dependent RNA polymerase. The two strands are held together by hydrogen bonds to form double-stranded (ds) RNA. The dsRNA is cut by a Dicer enzyme to produce siRNA. siRNA forms a complex with a protein, and one of the siRNA strands is destroyed.

What is the name of the siRNA-protein complex?

RISC

What does RISC do?

The siRNA molecule in the RISC guides the enzyme to an mRNA molecule by pairing up its bases with the complimentary ones on a section of the mRNA molecule. The RISC then cuts the mRNA into smaller sections.

This leads to the mRNA being broken down to prevent any further polypeptide synthesis; the gene has been blocked.

What are the two types of tumour?

benign and malignant

What are tumor supressor genes?

these genes slow down cell division, repair mistakes in DNA, and can cause apoptosis (programmed cell death)

What happens if tumor supressor genes are switched off?

if these genes become switched off, the inhibition of cell division is removed

most mutant cells die but a few that survive can make clones of themselves and grow out of control and cause a tumor.

What are oncogenes?

mutations of proton-oncogenes.

What do proto-oncogenes do?

stimulate a cell to divide when growth factors attach to a protein receptor on its cell-surface membrane.

What happens when a proto-oncogene mutates into an oncogene?

It can become permanently activated for two reasons:

- the receptor protein on the cell surface membrane can be permanently activated, so that cell division is switched on even in the absence of growth factors.

- the oncogene may code for a growth factor that is then produced in excessive amounts, again stimulating cell division

this results in cells dividing too rapidly and becoming out of control so that a tumor develops.

What are some examples of tumour supressor genes?

TP53, BRCA1 and BRCA2

What is hypermethylation?

increased methylation

How can hypermethylation cause cancer?

- hypermethylation occurs ina. specific region (promoter region) of tumour supressor genes

- this leads to the tumour suppressor gene being inactivated

- transcription of the promoter regions of tumour suppressor genes in inhibited

- the tumour suppressor gene is therefore silenced

- as the tumour suppressor gene normally slows the rate of cell division, its inactivation leads to increased cell division and the formation of a tumour

Why does a woman's chance of developing breast cancer increase after menopause?

- there is a reduction in the production of oestrogen from the ovaries, fat cells in the breast produce more oestrogen after menopause, which triggers the development of a tumour.

- the tumour attracts white blood cells that increase the concentration of oestrogen production increasing the tumour size.

How does oestrogen promote transcription?

- oestrogen is lipid soluble to can easily diffuse throught the phospholipid portion of the cell membrane

- once inside the cytoplasm of a cell, oestrogen bidns with a site on a receptor molecule of the transcriptional factor. The receptor and oestrogen are complimentary to one another.

- By binding with the site, the oestrogen changes the shape of the DNA binding site on the transcriptional factor, which can now bind to the DNA (it is activated)

- the transcriptional factor can now enter the nucleus through a nuclear pore and bind to specific base sequences on DNA.

- the combination of the transcriptional factor with DNA stimulates transcription of the gene that makes up the portion of DNA

What needs to occur for transcription to begin?

the gene needs to be switched on by specific molecules that move from the cytoplasm tothe nucleus, called transcriptional factors. Each transcriptional factor has a site that binds to a specifc base sequence of the DNA in the nucleus.

What are the differences between benign and malignant tumours?

- malignant grow more quickly

- Cells in malignant tumors become de-differentiated, whereas cells in benign tumors are often well differentiated

- cells in malignant tumors do not produce adhesion molecules like those in benign tumors, so can metastisise

- benign tumors are surrounded by a capsule of dense tissue so remain as a complex structure, but malignant tumore are not so can grow finger-like projections into surrounding tissue

What is recombinant DNA technology?

Techniques that allow the transfer of fragments of DNA from one organism, or species, to another.

Why can we transfer DNA from one species to another?

The genetic code is universal, meaning so are transcription and translation mechanisms. The transferred DNA can be translated within cells of the recipient organism. W

What is the name for the recipient organism in a DNA transfer?

a transgenic organism

What are the three methods for producing fragments of DNA?

- conversion of mRNA to complimentary DNA (cDNA) using reverse transcriptase

- using restriction enzymes to cut a fragment containing the desired gene from DNA

- creating the gene in a 'gene machine'

Describe conversion of mRNA to cDNA using reverse transcriptase.

- a cell that readily produces the protein is selected (e.g. the B cells of the islets of Langerhans from the pancreas are used to produce insulin.

- these cells have large quantities of relevant mRNA, which is therefore more easily extracted

- reverse transcriptase is then used to make DNA from RNA. This DNA is known as cDNA because it is made of nucleotides complimentary to the mRNA

- to make up the other strand of DNA, DNA polymerase is used. This double strand of DNA is the required gene.

Describe in vivo gene cloning

- the DNA is cut at specific base sequences using restriction endonuclease enzymes called recognition sites, leaving the DNA with staggered or sticky ends.

- the same restriction endonuclease is used to cut open the vector, usually a plasmid. The gene is inserted using DNA ligaments and complementary base pairs join up, forming a recombinant plasmid.

Describe transformation

- this is the process of uptake of the recombinant plasmids by the bacteria. They are usually mixed in a cold solution of calcium chloride, which is warmed up.

