UNIT 8 - Gene expression and DNA technology

what are gene mutations

changes in the sequence of nucleotide bases in DNA resulting in a formation of a new allele

Explain why some mutations may not affect the function of the protein coded for by the gene affected. 

 

Genetic code is degenerate (so amino acid sequence may not change);​ 

Does change amino acid but no effect on tertiary structure

• the mutation occurs in the intron (so sequence may not change

• the mutation may occur in a recessive allele so its not expressed

A gene encoding an enzyme has mutated and a frame shift has occurred. Define frame shift and explain why the enzyme becomes non-functional.

Insertions and deletions result in frame-shift mutations. 

Triplets of bases/Codons change after mutation.  

Sequence of amino acids changes (primary structure), this changes the position of the bonds between R groups (ionic, hydrogen and disulphide bridges), tertiary structure of active site changes. Enzyme-substrate complex can no longer form.  

name and describe all the different types of mutations

• Substitution mutation :

 A substitution mutation occurs when one or more base is changed for another 

• Deletion mutation :

 deletion mutation occurs when one or more base is removed. 

• Addition mutation :

 An insertion mutation occurs when one or more base is added into the DNA. 

• Inversion mutation 

 An inversion mutation occurs when a sequence of bases is reversed 

• Duplication mutation 

 A duplication mutation occurs when one or more bases are repeated 

• Translocation mutation 

A translocation mutation occurs when one or more base is moved from one location in the genome to another. This could be within the same or a different chromosome.  

what is a frame shift

alteration in all the base triplets after the point of mutation

what are properties of mutations

• Gene mutations occur spontaneously.

• Gene mutations might arise during DNA replication.​

• The mutation rate is increased by mutagenic agents e.g. X-rays, benzene​

• Mutations can result in a different amino acid sequence in the encoded polypeptide.

• This is due to the altered base sequence coding for a different sequence of amino acids.

what causes mutations

• incorrect base pairing during DNA replications

• mutagenic agents

  • X-rays/UV light​

  • Chemicals (benzene nitrous oxide)​

  • Βeta particles​

  • Gamma rays​

what are mutagenic agents

factors that increase the rates of mutations

are mutations passed onto off spring?

• Mutations in sex cells (gametes) are passed onto the next generation​

  • as the mutation has to change every single cell in the body

• Mutations in body cells (somatic) are NOT passed onto the next generation.

what are the effects of gene mutations?

• May change only one triplet code (substitution). ​

• May not change encoded amino acid - due to the degenerate nature of the genetic code.​

• Some gene mutations change the nature of all base triplets downstream from the mutation, i.e. result in a frame shift​

how do mutations affect protein synthesis

• DNA Base sequence changes​

• So mRNA codons change​

• So different tRNA molecules come to the ribosome ​

• So amino acid sequence changes​

• So tertiary structure of protein changes

what two genes is the rate of cell division controlled by?

• tumour suppressor - (code for proteins that slow down cell division)

• proto- oncogene -(code for proteins that stimulate cell division)

how can a mutation in a tumour suppressor gene result in a tumour?

the gene can be inactivated if a mutation occurs in the base sequence.

Mutation would stop the production of proteins (that slow down cell division) so the cell divides uncontrollably - leading to a tumour

how can a mutation in the proto- oncogene result in a tumour

A mutation can cause a proto-oncogene to form an oncogene.

• mutation could code for proteins that further increase cell division - by causing them to divide too quickly

• The cell divides uncontrollably (by mitosis) resulting in a tumour

what is cancer

group of diseases caused by alterations in the genes that regulate mitosis and the cell cycle

what are tumours

masses of dividing cells

what are the two types of tumours

• benign

• malignant

what are the differences between benign and malignant tumours

Benign tumours​	Malignant tumours​
Grow slower than malignant tumours​	Grow faster than benign tumours​ ​
Cells do not spread (metastasize) to other tissues as the tumour is enclosed by fibrous tissue ​ ​	Cells can break off and spread to other parts of the body as the tumour is not enclosed.​
Cells often remain differentiated (specialised)​ ​	Cells often become undifferentiated (unspecialised)​
Non-cancerous​ ​	Cancerous​
Cell nucleus has a relatively normal appearance​ ​	Cell nucleus is larger and darker​what

what turns genes on and off?

