3.8 Control of Gene Expression Flashcards
3 effects of substitution
Formation of a stop codon which will stop production of the polypeptide prematurely so protein produced will not be functional
Formation of a codon for a different amino acid so the polypeptide may differ in shape and be dysfunctional
The different codon produces the same amino acid because code is degenerate so the polypeptide produced is the same
effect of deletion
Creates a frame shift so different amino acids will be coded for creating a non-functional protein
effect of addition
If less than three extra bases are added, it will cause a frameshift so a different protein will be produced
If three extras are added, the polypeptide will not be different to such an extent as it would be if there was a frameshift
effect of duplication
frame shift to right
effect of inversion
Base sequence of the affected portion is reversed which affects the resulting amino acid sequence
effect of translocation of bases
Lead to an abnormal phenotype ie development of some cancers or reduced fertility
define inversion and translocation
Inversion - a group of bases become separated from the DNA sequence and rejoin at the same position but in the reverse order
Translocation - a group of bases become separated from the DNA sequence on one chromosome and become inserted into the DNA sequence of a different chromosome
two mutagenic agents
high energy ionising radiation, chemicals
4 sources of stem cells
embryonic, umbilical cord blood, placental, adult stem cells
embryonic stem cells
embryos in early stages of development and can differentiate into any type of cell
umbilical cord blood stem cells
similar to adult stem cells
placental stem cells
develop into specific types of cells
adult stem cells
body tissues of the foetus through to the adult and are specific to a particular organ/tissue, maintain and repair tissues through an organism’s life
totipotent
Can divide and produce any type of body cell
Zygotes
Occur in early mammalian embryos for a limited time (the first few cellular divisions)
During development, they translate only part of their DNA, resulting in cell specialisation
pluripotent
Found in embryos after the first few cellular divisions
Can divide in unlimited numbers
Any cell excluding the placenta
Used in treating human disorders
unipotent
Found in mature mammals
Can only differentiate into one type of cell
Classed as adult stem cells even though the organism is not adult yet
Derived from multipotent and are made in adult tissue
multipotent
Found in mature mammals
Classed as adult
Forms a limited number of different cell types
cardiomyocytes
unipotent heart cells, which may be able to replace old or damaged cardiomyocytes
induced pluripotent stem cells
Produced from unipotent stem cells using protein transcription factors
Capable of self renewal so can replace embryonic stem cells, combatting the ethical issues of embryo use in research
The adult stem cells express transcription factors characteristic of pluripotent stem cells
Inject with a virus with genes coding for the desired transcription factors so the host cell produces them
ethics of embryonic stem cells
Could develop into a foetus, denying the right to life
However can become any type of cell so its useful
An embryo not used in IVF will be destroyed anyway
ethics of adult stem cells
Does not destroy an embryo
But can only become a limited number of cells
ethics of unfertilised egg stem cells stimulated to divide
No right to life involved as no embryo
Wouldn’t produce a foetus if implanted in the womb
benefits of stem cell medicine
Improves QOL for many
Can use a patient’s own cells so eliminates need for donor and risk of rejection/immunosuppressants
Costly for NHS
transcription factors
proteins which control gene transcription
how do TSF work
Eukaryotic transcription factors move from the cytoplasm to the nucleus via diffusion
Each factor has a site which binds to a specific base sequence at the beginning of the gene (promoter)
Once bound, transcription of the DNA begins and mRNA is produced so the information can be translated into a polypeptide.
TSF control the gene expression by controlling rate of transcription
activators (TSF)
stimulate or increase the rate of transcription by helping RNA polymerase bind to the promoter region of the target gene
repressors (TSF)
inhibit or decrease the rate by binding to the promoter region preventing RNA polymerase from binding
what happens to the TSF if a gene is switched off
the site of the TSF specific to that DNA is inactive because an inhibitor is attached. This means it cannot cause transcription or polypeptide synthesis.
