Unit 8: control of gene expression Flashcards
what are mutations
changes in the sequence of nucleotides in DNA nucleotides
what are the different types of mutations
insertion/deletion mutations
duplication
inversion
translocation
what are insertion/deletion mutations and what are the effects of this mutation
where one or more nucleotide pairs are inserted or deleted from the sequence. this type of mutation alters the sequence of nucleotides after the insertion/deletion point, known as frameshift
3 effects of substitution
-formation of stop codon, which will stop production of the polypeptide prematurely, protein produced wont be functional
-formation of a codon for a different amino acid, polypeptide may differ in shape and be dysfunctional
-different codons produces the same amino acid because code is degenerate so the polypeptide produced is the same
what is a duplication and what is the effect of this mutation
one or more bases are repeated and therefore produces a frameshift
what is inversion and what effect does this mutation have
a group of bases become separated from the DNA sequence and then rejoin at the same position but in reverse order, this therefore affects the amino acid that is produced
what is translocation and what effect does this mutation have
a group of bases become separated from the DNA sequence on one chromosome and are inserted into the DNA sequence on another chromosome, this can often lead to significant effects on the phenotype
what is the effect of translocation on bases
leads to an abnormal phenotype
e.g. development of some cancers or reduced fertility
what is ‘frameshift’ mutation
a genetic alteration resulting from the insertion or deletion of nucleotides in a DNA sequence that is not a multiple of 3.
-it impacts all the base triplets downstream from the mutation, causing a shift in the reading frame and potentially resulting in different amino acids being coded
what are mutagenic agents and examples
factors that increase the rate of mutations occurring
e.g. radiation (UV), chemicals (benzene) and some viruses (HPV)
how can mutagenic agents increase the rate of mutations
deleting bases or changing their chemical structure so that they pair with bases that they wouldn’t normally do so.
-they can also change the structure of the dna itself, which can cause problems during dna replication
what are acquired mutations
mutations which occur after fertilisation
-if these occur in the genes that control mitosis, can cause uncontrolled cell division and hence may result in a tumor
benign tumors
non invasive
-usually grow slowly
malignant tumors
invasive
-grow rapidly
-invade and destroy surrounding tissue
-cells can break off and travel around in the blood or lymphatic system
what role do tumor suppressor genes have in the development of tumors
‘suppress’ cell growth
-slow cell division by producing proteins which will either stop cell division or cause the cell to self destruct
-if a mutation occurs, the protein will not be produced and the cells divide uncontrollably
what role do proto-oncogenes have in the development of tumors
a proto-oncogene is a gene that stimulates cell division by producing proteins that make cells divide. When a mutation occurs in a proto-oncogene, the gene can become overactive causing the cell to divide uncontrollably and resulting in a tumour (a mutated proto-oncogene is known as an oncogene)
hypermethylation of tumor suppressor genes
the gene is not transcribed or translated so no protein is produced to stop cell division
hypomethylation of proto-oncogene
not methylated enough
-can cause them to act as oncogenes
-this increases the production of cell division stimulating proteins causing uncontrolled cell division
how can increased oestrogen concentration lead to tumors forming
oestrogen stimulates breast cells to divide more frequently which increases the probability of mutation
-helps cancerous cells divide faster so tumor growth is rapid
-some research indicates that oestrogen can add mutations directly into the DNA of certain breast cells increasing the risk of them being cancerous
why can it be difficult to interpret data on the risk factors of cancer
-some cancers are polygenic- triggered by more than one gene
-some cancers triggered by many environmental factors- difficult to know which environmental factors are having the greatest effect
-can have a control group of lab animals but not people
why is understanding mutations and methylation levels important in preventing and treating cancer
prevention:
possible to screen for certain cancers and look for a mutation to their DNA
new, more sensitive tests are being developed which will diagnose the disease earlier, giving a better prognosis#
treatment:
mutations to a proto-oncogene can be treated with a drug that inhibits the enzyme produced by the mutation so the cells stop expressing it and the mutation does not spread, tumor does not grow
what are stem cells