• Protein transcription factors found in the cytoplasm of cells.​

• A transcription factor, when activated, moves into the nucleus and attaches to a promoter region close to the target gene or genes which it affects. ​

• This attachment to the promoter region stimulates the transcription of the targeted gene/s by activating RNA polymerase.​

• The expression of different genes results in different proteins being coded for resulting in different specialised cells being produced i.e. cell differentiation.​

how does a transcription factor actually work?

they work by promoting (as an activator) or blocking (repressor) the recruitment of RNA polymerase

what are stem cells

undifferentiated cells that can divide by mitosis and differentiate into different types of cells.

name and describe the functions of all the different stem cells.

• totipotent

  • occur for a limited time in mammalian embryos

  • differentiate into any type of body cell - (extra

• pluripotent

  • Develop from totipotent cells ​

  • Are able to differentiate into almost all types of cells ​

  • But they cannot produce the cells of extra-embryonic tissue. (placenta)

• multipotent

  • found in mature mammals usually the bone marrow

  • can differentiate into a few, limited types of specialised cells (somatic stem cells)

• unipotent

  • found in mature mammals

  • can only differentiate into one type of cell

what is iPS cells

INDUCED PLURIPOTENT STEM CELLS

• pluripotent stem cells produced from unipotent stem cells

• Uses appropriate protein transcription factors​

• The transcription factors are used to express (‘turn on’) or inhibit genes so that these cells now develop similar characteristics to embryonic stem cells.​ (differentiate into a particular type of cell)

• These iPS cells can then be used to develop a wide range of different types of tissue used to treat people with diseases

how does an inhibitor prevent gene expression

the inhibitor could bind to the transcription factor preventing it from binding it to the promotor region of the target gene in result preventing the stimulation of RNA polymerase overall.

describe and explain how transcription factors can cause cell differentiation

• transcription factors are protein

• bind to the promotor region of gene

• TF complementary to the base sequence on the promotor region

• allows binding of RNA polymerase

• may inhibit binding of RNA polymerase

• transcription of gene occurs

• resulting in synthesis of certain protein

how do hormones regulate gene expressions

some hormones are able to enter the target cell. once inside the cell, they function by stimulating the expression of a particular gene in the target cell.

describe and explain the role of oestrogen in gene expression

1. oestrogen is lipid soluble (steroid hormone) - diffuses easily through the cell membrane

2. oestrogen binds specifically to a receptor protein that is part of the transcription factor

3. binding of oestrogen changes shape of transcription factor, allowing it to bind to the promotor region of a particular gene specifically

4. transcription moves into the nucleus

5. this allows RNA polymerase to attach to the gene and catalyse the transcription of the gene

6. mRNA is transcribed from the gene

7. this mRNA is translated into protein

therefore oestrogen increases the expression of particular genes - stimulating production of particular proteins

how could oestrogen lead to breast cancer?

• some tissues oestrogen increases expression of genes associated with cell division (proto- oncogenes/ tumour suppressor)

• This means that high blood concentrations of oestrogen over a period of time can increase the risk of uncontrollable cell division therefore cancer, especially breast cancer

what is an example of a drug that helps with breast cancer (in relation to oestrogen) and how does it work?

• Tamoxifen

• in the body, tamoxifen is converted to endoxifen, a molecule similar in structure to oestrogen.

• Endoxifen competes with oestrogen for binding to the oestrogen receptor, inhibiting the effect of oestrogen.

how can translation of mRNA be inhibited in eukaryotes and prokaryotes?

by RNA interference (RNAi),

involves small interfering RNA or siRNA

describe siRNA molecules

short, double-stranded sections of RNA, usually only 20-25 base pairs long.

how does siRNA regulate gene expression?

causing mRNA to be broken down after transcription, thereby preventing translation.

explain the process on how siRNA prevents translation

1. Longer, double-stranded molecules of RNA are hydrolysed (by an enzyme) into shorter molecules.

2. RNA becomes single-stranded siRNA

3. siRNA binds to an enzyme that hydrolyses mRNA

4. The siRNA binds to a specific molecule of mRNA by complementary base-pairing. Thus siRNA ‘guides’ the hydrolytic enzyme to a target molecule of mRNA.

