Biology unit 8

gene mutation

Alteration of a base sequence in DNA that may result in an altered polypeptide, occur continuously and spontaneously

facts about gene mutation

-occur spontaneously, but the frequency of this occurring can be increased by factors called mutagenic agents

-likely to occur during DNA replication (interphase)

-because they alter the gene, they can result in a different amino acid sequence in the encoded polypeptide

how is a non-functioning protein formed

-if the amino acid sequence changes (mutation) when the protein is modified into the tertiary structure

-it will form hydrogen and ionic bonds in different places and fold differently

-resulting in a 3D shape and therefore a non-functioning protein

Mutagenic agents

-high energy and ionising radiation

-carcinogens

exposure increases risk of mutations, higher frequency of spontaneous mutations

high energy and ionising radiation

-mutagenic agents such as alpha and beta particles

-x rays and gamma rays

carcinogens

-The term given to chemicals that can alter the structure of DNA and interfere with transcription

-in tobacco smoke and peroxides

6 types of gene mutations

-addition

-deletion

-substitution

-inversion

-duplication

-translocation

Addition mutation

-adding one base so that all subsequent codons are altered

-frameshift mutation

-can be very harmful as the new codons could potentially code for new amino acids

-resulting in a different sequence of amino acids resulting in a non-functioning protein

deletion mutation

-deletion of a base in a sequence

-frameshift left

-could result in a different polypeptide chain and non-functioning protein

substitution mutation

-bases swapped for another

-no frameshift as no. of bases remain the same

-only one codon changing, as genetic code is degenerate it may still code for the same amino acid (silent mutation)

Inversion mutation

-section as bases detach from the DNA sequence but when they re-join they are inverted

-section of code is back to front

-results in different amino acids being coded for in this region

translocation mutation

-a section of bases on one chromosome detaches and attaches onto a different chromosome

-this is a substantial altercation with significant impact on gene expression therefore the resulting phenotype

Duplication mutation

-a section of DNA bases duplicated within the DNA sequence

stem cells

cells which are undifferentiated that can continually divide and specialise

differentiation is the process by which stem cells become specialised

totipotent stem cells

-stem cells that can divide and produce any type of body cell

-during development they only translate part of their DNA, resulting in cell specialisation

-they occur only for a limited time in early mammalian embryos

pluripotent stem cells

-found in embryos and can become almost any type of cell

-used in research with the prospect of using them to treat human disorders (e.g regrowing damaged cells)

-there are issues with this as sometimes the treatment does not work, or the stem cells continually divide to make tumours

-ethically questionable whether it is right to make a therapeutic clone to make an embryo to get the stem cells to cure the disease then destroy the embryo

multipotent and unipotent stem cells

-stem cells that are found in mature mammals and divide to form a limited number of different cell types

-multipotent (e.g inside bone marrow) can differentiate into a limited number of cells

-unipotent can only differentiate into one type of cell

sources of stem cells in mammals

-embryos up to 16 days after fertilisation (pluripotent)

-umbilical cord blood (multipotent)

-placenta (multipotent)

-bone marrow

induced pluripotent stem cells (iPS)

-can be produced from adult somatic cells using appropriate protein transcriptional factors to overcome some of the ethical issues with using embryonic stem cells

how iPS work

-created from adult unipotent cells

-they are altered in a lab to return to a state of pluripotency

-to do this the genes that were switched off to make the cells specialised must be switched back on

-this is done using transcription factors

-similar to embryonic pluripotent stem cells but do not involve the destruction of an embryo and the adult can give permission

-they have shown a self renewal property in which they can divide indefinitely to give limitless supplies (useful in medical treatment)

epigenetics

heritable change in gene function without changing the DNA  base sequence

caused by changes in the environment and can inhibit transcription

control of gene expression

-factors such as diet stress and toxins can add epigenetic (chemical tags) to the DNA, can control gene expression in eukaryotes

