the control of gene expression Flashcards
what is a mutation
change in the arrangement of bases in an individual gene or in the structure of a chromosome (which changes the arrangement of genes)
what causes a frameshift
- deletion
- addition
what causes a point mutation
substitution mutation
so only results in a change of one base/amino acid
nonsense mutation
results in a stop codon being inserted and the polypeptide chain being truncated (shortened)
missense mutation
still codes for an amino acid but it is an incorrect one which alters the functioning of the protein
silent mutation
has no apparent effect on a phenotype. the substitution does not change the amino acid sequence
types of gene mutations
- base duplication
- inversion
- translocation
what is a base duplication
several bases are repeated causing a frameshift to the right
what is an inversion of bases
number of bases are reversed
what is translocation within bases
several bases are removed from one chromosome and become inserted into a different one
what is a whole chromosome mutation
an entire chromosome is lost or repeated during cell division
e.g. down syndrome - has an extra chromosome 21
the effect of mutations
- production of advantageous alleles
- neutral mutation
- production of disadvantageous proteins
mutation effect of the production of advantageous alleles
gain a reproductive advantage
mutation effect of a neutral mutation
no change
mutation effect of the production of disadvantageous alleles
fatal or causes disease
what are stem cells
an undifferentiated cell
what can stem cells do
- self renew - to ensure a constant supply, maintain a stem cell pool
- differentiation - replaces dead or damaged cells throughout the life of the organism
features of totipotent
- can divide and produce any body cell
- occur only for a. limited time in early mammalian embryos
the process of totipotent cells
- fertilised egg has the potential to form any human cell - it’s totipotent
- as fertilised egg divides the specific genes are turned off so only certain genes are made
- protein synthesis is prevented by preventing transcription or translation
- once cells are specialised only the relevant genes will be translated from the DNA
features of pluripotent
- found in embryos
- can divide into unlimited numbers
- is used in treating human disorders
- can divide into almost every cell but not placenta cells
features of mulitpotent
- found in mature mammals (adult cells)
- divides to form a limited number of different cell types
e.g bone marrow stem cells make platelets, red, and white blood cells
features of unipotent
- found in mature mammals (adult cells)
- used to make cardiomyocytes
features of Induced pluripotent stem cells (IPS cells)
produced from adult somatic cells using appropriate protein transcription to overcome some ethical issues with using embryonic stem cells
how to produce IPS cells
- take a sample of adult cells from the liver
- switch the genes off that make the cell specialised
- turns the cell into a pluripotent state
- switch genes back on
- this is done through transcriptional factors
features of embryonic stem cells
- ES cells can be isolated from the early embryo (<14 days) and grown in culture
- using the correct growth factors and nutrients ES cells can be made to differentiate into many types of specialised cells
positives and negatives of embryonic cells
- need embryo - unethical
- uncontrollably divide
- differentiate into anything
- used within 14 days
- chance of rejection
- can self-renew
- divides definitely
- doesn’t have a gene added
positives and negatives of IPS cells
- no need for embryo
- uncontrollably divides
differentiate into anything - no chance of rejection
- can self-renew
- divides indefinitely
- have a gene added
plant stem cells
- plants unlike animals maintain totipotency in the form of meristem cells
- cuttings/ tissue culture can be used to produce whole plants or plant organs under the right growth conditions
why do you regulate the transcription of genes
it ensures that cellular resources are not wasted
how is cancer formed
when genes are not correctly regulated
what are transcription factors
proteins that bind to DNA at promoter regions. a specific combination of transcription factors is needed for the transcription of a gene to occur
are all transcription factors the same?
no
different transcription factors are present in different cells and at different stage of development
different transcription factors
- activator molecules - promote
- repressor molecules - prevent
what happens when a gene does not need to be transcribed?
an inhibitor molecule blocks the DNA binding site on the transcription factor. this prevents transcription
activated oestrogen process
- oestrogen is lipid soluble so it passes easily through the cell surface membrane
- oestrogen combines with a receptor site on the transcription factors (complimentary binding)
- the binding of oestrogen changes the shape of the transcription factor causing it to release the inhibitor molecule
- the transcription factor enters the nucleus and combines with DNA. transcription is activated
what is oestrogen effect on cancer
linked to breast cancer. cells that form breast cancer have receptors that are complimentary to oestrogen
what is epigenetics
the study of heritable changes in gene function that does not involve change to the base sequence of DNA
what is the epigenome determined by
environmental factors
what determines the shape of the histone-DNA complex
histones and DNA get covered in chemical tags. this is the epigenome.