5. The enzyme hydrolyses the mRNA molecule. This prevents the translation of mRNA into protein

what is epigenetics and how could this be resulted in?

Epigenetics refers to changes in gene function without changes in the base sequence of DNA

• changes in gene function may be caused by aspects of environment - e.g. stress, diet, exposure to toxins etc.

what are examples of epigenetics?

• increased methylation of DNA or

• decreased acetylation of associated histones.

describe results of increased methylation of DNA

• This occurs when a methyl group (CH3) attaches to the DNA sequence of a gene.

• The methyl group always attaches to a cytosine (C) base when it is next to guanine (G), (known as a CpG site, the p representing the phosphate between the 2 bases)

• Increased methylation inhibits transcription by preventing the binding of transcription factors to the promoter sequence so that the gene is not expressed. (sites are already occupied)

describe results of decreased acetylation of histones

• In eukaryotes, the DNA is wrapped around proteins called histones to form chromatin. These histones can be epigenetically modified by the addition or removal of acetyl (COCH3) groups.

• When histones are more acetylated (acetyl groups added), the chromatin is less condensed (more loosely packed). Transcription of genes is more likely as the genes (DNA) are now more accessible to transcription factors.

• When histones are less acetylated (removal of acetyl groups) the chromatin is more condensed. Transcription is inhibited as the genes are not accessible to transcription factors.

how can epigenetics lead to disease

by causing abnormal activation or inhibition of genes.

how can epigenetics cause cancer?

• The hypermethylation (too much methylation) of tumour suppressor genes so that these genes are not transcribed.

• The proteins that slow down cell division are not produced leading to uncontrolled cell division and the development of a tumour.

• The hypomethylation (too little methylation) of proto-oncogenes so these genes are continually transcribed.

• This increases production of proteins involved in stimulating cell division. This leads to rapid, uncontrolled cell division and the development of a tumour

what are the tools that DNA technology typically uses?

• Restriction Enzymes

• Electrophoresis

• PCR

• DNA Primers & DNA Probes

what are the benefits of gene technologies?

allow the study and alteration of gene function, giving a better understanding of organism function and the design of new industrial and medical processes.

describe and explain how restriction endonucleases work

• fragments

• These enzymes hydrolyse the phosphodiester bonds in DNA or RNA, producing smaller fragments at recognition sites (specific base sequences)

• They hydrolyse the phosphodiester bonds in both strands of DNA.

• Some restriction enzymes hydrolyse the DNA at different locations, producing ‘sticky ends’. Some hydrolyse at the same position in both strands, producing ‘blunt ends’:

how do sticky ends benefit?

enable the DNA to be joined or spliced onto a different piece of DNA more easily because complementary base pairing can occur between the sticky ends.

describe and explain how gel electrophoresis works

• separation

• smaller DNA fragments will travel faster and therefore further through the gel when an electric charge is applied.

• Negatively charged DNA fragments move towards the positively charged terminal.

• The DNA fragments in each sample are separated according to size as smaller fragments moved further in the gel when an electric charge is applied.

• After electrophoresis, the DNA fragments are transferred to a nylon membrane, then radioactively labelled DNA probes are added.

• nylon membrane is placed on X-ray revealing the labelled fragments

• these are compared to a DNA ladder which has DNA fragments of known sizes (base number) which can be used to compare to the unknown sample.

describe and explain how PCR works + state what the reaction requires

• amplification

• This technique enables multiple copies of identical fragments of DNA or genes to be produced (or amplified) from a small sample

• reactants:

  • DNA to be copied

  • primers

  • ‘Free’ DNA nucleotides (complementary)

  • thermostable DNA polymerase

• Stage 1- Reactants are mixed together and heated at 95 ºC for 5 minutes. This breaks hydrogen bonds in the DNA

• Stage 2 - The mixture is cooled to 50-60 ºC for 2 minutes. This allows the primers to join (anneal) to their specific complementary target sequence. ‘Free’ DNA nucleotides align to the DNA strands by complementary base-pairing.