undifferentiated cells which can differentiate into specialised cells
what are four sources of stem cells
embryonic, umbilical cord blood, placental, adult stem cells
embryonic stem cells
taken from embryos in early stages of development
-can differentiate into any cell
umbilical cord stem cells
taken from umbilical cord blood straight away
-similar to adult stem cells
placental stem cells
taken from placenta after birth
-can only specialise into specific cells
adult stem cells
body tissues of the foetus through to the adult and are specific to a particular organ/tissue within which they maintain and repair tissue through an organisms life
totipotent
can divide and produce and type of cell
-during development, totipotent cells translate only part of their DNA, resulting in cell specialisation
-only occur for a limited time in early mammalian embryos
pluripotent
found in embryos
-can divide to form a limited number of different cell types
-can divide in unlimited numbers and can be used in treating human disorders
multipotent and unipotent cells
both found in mature mammals
multipotent:
-can differentiate into a few different types of cells
unipotent:
-only differentiate into one type of cell
cardiomyocytes
unipotent cardiomyocyte heart cells may be able to replace old or damaged cardiomyocytes
induced pluripotent stem cells
(iPS cells)
-can be produced from adult somatic cells that are genetically altered to acquire characteristics of embryonic stem cells
-involves switching on certain genes within the cells to induce the expression of genes and transcription factors
transcription factors
proteins that control the rate of transcription
-their function is to regulate- turn on and turn off genes- in order to make sure they are expressed in the right cell at the right time
ethics of embryonic stem cells
-can become any type of cell
-has a right to life
-destruction of embryo that could develop into foetus in the womb
-however, an embryo not used in ivf would be destroyed anyway
ethics of adult stem cells
-does not destroy an embryo
-only becomes a limited number of cells
ethics of unfertilised egg 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 therapy
-improve the quality of life for many people
-using a patients own cells to grow organs and tissues which elimates risk of rejection or requirement of immunorepresent drugs
-costly for the NHS
how do transcription factors 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 the rate of transcription
activators in transcription factors
activators stimulate or increase the rate of transcription by helping RNA polymerase bind to the promoter region on the target gene
repressors in transcription factors
repressors inhibit or decrease the rate by binding to the promoter region preventing RNA polymerase from binding
what happens to the transcription factor if a gene is ‘switched off’
the site of the transcription factor is specific to that DNA is inactive because an inhibitor is attached
-this means it cannot cause transcription
how does oestrogen help with transcription
-lipid soluble nature of oestrogen means it can freely diffuse across the cell membrane where it binds to a receptor molecule on a transcription factor
-the binding alters the shape of the DNA binding site on the transcription factor and makes it able to bind to the DNA
-TSF therefore enters the nucleus via nuclear pore where it binds to the promoter sequence and initiates transcription
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 a complementary sequence of mRNA
-cell detects the double stranded form of mRNA and views it as abnormal. therefore, the enzyme is broken down by enzymes preventing translation
how does miRNA work in mammals
miRNA is not fully complementary to mRNA which means it can target more than one RNA molecule
-when miRNA is first transcribed, it creates a long, folded strand. which 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
the study of how environmental cues can cause heritable changes to an organisms phenotype without changing the DNA/genotype
what are epigenetic marks
chemical groups or DNA or histone proteins that regulate the activation or silencing of genes through modulation of the intermolecular interactions between the DNA strands and the protein machinery
-do not alter base sequence of DNA
-they alter how easy it is for enzymes and other proteins needed for transcription to interact and transcribe the DNA
heritable changes of the epigenome
-epigenome is considered flexible, the tags respond to environmental cues
-the epigenome is an accumulation of these tags during a lifetime
-most tags are removed in the early foetus so do not get passed between generations
-those that escape removal are passed onto offspring. this means that the expression of some genes can be affected by environmental changes which affected their parents/grandparents
effect of acetylation on histones
DNA acetylation changes DNA structure.