how does oestrogen help with transcription
Lipid soluble so diffuses easily through the phospholipid bilayer of the cell surface membrane
Once in the cytoplasm, it binds to the oestrogen receptor and forms an OE-OER complex
This changes the shape of the DNA binding site of the TSF causing the inhibitor to be released
The transcription factor is now activated and enters the nucleus through a nuclear pore to bind to the promoter sequence and initiate transcription
what is gene silencing
Small double stranded RNA molecules stop mRNA from being translated into proteins
characteristics of SiRNA
Double stranded
Taken up by cells via vectors
Not in mammals, in lower animal + plant kingdoms
Binds perfectly so can only inhibit translation of specific mRNA sequences
characteristics of MiRNA
Single stranded
Made inside the cell within the introns of larger RNA molecules
In all animals + plants
Pairing is imperfect so can inhibit the translation of many different mRNA sequences
how does siRNA work
Unwinds in the cytoplasm, one strand is selected and the other is degraded
The single selected strand binds to mRNA
Proteins cut mRNA so it cannot be translated, the pieces of mRNA are then degraded
how does miRNA work
The strand is not fully complementary to mRNA so it can target more than one mRNA
Creates a long folded strand when first transcribed
This is then processed into a double strand then single by enzymes
One strand binds to mRNA, blocks translation instead of cutting it into pieces
The mRNA is either stored or degraded
define epigenetics
heritable changes in gene function without causing changes to the base sequence of DNA
heritable changes of the epigenome
The epigenome is considered flexible, the tags respond to environmental cues
It is the accumulation of these tags during a lifetime
Most tags are removed in the early fetus so do not get passed between generations
Some escape removal so are passed onto offspring, so expression of some genes are affected by environmental changes which affected the parents or grandparents of the offspring
effect of acetylation on histones
Acetylation makes the chromatin LESS condensed so it’s accessible to enzymes and can be transcribed
This switches the gene on
effect of deacetylation of histones
Deacetylation makes the chromatin MORE condensed because of the increased attraction of histones to phosphate groups of DNA so enzymes cannot access it for it to be transcribed
This switches the gene off
effect of methylation of DNA
Addition of a methyl group to the cytosine bases of DNA
This prevents the binding of TSF to the DNA
It can alternatively attract proteins that condense the DNA-histone complex (inducing deacetylation of the DNA) making DNA inaccessible to TSF
This switches the gene off
what happens in the early stages of cancer
High levels of methylation on a TSG inactivates the promoter regions so DNA cannot be transcribed and the TSG itself is inactivated, early stages of cancer
Increased methylation of protective genes means mutated DNA cannot be repaired and mutations spread, leading to cancers
how can knowledge of histone acetylation and DNA methylation be used to treat disease
Use drugs to inhibit enzymes that cause methylation, which can reactivate genes that have been silenced
Drugs to inhibit enzymes involved in acetylation as well
Genes must be specifically targeted to prevent switching on/off genes being read correctly which will cause a secondary cancer
Tests to identify the level of DNA methylation and histone acetylation to indicate an early stage of disease for the patients to seek early treatment and have better chance of cure
compare benign to malignant tumours
Benign tumours
Malignant tumours
Grow to a large size
Grow to a large size
Grow very slowly
Grow rapidly
Cell nucleus appears relatively normal
Cell nucleus is often larger and darker (due to abundance of DNA)
Cells are often differentiated/specialized
Cells become de-differentiated/unspecialized
Cells produce adhesion molecules that makes them stick together so they remain within the tissue from which they arise/ primary tumours only
Cells do not produce adhesion molecules so they tend to spread to other regions of the body (metastasis)/form secondary tumours
Surrounded by dense tissues remain compact
Not surrounded by a capsule so grow finger-like projections into surrounding tissue
Less likely to be life-threatening, can disrupt a vital organ functioning
More likely to be life-threatening as abnormal tissue replaces normal
Localized effects
Systemic effects such as weight loss and fatigue
Removed by surgery alone
Needs radio or chemotherapy as well as surgery
Rare reoccurrence
Frequently recur
role of TSG in tumour formation
If a mutation occurs in a TSG, the protein to stop cell division or cause apoptosis will not be produced. The cells will divide uncontrollably, forming a tumour
role of a proto-oncogene in forming a tumour
If a mutation occurs in a proto-oncogene, more of the protein to make cells divide will be produced so the cells will divide uncontrollably. This is now an oncogene.
hypermethylation of tumour suppressor genes
the gene is not transcribed or translated so no protein is produced to stop cell division/cause apoptosis
hypomethylation of proto-oncogenes
too many proteins will be produced and it now acts as an oncogene
how can increased oestrogen concentration lead to tumours forming
Estrogen stimulates breast cells to divide more frequently which increases the probability of a mutation
Helps cancerous cells divide faster so tumour growth is rapid
Can add mutations into DNA of breast cells so increases the risk of them becoming cancerous
why is it difficult to interpret data on risk factors
Polygenetic cancer are triggered by more than one gene so cannot be sure how one gene being present affects risk
Can be triggered by environmental factors, in which case the risk genes are not directly correlated to cancer
Difficult to know which of the environmental factors have the greatest effect
Cannot have a control group of people because it is unethical
how can understanding of mutations and levels of methylation be used for prevention + treatment of cancer
Can screen for certain mutations in DNA for certain cancers
Knowledge of increased risk means preventative steps can be taken
More sensitive tests can diagnosis earlier for a better prognosis
Mutations to a proto-oncogene can be treated with a drug which inhibits the enzyme produced by the mutation so the cells stop expressing it and the mutation does not spread so the tumour does not grow
what did the human genome project do
The human genome project created a sequence of the average composite derived of several individuals
More samples were collected than used and no names to remain anonymous
Sequenced yeast and zebra fish too
whole genome shotgun sequencing
cutting the DNA into smaller sections with overlapping ends and using computer programs to assemble them into the entire genome
genome sequencing in smaller organisms
There are only a small number of introns so determining the genome allows the proteome to be determined. This is useful to determine antigens on disease causing bacteria for vaccine production, also helps determine antibody resistance for better management and disease outbreaks monitored.