• Stage 3 - The temperature is increased to 72 ºC, which is the optimum for DNA polymerase. This enzyme joins the individual nucleotides of a strand together to form a new complementary strand.

• Many cycles of heating and cooling produce exponential numbers of DNA molecules.

how do you calculate the number of DNA molecules produced?

2ⁿ

n = no of cycles

during PCR why could the production of DNA be eventually stopped?

• enzyme eventually denatures due to the extreme fluctuations in temp

• primers are used up

• free nucleotides are also used up

what are DNA primers and how do they work

(starting)

DNA primers are short, single-stranded molecules of DNA.

• They provide a starting sequence for DNA polymerase. This is important because DNA polymerase cannot begin at a single-stranded starting point. (essentially make the sequence “double stranded)

• They also help to prevent the original DNA strands from simply joining back together

what are DNA probes and what are they used for?

(finding)

• DNA probes are short, single-stranded molecules of DNA that are radioactively or fluorescently labelled.

• They are used to identify or locate known sequences of DNA.

what is recombinant DNA technology

• Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another.

what is a transgenic organism?

organism that has received transferred DNA.

how can the transferred DNA be translated within cells of the recipient (transgenic) organism.

because the genetic code is universal, as are transcription and translation mechanisms

what are methods used to obtain required fragment/ gene?

• Using Reverse Transcriptase

• Using Restriction Endonucleases

• Using a ‘gene machine’

describe and explain how using reverse transcriptase can be used to obtain a gene

• mRNA which has been transcribed from the gene is removed from cells and used (instead of DNA)

• The mRNA is used as a template to produce the required gene or required fragment of DNA.

• For example, to produce a gene for insulin production, mRNA complementary to the insulin gene is isolated from pancreatic cells.

• This mRNA is mixed with free DNA nucleotides and the enzyme reverse transcriptase.

• The free DNA nucleotides align next to their complementary bases on the mRNA template.

• Reverse transcriptase then joins the DNA nucleotides together to produce a fragment of DNA (gene) for insulin production.

• The DNA strand produced by this technique is known as complementary DNA (cDNA).

• Double-stranded DNA is produced from this cDNA using DNA nucleotides and the enzyme DNA polymerase.

why is mRNA actually used to produce the DNA

The absence of introns means these fragments can be transcribed by bacteria (usually they cannot)

• mRNA is a lot more accessible in cytoplasm

• present in abundance in cells making proteins

what could be a disadvantage of using restriction enzymes to obtain a gene fragment

A gene or a fragment obtained in this way from a eukaryotic organism will contain introns.

how can a gene machine be used for production of fragments

• doesn’t need pre-existing DNA or mRNA as a template.

• the amino acid sequence of a protein is used as a template to determine the sequence of DNA nucleotides for a specific gene.

• This is an automated process where the required nucleotide sequence is programmed into the gene machine

• absence of introns

what is the promotor region

initiate transcription of the gene by promoting the binding of RNA polymerase

what is the terminator region

marks the end of a gene and triggers the release of the mRNA transcribed.

in what ways can DNA be amplified

• In vivo – where the copies are made inside a living organism.

• In vitro – where the copies are made outside a living organism (usually by PCR).

how can DNA be transferred

via the use of vectors

give examples of vectors

• plasmids (bacteria)

• viruses

• liposomes

what are bacteria widely used for?

• Produce a protein coded for by a transferred gene (e.g. human insulin)

• Clone genes or fragments. This is known as in vivo cloning. The rapid reproduction rate of bacteria enables a transferred gene to be quickly copied so that a large amount of gene product can be obtained.

how can we insert a gene into a host for use in recombinant DNA tech?