-acetylation makes the chromosomes LESS condensed so its accessible to enzymes and can be transcribed, this switches the gene on
effect of deacetylation on histones
deacetylation is where an acetyl group is removed from a molecule
makes the chromatid MORE condensed, this means that the enzymes cannot reach it, transcription cannot take place. this switches the gene off
effect of methylation of DNA
DNA methylation is a process by which methyl groups are added to the cytosine bases of DNA. methylation inhibits the transcription of genes by:
-preventing the binding of TSF to the DNA
-attracting proteins that condense the DNA-histone complex.
this makes the nucleosome (DNA-histone complex) pack more tightly together
DNA inaccessible to transcription factors so enzymes (RNA polymerase)/proteins cannot interact with the gene
gene is not transcribed
-this switches the gene off and so it is not expressed
how can knowledge of histone acetylation and DNA methylation be used to treat diseases
-use of drugs to inhibit enzymes that cause methylation, which can re-active genes that have been silenced
-genes must be specifically targeted to prevent switching on/off genes being read incorrectly 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 a better chance of being cured
genome
the genetic constitution of an organism, the complete set of genes
proteome
all the proteins a cell/the genome can produce
-protein only produced when the cell is switched on
what did the human genome project do
determined the sequence of bases of a human genome
-more samples were collected than used and no names used to remain anonymity
whole genome shotgun sequencing
cutting the DNA into smaller sections with overlapping ends
-use of computer programs to assemble them into an entire genome
genome sequencing in simple organisms
-organisms such as bacteria, have only a small number of introns so it is relatively easy to determine the proteome
-can be very useful in medical research (determining the protein antigens on a disease-causing bacteria can help to develop new vaccines)
-also allows for diseases to be monitored during outbreaks
genome sequencing in complex organisms
-the presence of more 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 two different organisms that has been combined
-these organisms are known as transgenic/genetically modified
recombinant DNA technology
involves the transfer of fragments of DNA from one organism or species to another
how is it possible that the DNA of one organism is not only accepted by a different species but also functions normally when it is transferred
genetic code is universal (same in all organisms)
mechanisms of transcription & translation are essentially the same in all living organisms
-so transferred DNA can be transcribed & translated within the cells of the recipient organism and the proteins it codes for can be manufactured in the same way as they would be within the donor organism
reverse transcriptase to make DNA fragments
reverse transcriptase is an enzyme that catalyses the production of complementary DNA to form mRNA
PROCESS:
-cell that naturally produces the protein of interest is selected
-these cells have large amounts of mRNA for the protein so it is more easily extracted
-mRNA acts as a template for the reverse transcriptase enzyme
-this joins free DNA nucleotides with complementary bases to the mRNA sequence
-single stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme
-double stranded DNA is then formed on the cDNA template using the enzyme DNA polymerase
-this double strand of DNA is the required gene
-the cDNA is intron free because it is based on the mRNA template
using restriction endonuclease enzymes
restriction endonuclease are enzymes that cut up DNA
-each restriction endonuclease cuts DNA at a specific sequence of bases (a recognition sequence) where it has an active site complementary to the recognition sequence
-the recognition sequence is often specific palindromic sequences
-the DNA is incubated with the specific restriction restriction enzyme
-the restriction enzyme hydrolyses the DNA
-this leaves sticky ends after the cut which are used to anneal the DNA fragment with other pieces of DNA with complementary base pairs
using a gene machine
synthesis of DNA fragments from scratch without the need for a pre-existing DNA template using a compterised machine
-desired sequence is made in the machine if it does not already exist
-the first nucleotide is fixed with support
-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
-short sections of DN called oligonucleotides are produced by breaking off the support and supporting groups. they are then joined together to form long sections of DNA from each short section
advantages of using the gene machine
-any sequence of nucleotides can be produced
-in a very short time
-with great accuracy
advantages of using mRNA to make DNA fragments instead of restriction enzymes to cut gene from DNA
-more mRNA in cell than DNA- easily extracted
-introns removed by splicing (in eukaryotes) whereas DNA contains introns
-bacteria cant remove introns
what is in-vivo cloning
where copies of genes are made within a living organism
-as organism grows, it replicates the DNA
what is in-vitro cloning
where copies of the genes are made outside a living organism using the polymerase chain reaction
process of in-vivo cloning
-insert the DNA fragment into a vector