what happens if DNA is tightly coiled
genes are inaccessible for transcription
what types of chemical tags are there
- acetyl group on histones
- methyl group on cytosine bases
what is acetylation
adding acetyl groups
- acetylation of histone groups leads to transcription of genes
- removal of acetyl groups prevents transcription of genes as DNA coils tightly
what is methylation
- low methylation promotes transcription
- high methylation inhibits transcription
treating diseases with chemical tags
drugs are being developed to inhibit the enzymes involved in acetylation or methylation
what is small interfering RNA
when gene expression can be prevented by breaking down mrna before it is translated into a polypeptide. occurs in both prokaryotes and eukaroytes
process of small interfering RNA
- an enzyme cuts large double-stranded molecules of RNA into small interfering RNA
- the siRNA combines with an enzyme and is separated to give single strands
- the siRNA molecule allows the enzyme to act only on the complimentary mrna sequence
- once in position the enzyme cuts the mrna into smaller section. the mrna can no longer be translated into a polypeptide. the gene is not expressed
what is a tumour
abnormal cells that undergo rapid cell division
carcinogens
these are cancer-causing agents that cause changes to genes that control cell division. This leads to uncontrolled growth of the abnormal cells and results in a tumour
features malignant tumours
- cells become unspecialised
- grow rapidly
- cells spread to other regions - aka metastasis
- effects the whole body
- treatment generally chemo/radiotherapy
features of benign tumour
- grow very slowly
- cells remain specialised
- tend to have localised effects
- the tumour is surrounded by a capsule so forms a compact structure
- Tumours stay within the tissue they originate in
what is the rate of cell division (mitosis) controlled by
proto-oncogenes - they stimulate cell division
how does a proto-oncogene function
a specific proto-oncogene needs to be switched on by a growth factor or hormone to cause a cell to grow and divide
tumour suppressor genes function
They control cell division by inhibiting the proto-oncogenes, repairing DNA and activating apoptosis (cell death)
what happens when a proto-oncogene mutate
- it can mutate into an oncogene
- mutations are mainly acquired
- results in gene activation (transcription) in the absence of a growth factor
effect of a proto-oncogene mutation
one copy of a mutate proto-oncogene is enough to stimulate cells to divide out of control
what happens when the tumour suppressor gene mutates
- it becomes inactivated
- allows the rate of cell division to increase or DNA not be repaired
- both copies of the gene needs to be mutated to cause cancer
what can tumours be associated with (in terms of proto-oncogenes & tumour suppressor genes)
- reduced methylation of proto-oncogenes
- increased methylation of tumour-suppressor genes
why are introns the problem in genetic sequencing
the presence of non-coding DNA and
of regulatory genes means that knowledge of the genome cannot
easily be translated into the proteome
Identifying the sequences of proteins
Determining the genome of organisms allows the sequences of proteins that derive from the genetic code (proteome) to be determined. This is used in the identification of potential antigens for vaccine.
What is gene sequencing
Identifying the order of the base pairs in a segment of DNA
Sanger sequencing/ chain termination
step 1 and 2
- 4 test tubes labelled A,T,C, G. to all test tubes, add: DNA sample, radiolabeled primer (allows or visualization of DNA), DNA polymerase
- Add a modified nucleotide (conc 1%) into each test tube. Modified nucleotides can’t form phosphodiester bonds so if inserted, DNA synthesis stops
Sanger sequencing/ chain termination
step 3 and 4
- DNA polymerase synthesises multiple copies of the DNA sample. In tube A, all the fragments terminate at A. A full range of DNA molecules will be synthesised, varying from full length to a single base.