how is sequencing diseased rats helpful
comparing to the human genome identifies disease genes common to both so we can then identify how to treat the disease
genome sequencing in complex organisms
The presence of large numbers of introns and regulatory genes makes it difficult to find the protein-coding sections among them. This means the genome cannot be easily translated into the proteome.
recombinant DNA
DNA from 2 different organisms that has been combined
transgenic/genetically modified organisms contain recombinant DNA
why can DNA be transferred from one organism to another
the genetic code, translation and transcription mechanisms are universal.
reverse transcriptase to make DNA fragments
mRNA from the cell that produces the desired protein/gene is isolated and mixed with free nucleotides + reverse transcriptase
Reverse transcriptase uses mRNA as a template to make complementary DNA (cDNA)
To make the other strand of DNA, DNA polymerase is used to build up the complementary nucleotides on the cDNA template
The method targets mRNA instead of the gene because it is more abundant so easier to target
using restriction endonuclease enzymes
Restriction endonuclease enzymes recognise palindromic sequences and digest/hydrolyse the DNA
This leaves sticky ends after the cut which are used to anneal the fragment to another DNA fragment with sticky ends with complementary bases
Palindromic sequences can also be known as recognition sequences
using a gene machine
Desired sequence is made in the machine if it does not already exist
The first nucleotide is fixed with support i.e a bead
Nucleotides are then added one by one with a protecting group to make sure they are joined at the correct place with no unwanted branching
Oligonucleotides are produced by breaking off the support and protecting groups. They are then joined together to form long sections of DNA from each short section.
This method allows for DNA fragments to be made from scratch without needing a template
in vivo cloning
Insert the DNA fragment into a vector
Vector DNA cut open by restriction endonucleases to ensure sticky ends will be complementary to the DNA fragments
Vector + fragment DNA are mixed together with ligase which joins the ends together via ligation
Recombinant DNA is created
Either the vector DNA or the DNA fragment must have specific promoter + terminator sequences for the desired particular protein
The vector containing recombinant DNA transfers the gene into host cells
If a plasmid is used, a change in temperature and use of certain chemicals will encourage the cell to take it in
If a bacteriophage is used, it will inject the recombinant DNA into the host cell so target DNA is integrated into the bacterial DNA
The host cells taking up the vectors with the gene are transformed
Identify transformed cells
Marker genes are inserted into the vector with the DNA fragment
It will either code for antibiotic resistance or make the transformed genes fluoresce under UV light
Only those who are resistant to the specific antibody will be able to survive and replicate so those who grow are transformed
in vitro cloning/PCR
Create a mixture containing the DNA fragment, free nucleotides, DNA polymerase and primers
Heat the DNA mixture to 95c in order to break hydrogen bonds
Cool the mixture to 50-65c for primers to bind to the strands
Heat the mixture to 72c in order for the DNA polymerase to work
The DNA polymerase lines up free nucleotides along each template fragment strand to create complementary strands
PCR doubles the number of strands in each cycle. 4 strands are created in each one.
genetically modifying plants
Desired gene is inserted into a vector of either a plasmid or bacteria
The bacteria infects the plants and inserts its DNA into the genome
The plant produces the protein if the correct promoter gene is present
Can be used for added nutrients or to cause resistance to pests
A bacterial vector can infect the plant and cause it to develop the disease
genetically modifying animals
Desired gene is added into egg cell or early stage embryo
Modifying the egg means altering the genes of the germ cells which mature into GM egg + sperm
Germline editing is only of reproductive cells
This means it is possible to correct disease genes and pass them onto future generations
how to ensure the protein is made in the right area of the body
insert a promoter gene only activated by certain cells present in that area.