1. Isolate the desired gene using either restriction endonucleases, reverse transcriptase or gene machine methods. 

2. Amplify the gene using PCR.  (could also be amplified by bacteria - in vivo)

3. Insert the DNA fragment into a suitable vector, either virus, liposome or plasmid. 

4. Transfer the DNA into a suitable host cell  

5. Identify the gene has been taken up using gene markers. 

how are plasmids used as vectors to transfer genes into prokaryotic cells

• A plasmid is cut using the same restriction endonuclease used to cut the gene.

• The plasmid DNA and the ‘foreign’ DNA join by base-pairing as they have complementary sticky ends.

• The enzyme ligase is used to form the phosphodiester bonds.

• The plasmid with the foreign DNA is referred to as a recombinant plasmid.

• These plasmid vectors are added to a culture of bacteria, some of which take up the recombinant plasmid by a process called transformation

why is the use of vectors not guaranteed to work?

• The cells may not take up the vector at all.

• The cell may take up the vector, but the vector may not contain the gene. e.g. the plasmid may have joined back together without the ‘foreign’ gene/DNA being taken up.

how are bacteria encouraged to take in the plasmid?

bacteria treated with calcium chloride and heat shocked with high voltage - which makes the bacterial membrane more permeable

what are marker genes used for?

to check that the gene or fragment has been successfully transferred from vector to recipient

name an example of a marker gene and describe how it works

• GFP gene - which codes for a green fluorescent protein that lights up

• The GFP gene is added to the gene being transferred.

• Successfully transformed bacteria or eukaryotic cells can then be identified as they fluoresce when viewed with UV light under a microscope.

evaluate the uses of recombinant technology

• humanitarian

• environmentalist

• anti- globalisation activists

BENEFITS FROM HUMANITARIANS

• reducing famine and malnutrition by developing GM plants or animals which produce high yields and are resistant to disease

• producing vaccines and drugs

• treating genetic diseases by gene therapy

OPPOSITION FROM ENVIRONMENTALISTS AND ANTI GLOBALISATION ACTIVISTS

• possible transfer of foreign genes to non-target organisms, including humans

• it is an irreversible process with no certainty of economic benefits

• ethical considerations with regard to permanently altering the genome of animals

• long term ecological and evolutionary consequences are unknown

• large companies having a monopoly leading to lack of choice and forcing smaller companies out of business

what is gene therapy

Using recombinant DNA technology for the treatment of genetic diseases.

• It involves the introduction of functional copies of an allele into an organism which possesses defective alleles of the same gene.

what are some stages involved in gene therapy?

• Identifying the gene causing the disease

• Obtaining and cloning copies of the functional allele

• Transferring these functional alleles into the patient e.g. by the use of a vector

• Ensuring that the alleles reach their target cells and function normally.

what are limitations with gene therapy?

• vector may not reach cell

• allele may not enter the nucleus

• allele may not be incorporated into DNA of nucleus and not be further transcribed

• patient may mount an immune response against the virus

• modified cell containing containing normal gene can die anyways

• normal gene may be inserted into another gene - disrupting its function or could lead to tumours

briefly describe how gene therapy is used for cystic fibrosis

• Cystic fibrosis is an inherited disorder that develops in individuals who are homozygous recessive

• gene codes for chloride ion channel that helps move chloride ions in and out of cells

• affected individuals secrete large amounts of abnormally, thick and sticky mucus by epithelial cells particularly in the lungs and pancreas. This can have severe consequences.

• gene therapy is being attempted to alter epithelial cells in the lungs of affected individuals

what are DNA sequencing projects

involves techniques to determine the sequence of DNA nucleotide bases in an organism – the genomehow

how can the proteome be determined and why does this not work in more complex organisms?

• Determining the genome of simpler organisms allows the sequences of the proteins that derive from the genetic code (the proteome) of the organism to be determined.

• In more complex organisms the presence of non-coding DNA and of regulatory genes means that knowledge of the genome cannot easily be translated into the proteome

what are applications of genome projects

• identification of potential antigens for vaccine production

• spot changes in genes and association with diseases and phenotypes

• identify individuals in forensic science

• study the genome and the proteins it produces

• identify disease causing mutations in an organism

• detection of specific cancer alleles

• personalised medicine

what are DNA probes and DNA hybridisation used for?

screen individuals for specific alleles.

how can we use DNA probes to screen for DNA sequences?