- The contents of 4 tubes are run on an electrophoresis gel, and the DNA bands are visualised. fragments sorted by length & sequence is read starting with the smallest fragment at the bottom and reading upwards
How does gel electrophoresis work
- DNA has a -ve charge so travel towards +ve electrode
- the distance travelled is determined by the size of the fragment- smaller moves further
- Fragments are fluorescently labelled or radiactively labelled
what is cycle sequencing
- Automates and speeds up the sanger method
- The 4 modified nucleotides are fluorescently labelled, each with a different colour, and the reaction is done in a single tube
- Fragments are separated using a capillary electrophoresis
- The fluorescence is detected and converted into a DNA sequence by a computer program
what does recombination DNA technology involve
the transfer of fragments of DNA from one organism to another
How are fragments of DNA (genes) produced
- using reverse trnascripase
- using restriction enzymes
- gene machine
using reverse transcriptase process
- mRNA is extracted form a cell that readily produces the protein of interest
- Incubate the mRNA with reverse transcriptase to synthesize cDNA strand (complementary strand to the mRNA)
- when cDNA is complete the mRNA is hydrolysed
- DNA polymerase synthesises the second strand of the DNA to form double-stranded DNA
DNA produced from using reverse transcriptase
DNA produced is a copy of the original gene but without the introns
using restriction enzymes in bacteria
Bacteria contain restriction enzymes to protect themselves from the phage virus. Bacteria use them to cut up viral DNA. The enzymes cut the DNA at restrictive sites
how od restrictino enzymes work
Isolate a specific gene from a larger DNA molecule. Restriction enzyme recognises a specific palindromic sequence and cuts the sugar phosphate backbone of both strands
sticky ends
At the end of the DNA fragments, there are exposed bases. sticky ends enable two strands of DNA to be specifically joined together by complimentary base pairing
What can some restrictions result in
blunt ends - these do not have exposed bases
gene machine
- Artificial synthesis using a “gene machine”
- scientists use computers to generate the nucleotide sequence to produce the gene
- Short fragments of DNA are first produced which join to make longer sequences of nucleotides and are inserted into vectors
- This creates genes contained in vaccines and synthesises new bacteria genomes
Advantages of the gene machine
- create any sequence of nucleotides that are required
- DNA fragments produced do not contain introns
- can be transcribed and translated by proakryotes
- ngates the need for restriction enzymes which require there to be a ocnenient restriction sit
The polymerase chain reaction (in-vitro gene cloning) PCR features
- PCR can clone and amplify DNA samples as small as a single molecule
- useful for producing large quantities of fragments
whats in the PCR test tube
- TAQ polymerase
- primer
- free nucleotides
- DNA
has to be stable at high temperatures
PCR Process
- DNA strands are separated by briefly heating to 95°C. This break’s the hydrogen bonds
- The DNA is cooled to 55°C to cause the primers to anneal to the DNA
- The temperature is increased to 72c, the optimum temperature for the polymerase. It extends the primers and replicates the remaining DNA
- Cycles can be repeated 20-30 times depending on the quantity needed
why are sticky ends important
DNA from different sources can be joined together with the same sticky ends. This occurs if they have been cut using the same restriction enzyme
how can sticky ends be joined together
by DNA ligase, this joins the sugar phosphate backbone together, and the new DNA molecule is called recombinant DNA
what is a vector
used to transport DNA into a host cell
what are plasmids
most commonly used vector. They are useful as they nearly always contain genes of antibiotic resistance
plasmid features
- contains 2 antibiotic resistance genes
- 1 is disrupted when the restriction enzyme cuts open the plasmid
- the other is used to select cells which have taken up the plasmids
in-vivo gene cloning
Introduction of DNA into host cells/ transformation
- plasmids reintroduced into host cells
- The bacteria, plasmids and Ca2+ are mixed
- by altering the temperature, the membrane becomes permeable and the plasmid moves through
in-vivo gene cloning
Identification of bacteria containing a plasmid with the DNA fragment
- gene markers are used to identify which palsmdsi have taken up the DNA fragment
- gene markers can be: resistant to an antibiotic, fluorescent protein, enzyme whose action can be identified
- the gene marker is disrupted if the DNA fragment is present
in vivo gene cloning
identification of bacteria containing a plasmid - antibiotic
- donor DNA si inserted into the middle fo one of the antibiotic resistance genes
- This disrupts the gene and stops it being expressed i.e. no resistance to that antibiotic
- Replica plating is used to identify the bacteria with the reocmibannt plasmid
- Bacteria are transferred to identical positions on plates containing ampicillin and hen tertraceyline
- The bacteria with the donor DNA will be able to grow on ampecillin, but not tetracyline
fluorescent markers
- the generator green lfuroeoesnct protien (GFP) can be inocrpototpated into a plasmdi
- if the donor DNA is inserted into the GFP gene, the bacteria will not grow and can be identified
enzyme markers
- the enzyme lactase turns a colourless substrate blue
- If the donor DNA is inserted into the gene for lactase, the substrate will remain colourless
what is gene therapy
treatment of genetic diseases by providing the sufferer with a corrected copy of their defective gene
feautres of somatic cell therapy
- copies of the corrected gene are inserted into the body cells of sufferers
- doesn’t prevent the disease from occurring in the next generation
- Repeated many times as the effects don’t last long
- if inserted into the wrong location, it disrupts the functioning of other genes
Features of germ-line therapy
- the corrected gene is inserted into a fertilised egg by IVF
- If successful, all the cells of the embryo contain the corrected gene due to mitosis
- germ cell therapy is permanent and ensures offspring inherit the corrected gene
- currently illegal
why is gene therapy more difficult when targeting the lungs
a vector is needed to introduce the corrected gene into the cells of the lung
liposomes or viruses
Liposomes as vectors
- The functioning gene is isolated and inserted into plasmids
- Recombinant plasmids are placed back into bacteria and cloned
- Plasmids are extracted & wrapped in lipid molecules forming liposomes
- liposomes sprayed into patients’ airways
- liposomes fused with plasma membrane of target cells and into the nucleus
viruses as vectors
- adenviruses modified to make it harmless
- Adenoviruses are grown in cells in-vitro. Recombinant plasmids that contain the normal gene are added.