-vector DNA cut open by restriction endonuclease 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 recombinant DNA into the host cell so target DNA is integrated into the bacteria DNA
-the host cells take up 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
process of 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 the 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 in each cycle. 4 strands are created in each one
genetically modifying plants
-desired gene is inserted into a vector of either plasmid or bacteria
-the bacteria infects the plant and inserts its DNA into the genome
-the plant produces the protein if the correct promotor 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 and sperm
-germline editing is only of reproductive cells. this means it is possible to correct disease genes and pass them onto future generations
promotor gene
control what part of the body proteins are made in
-they are 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 recombinant DNA in agriculture
-crops larger and higher yield so prices will fall
-crops more resistant to disease
-increased shelf life
-crops can produce herbicides themselves, so cheaper
benefits of recombinant DNA in industry
-food production & cleaning can be done by enzymes made by recombinant DNA
-cheap production
-larger quantities made
risks of recombinant DNA in medicine
-companies may limit use of certain drugs by increasing charging prices because stock is limited
risks of recombinant DNA in agriculture
-plants could be infected with disease from vector
-decrease in biodiversity which damages food chains & ecosystem cohabitation
-seeds may blow into nearby farms and contaminate organic products
risks of recombinant DNA in industry
-no choice about eating GM food
-large companies control GM tech so small businesses are forced out because they cannot compete with lower prices
gene therapy
altering defective alleles to treat genetic disorders and cancer
somatic therapy
alters faulty alleles which are affected. doesn’t effect sex cells, so disorder can still be inherited e.g. CF
germ line therapy
alters the alleles in sex cells, this means that all the cells will be altered and the individual, as well as their offspring will suffer the disease- this is currently illegal
ethical issues of gene therapy
-who decides what traits are normal and which constitute a disability/disorder
-will the high costs of gene therapy make it available to the wealthy
-should it be allowed to enhance basic human traits (e.g. height, intelligence)
DNA probes
short, single stranded DNA molecule that can be used to locate specific alleles on genes
production of DNA probes
sequence the allele you want to screen for and then use PCR to produce multiple complementary copies of the allele
what do DNA probes also contain
markers
-show where the probe has bound
-most common markers are radioactive, detected using x-ray film or a florescent maker detected with UV light
process of using DNA probes to screen for an allele
- DNA sample digested into fragments using restriction enzymes. it is then separated using gel electrophoresis
- DNA fragments are then transferred to a nylon membrane and incubated with the fluorescent marker. if the allele is present, the DNA probe will hybridise to it
- UV light is shone on the nylon membrane, if the allele is present then the probe will have bound to the DNA fragment and will fluoresce
how can the use of DNA probes be helpful in genetic counselling
-used to give advice to people and their families about the options for genes to be screened
-can also advise if someone is a carrier for a mutant allele, what type of mutation it is and what treatments are an option
how can the use of DNA probes assist with personalised medicine
this means that it is personalised to their DNA
-doctors can predict the drugs you will respond best to and prescribe those
-this means that recovery time should be quicker and have a smaller impact on your life
genetic fingerprinting
technique that can detect differences in peoples DNA, it involves the use of variable number tandem repeats- which are short repeating sequences or bases
why are VNTRs useful in genetic fingerprinting
the probability of two individuals having identical VNTRs is extremely low therefore VNTR analysis can be used in genetic fingerprinting
process of making genetic fingerprints
-sample of DNA is taken and it is cut into fragments (using restriction enzymes) and PCR is used to make many copies of the areas of DNA within VNTR
-primers are attached to the sticky ends at either side of repeats and the whole repeat is amplified
-a fluorescent tag is attached, usually to the primer to be easily viewed under UV
-end result of fragments with different lengths which correspond to the number of repeats at a specific location
separating DNA fragments
fragments are placed into a well of agarose gel and covered in a buffer solution which conducts electricity. an electric current is passed through the gel
-DNA is negatively charged and so will move towards the positive electrode
-shorter, lighter fragments move further whilst longer, heavier wont move as far
analysis of separating DNA fragments
-gel is viewed under UV light and the DNA fragments are seen as bands
-2 or more individual bands can be compared, if they have the same bands then they have the same length of VNTRs at same location so its a match
why are ladders added to genetic fingerprinting when analaysing DNA fragments
ladder contains DNA fragments of known lengths, this allows you to calculate the length of the fragments that appear