benefits of recombinant DNA in medicine
Cheaper production of treatments
Quicker process
Larger quantities
benefits of rDNA in agriculture
Crops larger and higher yield so prices will fall
Crops more resistant to disease
Increases shelf life
Crops can produce herbicides themselves so cheaper
benefits of rDNA in industry
Food production + cleaning can be done by enzymes made by recombinant DNA
Cheap production
Large quantities made
risks of rDNA in medicine
Conglomerates will limit use in order to charge higher prices because stock is limited
risks of rDNA in agriculture
Plants could be infected with disease from vector
Decrease in biodiversity which damages food chains + ecosystem cohabitation
Could breed with wild plants to produce superweeds
Seeds may blow into nearby farms and contaminate organic products
risks of rDNA in industry
No choice about eating GM food
Large companies control GM technology so small businesses are forced out because they cannot compete with lower prices
if an addition occurs at the end of the sequence
few, if any, amino acids they code for will differ and the resultant polypeptide will be normal/near normal
explain the effect if 3 duplicated bases are consecutive
frame shift is 3 bases long so subsequent codons are not affected. The resulting polypeptide will have an additional amino acid but otherwise unchanged.
explain the effect if 3 duplicated bases are inserted into separate locations on the DNA
codons after each duplication will change so polypeptide may have different amino acids but not all different due to degenerate code. After the third duplicate base, the codons will be unchanged.
when might a polypeptide not change from a frame shift
if the replacement codons are the same as the originals
why is mRNA unlikely to be cut by the enzyme attached to SiRNA if there are 2 siRNA strands
the second will have comp bases and it is unlikely these will complement a sequence on the miRNA
how could siRNA be useful for preventing disease or observing effects of mutations
siRNA that blocks a certain gene could be added to cells, by observing the effect we could determine the role of the blocked gene. Could also be used to prevent disease by blocking the gene that causes it
effect of a single base mutation
a change in the sequence of amino acids so a change in the hydrogen bonds which results in an alters tertiary structure
what do oestrogen, methyl groups, and acetyl groups bind to
oestrogen with protein + DNA, methyl binds with DNA, acetyl binds with protein
how to find median value
rank all values in ascending order and find the value with the same number of people above and below
how is a stem cell transplant an effective treatment for a cancer that prevents production of healthy blood cells
the patient will produce healthy blood cells, there will be no cancerous blood cells present. the stem cells will be able to divide and replicate to increase numbers of healthy blood cells.
suggest how iPS cells could correct red-green colour blindness
iPS cells divide and differentiate into green sensitive cones
Suggest how the growth of new blood vessels into damaged heart tissues
could increase the rate of repair of tissues
Greater blood supply (to damaged areas);
Bringing more oxygen / glucose for respiration;
Brings more amino acids for protein synthesis;
For cell repair / mitosis / division;
how can a virus become able to infect other species having previously been able to only infect one
Mutation in the viral DNA
Altered tertiary structure of the viral attachment protein
Allows the attachment protein to bind to receptors of other
species
techniques used to determine close relation of viruses by assessing viral DNA
gel electrophoresis, DNA fingerprinting, genome sequencing, PCR
Suggest two features of the structure of different proteins that enable them
to be separated by gel electrophoresis
mass/number of amino acids
charge
R groups differ
role of reverse transcriptase in RT-PCR
produces cDNA using mRNA
role of DNA polymerase in RT-PCR
joins nucleotides to produce complementary strands of DNA
why is DNA in the sample hydrolysed by enzymes before being added to the mixture in RT-PCR
to remove any DNA present because this DNA would be amplified/replicated
why does DNA replication eventually stop in PCR
there is a limited number of primers + nucleotides
inserting copies of desired gene into plasmid
Cut the plasmid with a restriction endonuclease;
(So that) both have complementary / sticky ends;
(Mix together) and add ligase to join the complementary / sticky ends
why are plasmids injected into eggs, and not directly into cells of the organism
the gene gets into all of the cells in the silkworm so gets into the cells that make silk
Radioactively labelled probes
identified using an X-ray film exposed by radioactivity
fluorescently labelled probes
fluoresce when bound to the target DNA sequence
Use of DNA probes to identify particular alleles
Sequence the allele you are screening for
Produce a fragment of DNA containing the complementary base sequence to the desired allele using restriction enzymes + separate with gel electrophoresis
Use PCR to produce multiple copies of the complementary DNA
Attach a marker to the DNA, making it a DNA probe
Sample DNA is heated to separate the two strands and cooled in a mixture containing the DNA probes
If the desired allele is present, the DNA probes will bind to it causing DNA hybridisation
Wash sample clean to remove unattached probes
The remaining hybridised DNA will fluoresce
screening for multiple genes
- Use a microarray with desired DNA probes attached