- Recombinant plasmids are taken up by adenovirus. gene becomes part of virus DNA.
- Viruses are isolated and sprayed into the nostrils of patients
- viral DNA inserts into epithelial cells of patients’ lungs
advnatges of viruses as vectors
- good entry into speicfic cells
- easy to modify
disadvnatges of viruses as vectors
- limited amount of genetic material
- may trigger an immune response
advantages of liposomes as vectors
- larger genes possible
- less liekly to trigger an immune response
Disadvantages of liposomes as vectors
- less efficient - as there are multiple physical barriers to overcome for gene delivery, i.e. plasma membrane, cytoplasm, nucleus
what can specific alleles being located identify
- inherited conditions
- those with particular health risks, e.g. mutated tumour suppressor genes
- those that respond to particular drug treatments
what is a DNA probe
short, single-stranded DNA with a label attached to i. so they’re easily identified
process of locating specific alleles of a gene
- Determine the sequence on nucleotides on mutated alleles using DNA sequencing techniques
- make DNA fragment with a complementary base sequence to the mutated allele’s one
- Use PCR to make lots of copies of the DNA probe
- Obtain DNA from the person with the allele and heat it - to separate strands
- Cool strands and mix with DNA probes
- if mutatated allele is present. DNA probe will bind to complementary base sequence
- Wash DNA to remove unattached probes
- The remaining hybridized DNA will be labelled with fluroscent markers
- detect with UV light & microscope
using a DNA microarray
- Fluorescently label a human DNA fragment
- Wash this over the array
- if any human DNA sequences match any of the DNA probes sequences, they will stick to the array
- Wash the array & remove unattached DNA
- expose to UV light. any spot that fluoresces has that specific mutant allele
what is genetic counselling
advising people about screening - looking for mutated alleles and explaining results of screening.
to enable people to make personal decisions about themselves and their offspring
what does genetic counselling involve
- researching family history
- advising parents of likelihood of it arising in children
- informs effects of a disease i.e. psychological, medical, social…
What does genetic fingerprinting rely on
Eukaryotic organisms DNA genome contains many VNTR. These are repetitive, non-coding bases of DNA
what happens if two individuals have similar VNTR
the individuals are more closely related
5 stages of creating a genetic fingerprint
- extraction
- digestion
- Separation
- hybridisation
- development
genetic fingerprint stages
1. extraction
- extract DNA sample from the rest of the cell
- it can be tiny, so need to increase quantity by using PCR
genetic fingerprint stages
2. digestion
- cut DNA into fragments using same restriction endonucleases
- fragments contain minisatellites (short region of DNA, with unique repeating sequences found in introns - not responsible for expression of proteins)
genetic fingerprint stages
3. Separation
- DNA fragments are separated according to size by gel electrophoresis
- immersed in alkali (denaturaiotn) to separate double into single strands
genetic fingerprint stages
4. hybridisation
- radioactive/fluroesecnt DNA probes added
- bind to VNTR’s if the base sequence of probes are complementary to VNTR’s
- different probes bind to different target DNA sequences
genetic fingerprint stages
5. development
- x-ray film is put over a nylon membrane & exposed by radiation
- These points correspond to a position of DNA fragments separated during electrophoresis; bars are revealed
- pattern of the bands is unique to every individual except identical twins
uses of genetic fingerprinting
- genetic relationship. and variability - paternity testing, genetic variability in the population
- forensic science - prescence of a person at a crime scene
- medical diagnosis
- plant & animal breeding - prevent undesirable inbreeding