-a single layer of chemical tags on the DNA is called the epigenome and this impacts the shape of the DNA-histone complex and whether the DNA is tightly wound (not expressed) or unwound (expressed)

-if the DNA is tightly wound then transcriptional factors can not bind, therefore the epigenome can inhibit transcription

methylation of DNA

-increased methylation of DNA inhibits transcription

-methyl groups added to the DNA

-this prevents transcriptional factors from binding and attracts proteins that condense the DNA-histone complex

-preventing a section of DNA from being transcribed

acetylation of histone proteins

-decreased acetylation of histone proteins on DNA inhibits transcription

-if acetyl groups are removed from the DNA then histones become positive and are attracted more to the phosphate group on DNA

-making DNA and histones more strongly associated and hard for the transcription factors to bind

heterochromatin

-”silent”, tightly packed or condensed DNA

-increased methylation (inhibits transcription)

-decreased acetylation of associated histones (inhibits transcription)

-DNA tightly coiled

euchromatin

-”active”

-transcription will occur

-decreased methylation

-increased acetylation of histone proteins

-not tightly coiled

tumour suppressor genes

-these genes produce proteins to slow down cell division and cause cell death if DNA copying errors are detected

-if a mutation occurs in the TSG and prevents it from carrying out it’s function then cell division would continue and mutated cells would not be identified or destroyed

abnormal methylation

-links to control of transcription, methylation can turn a gene on or off

-TSGs could be hypermethylated, meaning an increased number of methyl groups are attached to it, this results in the gene becoming inactivated and becomes turned off

-the opposite could occur in oncogenes, they may be hypomethylated, reducing the number of methyl groups attached, resulting in the gene being permanently switched on

why is this bad?

control of transcription

-In eukaryotes, transcription of target genes can be stimulated or inhibited when specific transcriptional factors move from the cytoplasm into the nucleus

-this can turn genes on/off so only certain proteins are produced in a particular cell

-this is what enables them to become specialised

transcriptional factors

-transcription of a gene will only occur when a molecule from the cytoplasm enters the nucleus and binds to the DNA in the nucleus

-these molecules are proteins called transcriptional factors and each one can bind to different base sequences on the DNA and therefore initiate transcription of genes

-once bound, transcription begins, creating the mRNA molecule for that gene which can be translated in the cytoplasm to create the protein

-without the binding of a transcription factor, the gene is inactive and the proteins wont be made

oestrogen

-steroid hormone that can initiate transcription

-it does this by binding to a receptor site on the transcriptional factors

-when it binds to the transcriptional factor, it causes it to slightly change shape

-making it complementary and able to bind to the DNA to initiate transcription

gene regulation definition

genes are regulated by transcription factors

steroid hormone

bind to transcription factors e.g oestrogen

RNA interference- definition

a form of post-transcriptional modification which occurs in the cytoplasm resulting in the silencing of gene expression

RNA interference-process

-double stranded RNA hydrolysed into siRNAs

-siRNAs bind to protein complexes which use energy from ATP to separate the two strands of siRNA

-exposing the nucleotide bases, allowing them to pair with mRNA bases

-complementary base pairing with mRNA

-mRNA cut by the enzyme associated with siRNA

-cut mRNA cant be translated therefore wont produce proteins

cancer

-mutations in genes that regulate mitosis

-if these genes mutate and non-functioning proteins are made then mitosis is not regulated

-results in the rapid uncontrollable division of cells and the creation of a tumour

benign tumours

-can grow very large but at a slow rate

-non-cancerous because they produce adhesion molecules sticking them together and to a particular tissue, they are often surrounded by a capsule so they can remain compact and be removed by surgery

-rarely return

-impact is localised and often not life-threatening depending on the location

malignant tumours

-cancerous and grow large rapidly

-cell nucleus becomes large and the cell can become unspecialised again

-they don’t produce the adhesive, instead metastasis occurs, meaning the tumour breaks off and spreads to other parts of the body

-tumour is not encapsulated and can grow projections into surrounding tissues and develop it’s own blood supply