- The calcium ions and the increased temperature improve the uptake of the recombinant plasmid (1%), some plasmids will close before the gene is taken up and the DNA fragments can join end to end to form their own plasmid.

when a bacterial cell has taken up a plasmid, it is said to be transformed

How do we identify cells that have taken up the DNA fragment?

We use antibiotic resistance genes found in bacterial plasmids that code for an enzyme that destroys the antibiotic. The R-plasmid contains two genes that are resistant to ampicillin and tetracycline.

This process is carried out by:

- growing the bacteria on a medium containing ampicillin

- those bacteria that have taken up plasmids containing the gene will survive as they are resistant

- cells that have not taken up the plasmids will die

However, some cells will have taken up plasmids without the required gene

How do we identify cells which took up plasmids without the required gene?

can be identified using marker genes. These could be:

- resistant to an antibiotic

- make a fluorescent gene using a gene called GFP

- may produce an enzyme whose action could be identified such as lactase (break down lactase in milk, so we would see clear zones around the bacteria instead of white)

Describe the "gene machine".

•The desired sequence of nucleotide bases of a gene is determined from the desired protein that we wish to produce. The amino acid sequence of this protein is determined. From this, the mRNA codons are looked up and the complementary DNA triplets are worked out.

• The desired sequence of nucleotide bases for the gene is fed into a computer.

• The sequence is checked for biosafety and biosecurity to ensure it meets international standards as well as various ethical requirements.

• The computer designs a series of small, overlapping single strands of nucleotides, called oligonucleotides, which can be assembled into the desired gene.

In an automated process, each of the oligonucleotides is assembled by adding one nucleotide at a time in the required sequence.

• The oligonucleotides are then joined together to make a gene.

This gene doesn't have introns or other non-coding DNA. The gene is replicated using the polymerase chain reaction (Topic 21.3).

• The polymerase chain reaction also constructs the complementary strand of nucleotides to make the required double stranded gene.

It then multiples this gene many times to give numerous copies.

• Using sticky ends (Topic 21.2) the gene can then be inserted into a bacterial plasmid. This acts as a vector for the gene allowing it to be stored, cloned or transferred to other organism in the future.

The genes are checked using standard sequencing techniques (Topic 20.6) and those with errors are rejected.

What are the three stages of the polymerase chain reaction? (PCR)

separation of the DNA strand

addition of the primers

Describe separation of DNA strands.

The DNA fragments, primers and DNA polymerase are put into a vessel in the thermal cycle. The temperature is increase to 95ºC , which causes the two strands to be separated as it breaks the hydrogen bonds in between the complementary base pairs.

Describe addition of the primers.

The temperature is reduced to 55ºC and the primers join/anneal to their complementary base pairs. The primers provide a start sequence for DNA polymerase to attach to nucleotides at the end of an existing chain. The primers also prevent the two strands of DNA rejoining.

What is the PCR (polymerase chain reaction)?

this is a method of copying fragments of DNA, which is an automated process, using a thermal cycle, that is very fast and efficient.

Describe the synthesis of DNA in the PCR.

The temperature is increased to 72ºC, which is the optimum temp for DNA polymerase as it is a thermostable enzyme. Complementary nucleotides align alongside the exposed bases on each strand, which begins at the primer and stops at the end of the chain.

So, the quantity of DNA doubles with each cycle, which increases the quantity of DNA very quickly.

What are the uses of in vitro gene cloning?

- forensic science

- enabling genetic conditions to be identified in a foetus

What two things condense the DNA-histone complex? What impact does this have?

- increased methylation

- decreased acetylation

This means that there is stronger association between the histone and the DNA so that it is inaccessible to transcription factors. This means that transcription is inhibited.

What are the benefits of in vitro gene cloning?

extremely rapid

does not require living cells

What are the advantages of in vivo gene cloning?

- useful to introduce a gene into another organism

- involves no risk of contamination

- very accurate

- cuts out specific genes

- produces transformed bacteria that can be used to produce large quantities of gene products

Why are bacterial cells able to produce human proteins from human DNA?

- genetic code is universal

- translation/ transcription is universal

Why may bacterial cells not be able to produce human proteins from human DNA?

they don't have golgi apparatus so cannot modify the proteins

they cannot carry out splicing so cannot remove introns

What is a DNA probe?

A short, single stranded length of DNA with an identifiable label which is either a:

- radioactively labelled probe, usually containing P-32 that is a radioactive isotope that is exposed on X-ray film.

- fluorescently labelled probe, which emits light

How are DNA probes used to identify particular alleles of genes?

- DNA probe is made complementary to DNA with our desired gene

- double stranded DNA is treated to separate its strands

- separated DNA strands are mixed with the probe

- probe binds to complementary base sequence = *DNA hybridisation*

- site where DNA probe binds can be identified

How is the DNA separated for use of a DNA probe?

It is heated, causing denaturation . The DNA cools and recombines (anneals) to reform the original strand.

What is DNA hybridisation?

When a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases.

Why are DNA probes useful?

- can locate a specific allele of a gene, which could code for a genetic disorder

- detection of oncogenes or mutated tumor suppressor genes

- this could help with genetic counselling

Describe personalised medicine.

Doctors can provide advice and healthcare based on a person's individual genotype.

Genetic screening could also determine if a drug may be less or more effective in treating a condition, or help with the dosage needed. G