- Wash a sample of fluorescently sampled DNA over the microarray and wash again to remove any unattached DNA
- Detect where fluorescence occurs to see where binding has taken place to know of any genetic disorders (and their alleles) present
personalised medicine
Screen a person’s DNA and tailor the drugs they recieve to ones they will respond to best
This helps for a quicker recovery time, lesser side effects, and more effective dosages which reduce NHS costs
what is genetic screening useful to detect
Oncogene mutations, determines type of cancer and hence the most effective drug/radiotherapy to use
Gene changes that predict which patients are more likely to benefit from certain treatments and give the best chances of survival
A single cancer cell among millions of normal cells, detecting risk of relapse
what do genetic counsellors do
inform people who are carriers of genetic disorders the likelihood of their future children having the condition
Advice on treatments or preventative treatments if genetic test is positive
Options on genes to be screened if people have a history of family illness
Variable number of tandem repeats are
The base sequences which don’t code for proteins but repeat over + over
Probability of two individuals having the same VNTRs is very low
what is genetic fingerprinting
comparing the VNTRs of two individual’s genomes
explain the process of making a genetic fingerprint
Extraction - separate the DNA from the rest of the cell sample and use PCR to increase the volume of the VNTRs
Digestion - using the same restriction endonuclease enzymes, cut the DNA into fragments
Separation - separate the fragments by gel electrophoresis, the agarose gel is immersed in alkali to separate the double strands
Hybridisation - DNA probes bind to the VNTRS because they have base sequences complementary to the base sequences of the VNTRs
Development - View the gel under UV or X-ray depending on the type of labelled probe, bands are revealed to show where the DNA probes have attached. The gel can contain a DNA ladder to compare lengths of the fragments which appear to alleles of lengths of known value to identify bands present
explain the results of gel electrophoresis and how it works
Shorter, lighter fragments move further and longer, heavier fragments do not move as far in the agarose
An electric current is passed through the gel, DNA is negatively charged so will move towards the anode
5 uses of genetic fingeprinting
to determine genetic relationships, determine genetic variability within a population, forensic science, medical diagnosis, and plant/animal breeding
genetic fingerprinting on determining relationships
each band on a DNA fingerprint of an individual should have a corresponding band in one of the parents DNA fingerprint
genetic fingerprinting on determining a population’s genetic variability
a population with very similar bands in their GFs show little genetic diversity and vice versa
explain the use of genetic fingerprinting in forensic science
can match the DNA fingerprint of a suspect to that of DNA found at the scene of the crime, however this does not prove they carried out the crime only that they were present at some point. The probability that someone else’s DNA might match that of the suspect (and therefore it may not have been the suspect) needs to be calculated e.g if their culture tend to have partners from within their own small community
how can genetic fingerprinting be used for medical diagnosis
can match fingerprints with people who have various forms of the disease or those without the disease to predict the probability of developing symptoms and when
how is genetic fingerprinting useful in plant and animal breeding
used to identify organisms with a desirable gene so they are selected to reproduce in order to increase the likelihood their offspring have the characteristic the desirable gene produces, also prevents undesirable breeding
how is DNA broken down into smaller fragments (2 marks)
use of restriction endonuclease enzymes which cut the DNA at the recognition site
explain the use of a DNA ladder (2 marks)
contains fragments of known sizes to compare positions of viral fragments
explain the importance of knowing the strain of virus infecting a patient (2 marks)
- to see if it is resistant to antibiotics in order to prescribe effective ones
- to see whether any vaccine works against the strain so potential contacts can be vaccinated and the spread stopped
why are sticky ends useful in genetic engineering
can join two pieces of DNA by complementary binding
why is mRNA a better start point than DNA for genetic engineering
mRNA more abundant than DNA so easier to target, mRNA has no introns
function of primers
to mark a region of DNA to be copied
useful because the enzyme needs a starting strand onto which attach nucleotides
describe and explain how mutation causing substances could cause colon cancer
change in base sequences due to addition, deletion or substitution
changes mRNA transcribed
therefore amino acid sequence changed so there is a loss of function in the protein leading to uncontrollable cell division
role of RENs in formation of plasmids containing donor DNA
they cut open the plasmid at the same base sequence
role of DNA ligase in production of plasmids containing donor DNA
anneal phosphodiester bonds
explain the adv of inserting gene into isolated plant cells rather than directly into cells within a whole plant
isolated cells divide by mitosis, there is a rapid production of toxin-producing plants and all cells will produce the toxin