-can be life-threatening and the removal often needs supplementary treatment (chemo/radiotherapy)

-recurrence more likely

oncogenes

-mutated proto-oncogenes

-proto oncogenes creates a protein involved in the initiation of DNA replication and mitosis when the body needs new cells

-oncogene mutations can result in this process being permanently activated making cells divide continually

increased oestrogen concentrations

-produced by the ovaries to regulate the menstrual cycle but after menstruation it stops

-instead fat cells in breast tissue can produce oestrogen and this has been linked with causing breast cancer in women post-menopause

-knock on effect as the tumour results in even more oestrogen production, increases the tumour size, attracts WBCs which increases the size further

-could be because oestrogen activates a gene by binding to a gene that initiates transcription, if this is a proto-oncogene then it may be permanently turned on, activating cell division 

the genome

entire genetic material in an organism in the nucleus of a cell (eukaryotes)

sequencing a genome means working out the DNA base sequence for all the DNA in a cell

sequencing methods

the methods used to sequence genomes are continuously improved and updated and has now become automated

genome sequencing-simpler organisms

-simpler organisms such as prokaryotes don’t contain introns in their DNA

-this means the genome can be used to directly sequence the proteins that derive from the genetic code (proteome) of the organism

-useful for identifying potential antigens to use in a vaccine

-more complex organisms have introns and regulatory genes in their DNA, therefore the genome can’t be easily used to translate the proteome

recombinant DNA technologies

-the combining of different organism’s DNA, which could enable scientists to manipulate and alter genes to improve industrial processes and medical treatment

-the 1st step is to produce or isolate fragments of DNA to be recombined with another piece of DNA

-there are 3 methods to do this:

reverse transcription

restriction endonucleases

gene machine

reverse transcription

-reverse transcriptase makes DNA copies from mRNA

-naturally occurs in viruses e.g HIV

-a cell that naturally produces the protein of interest is selected

-these cells should have large amounts of mRNA for the protein

-reverse transcriptase joins the DNA nucleotides with complementary bases to the mRNA sequence

-single stranded DNA is made (cDNA)

-to make the fragment double stranded, DNA polymerase is used

an advantage of this is cDNA is intron free as it is based on mRNA template

restriction endonucleases

-make cuts in the DNA at recognition sites (breaks phosphodiester bonds via hydrolysis)

-recognition site complementary to the enzyme’s active site

-recognise the palindromic sequence and cut on top and bottom strand, leaving sticky ends

gene machine

-scientists use computers to generate nucleotide sequence to produce the gene

-short fragments of DNA produced, joined to make longer sequences of nucleotides then are inserted into vectors (e.g plasmids)

pros of reverse transcriptase

mRNA present in a cell is from actively transcribed genes, so lots of the mRNA of interest present to make cDNA

cons of reverse transcriptase

more steps, more time consuming and difficult

pros of restriction endonucleases

sticky ends on DNA fragments make it easier to insert to make recombinant DNA

cons of restriction endonucleases

still contains introns

pros of gene machines

can design the exact DNA fragment you want

cons of gene machines

need to know the sequence of amino acids or bases

amplifying DNA fragments

once the DNA fragments have been isolated, they need to be cloned to create large quantities

this can be done in vitro or in vivo

in vitro cloning

fragments of DNA can be amplified by the polymerase chain reaction (PCR), this is done in an automated machine

PCR equipment list

-thermocycler

-DNA fragment to be amplified

-DNA polymerase-taq polymerase

-primers

-DNA nucleotides

PCR method

-temp increased to 95 degrees, breaks hydrogen bonds and split DNA into single strands

-temperature decreased to 55 degrees so primers can attach (annealing)

-DNA polymerase then attaches complementary free nucleotides and makes a new strand to align next to each template (synthesis)