1. A labelled DNA probe is made that is complementary to the DNA sequence of the allele being investigated.  

2. Multiple copies of the labelled DNA probe are made using the Polymerase Chain Reaction.  

3. The DNA that is being tested is extracted from the tissue sample and then broken down (by restriction endonucleases) into smaller fragments.  

4. The fragments are separated by gel electrophoresis.  

5. The DNA fragments are treated to split it into single strands which are transferred to a nylon membrane. 

6. The DNA probe (or probes if screening for more than one mutant allele) is added to the nylon membrane.  

7. The membrane is ‘washed’ to remove any unattached DNA probes.  

8. If the allele is present, the labelled DNA probe will bind to the complementary bases of one of its strands i.e. DNA hybridisation.  

9. The presence or position of the probe can be identified by the radioactivity or fluorescence it emits.

The use of labelled DNA probes and DNA hybridisation to locate specific alleles of genes enables patients to be screened for:

• heritable conditions e.g. sickle cell anaemia, cystic fibrosis, Huntington’s disease

• individual drug responses – people can respond differently to particular drugs due differences in their alleles. This can lead to personalised medicine – i.e. specific drugs being prescribed for certain individuals for particular diseases.

• health risks e.g. the BRCA1 gene increases the risk of developing breast cancer by between 50 to 85%

what is genetic counselling used for when mutant alleles are present

• help people understand the probability of them developing a disease.

• advise prospective parents who may be carriers of disease-causing alleles.

• to help decide the best course of drug treatment for genetic diseases

what are VNTRs

• variable number tandem repeats

• The genome of an organism contains many repetitive, non-coding sequences of nucleotide bases

how can VNTRs be used?

can be used to determine the relatedness between two individuals because, the more related two organisms are, the more similar their VNTRs will be

• as well as to match the identity of a DNA sample (for e.g. at a crime scene) to an individual.

how can we use VNTRs to produce a genetic fingerprint?

1. PCR is used to amplify the sample.   

2. The DNA is then cut into fragments, using restriction endonucleases. The endonucleases used cut DNA at sites close to, but not within, the VNTRs. This therefore gives a large number of DNA fragments. 

3. These fragments are separated by gel electrophoresis.   

4. The fragments are treated (using alkali) to form single strands.   

5. The single strands are transferred, in the positions they have moved to, on to a nylon ‘membrane’.   

6. Radioactive DNA probes are then added. These are complementary to the repeated sequences and allow the positions of the fragments to be identified. 

7. The excess DNA probe is washed off, leaving behind only the hybridised probe.  

8. The radioactive probes allow the position of the fragments to be identified when the ‘membrane’ is placed onto an X-ray film i.e. the genetic fingerprint is obtained. 

what is the position of the fragment dependent on?

number of nucleotides present. This will correspond to the number of repetitive sequences in each fragment

when two genetic fingerprints are compared what does it mean if they have a band in the same position?

If both fingerprints have a band at the same position on the gel it means that they have the same number of nucleotides and so the same number of repetitive sequences at that place. However, apart from identical twins the genetic fingerprints will not be the same.

why is the use of genetic fingerprinting important?

• Forensic science – compare DNA samples (extracted from blood, hair, semen) from a crime scene with the DNA of suspects.

• Medical diagnosis – certain diseases involve unique patterns of several alleles and can identified more readily by genetic fingerprinting.

• Determining genetic relationships and determining genetic variability within a population (more closely related species or organisms have more similar VNTRs)

• Animal and plant breeding – ensuring genetic diversity is maintained by screening organisms to prevent inbreeding between closely related individuals.

how can genetic fingerprinting be used in classification and paternity cases?

• In these cases, the child’s non-coding sequences (or bands) must have been inherited from one parent or the other.

• The mother presumably knows the child is hers. The child’s bands that do not correspond with the mother’s must have come from the father.