-for this stage the temperature is increased to 72 degrees, the optimum for taq polymerase 

advantages of PCR

automated-more efficient

rapid-100 billion copies of DNA can be made in hours

does not require living cells-quicker and less complex techniques needed

in vivo cloning stages

-create DNA fragments for gene of interest

-insert DNA fragment into a vector

-transform a host cell with the vector

-identify transformed cells

-grow the host cell

promoter and terminator regions-in vivo cloning

-restriction endonucleases are used to cut out the DNA fragment of interest

-these enzymes cut at recognition sites leaving sticky ends

-DNA fragments must be modified to ensure transcription can occur

-a promoter region must be added, added at the start of the DNA fragment, it is a sequence of DNA which is the binding site for RNA polymerase to enable transcription to occur

-a terminator region must be added, this is at the end of the gene, causes RNA polymerase to detach and stop transcription, so only 1 gene at a time is copied into mRNA

vectors

something to carry the isolated DNA fragment into the host cell

often a plasmid 

inserting DNA into a vector- in vivo cloning

-the plasmid is cut open using the same restriction endonuclease

-this creates the same sticky ends

-therefore the DNA fragment sticky ends are complementary to the sticky ends on the plasmid

-DNA fragment and cut plasmid combined and ligase sticks them together (anneals them)

-ligase catalyses the condensation reaction to form phosphodiester bonds between the nucleotides

transformation- in vivo cloning

-the vector (plasmid with recombinant DNA) next need to be inserted into the host cell where the gene will be expressed to create the protein required

-to do this, the cell membrane of the host cell must be more permeable

-to increase the permeability, the host cells are mixed with calcium and heat shocked

-enabling the vector to enter the host cell’s cytoplasm

identifying transformed cells- in vivo cloning

-not all host cells will successfully take up a recombinant plasmid

-3 issues can occur:

recombinant plasmid doesn’t get inside the cell

plasmid re-joins before the DNA fragment entered

DNA fragment sticks to itself rather than inserting into the plasmid

marker genes

-marker genes can be used to identify which bacteria successfully took up the recombinant plasmid

-antibiotic resistant genes

-genes coding for fluorescent proteins

-genes coding for enzymes

antibiotic-resistance marker genes

-insert into the bacterial plasmid a gene that makes the bacteria resistant to the antibiotic tetracycline and a gene for resistance to ampicillin

-insert the DNA fragment into the plasmid in the tetracycline gene

-disrupts the resistance to the tetracycline gene meaning the gene wont make a functional protein

-grow bacteria on agar

-transfer the bacterial colonies to a plate with ampicillin antibiotic in the agar

-transfer the bacterial colonies to a plate with tetracycline antibiotic in the agar

-bacteria transferred using sterile velvet on a block

-if bacteria grew on both, they must be resistant to both so they must be the original plasmid which does not contain the gene of interest

-if grew on ampicillin but not tetracycline then must be the recombinant plasmid, so can be taken and grown further

fluorescent markers

-jellyfish contain a gene which codes to create a green fluorescent protein (GFP)

-this can be inserted into the bacteria plasmid

-DNA fragment inserted into the middle of this gene, disrupting it and preventing GFP production

-only non-glowing colonies have taken up the recombinant plasmid under UV light

enzyme markers

-the enzyme lactase can turn a certain substance colourless to blue

-the gene for this enzyme is inserted into the plasmid

-DNA fragment inserted into the middle of the lactase gene, disrupting it and preventing lactase production

-bacteria grown on an agar plate with the colourless substance

-colonies that can’t turn it blue took up the recombinant plasmid

growing the host cell- in vivo cloning

-a fermenter is used to grow multiple copies of the host cell which have been identified as containing the recombinant plasmid

-this large, cloned population of the host cell can then produce the produce the protein coded for by the inserted DNA fragment

VNTRs

-95% of human DNA is made up of introns which consist of many variable number tandem repeats (VNTRs)

-the probability of 2 individuals having the same VNTRs is very low, the more closely related you are the more similar

-genetic fingerprinting is the analysis of VNTR DNA fragments, can be used to determine genetic relationships and genetic variability within a population

genetic fingerprinting step 1-collection and extraction

-sample of DNA collected from blood, body cells, ect

-if the sample is small, PCR is used to amplify the amount of DNA

genetic fingerprinting stage 2-digestion

-restriction endonucleases added to cut the DNA into smaller fragments

-enzymes which cut close to the target VNTRs are added

-maintaining the entire length of the VNTR

genetic fingerprinting stage 3-separation

-DNA samples loaded into small wells in agarose gel, the gel is placed in a buffer liquid with an electrical voltage applied

-DNA is negatively charged (due to phosphate group), so the DNA samples move through the gel towards the positive end of the gel

-this stage is gel electrophoresis

-the agar gel creates resistance for moving DNA, meaning smaller fragments move faster and further

-this is how different lengths of DNA (VNTRs) are separated

-an alkaline is then added to separate the double strands of DNA

genetic fingerprinting stage 4-hybridisation

-DNA probes are short, single stranded pieces of DNA complementary in base sequence to the VNTRs, the probes are radioactively are fluorescently labelled

-different DNA probes are mixed with the single stranded DNA VNTRs on the agar gel for them to bind (hybridise) 

genetic fingerprinting stage 5-development

-agar gel will shrink and crack as it dries, therefore the VNTRs and DNA probes are transferred to a nylon sheet

-the nylon sheet can then be exposed to x-rays to visualise the position of radioactive gene probes or UV light is fluorescent probes were used

genetic fingerprinting stage 6-analysis

-the position of DNA bands are compared to identify genetic relationships, the presences of a disease causing gene, or to match unknown samples from a crime scene

interpreting the results of gel electrophoresis

-paternity test analysis

-compare VNTRs of mother, child and potential fathers

-all of child’s VNTRs must have been inherited from the mother or father

uses of genetic fingerprinting

-medical diagnosis

-forensics at crime scenes

-ensure animals and plants are not closely related before breeding

DNA probes

-DNA probes are short, single-stranded pieces of DNA that are labelled radioactively of fluorescently so they can be identified

-this is used to locate specific alleles of genes and to screen patients for heritable conditions, drug responses or health risks

-DNA probes are created and have a complementary base sequence to the allele that is being screened for

-the patient’s DNA sample is treated to make it single stranded and it is then mixed with the DNA probes 

-if the patient had the allele then the DNA probe will hybridise and the label indicates the presence

DNA hybridisation

-the patient’s DNA sample is heated to make it single-stranded

-the heat causes the hydrogen bonds between the bases to break (denaturing)

-the patients single stranded DNA sample is mixed with the DNA probe and cooled, complementary sequences can align and form hydrogen bonds (anneal)

-some of the patients DNA samples will anneal back together, some with the DNA probe

locating specific alleles of genes

-DNA base sequence must be known to create the DNA probe

-this can be determined using DNA sequencing techniques

-the fragment of DNA can be produced using a gene machine

-fragment can be amplified by PCR

-the label is then added to either a radioactive nucleotide containing the isotope 32P, or a fluorescent label which emits light under UV light

-after hybridisation the DNA is washed so any unbound DNA probes are washed away

-the presences of the radioactive or fluorescent label therefore indicates that the allele of interest is present in the patient’s DNA

genetic screening

-the locating specific alleles of gene method can be used to screen for potential genetic disorders or the presence of caner causing oncogenes

-it is possible to screen for multiple diseases simultaneously using an array where multiple different DNA probes are attached

personalised medicine

-one key advantage to having your DNA screened

-certain drugs such as painkillers are more or less effective depending on your genotype

-it can also help determine the optimum dose, saving money and increasing effectiveness and safety

-vitamin E can be given to diabetics to decreased the risk of cardiovascular disease, but for others with a different genotype it can actually increase the risk

genetic counselling

-having DNA screened is an important decision that must be thought through carefully with clients so they can make an informed choice

-counselling is a type of social work, people can have their family history researched for the likelihood of them carrying certain alleles linked to disease

-examples include screening for cystic fibrosis or sickle-cell anaemia before starting a family