Biology Topic 8

substiution - one base swapped for another, different amino acid produced (can be avoided due to degenerate nature)

deletion - one base is deleted (causes frameshift and non-functional protein - changes final 3D shape of protein, detrimental to enzymes as it provents them from working)

addition - base is added (causes frameshift and non-functional protein)

duplication - base is repeated (causes frameshift and non-functional protein)

inversion - base sequence is reversed, different amino acid produced (can be avoided due to degenerate nature)

translocation - sequence of bases is moved from one location in genome to another (within chromosome or to another chromosome)

increases rate of mutation, e.g: 

- UV radiation

- ionising radiation

- carcinogenic agents e.g. nicotine 

achieves this by: acting as a base - base analogs substitute for a base during DNA replication e.g. agent substitues for thymine and pair with guanine (instead of adenine) which causes a substitution mutation

altering bases - chemicals can delete or alter bases e.g. alkylating agents can add an alkyl group to guanine which changes its tructure so that is pairs with thymine (instead of cytosine)

changing the structure of DNA - radiation can change structure of DNA e.g. UV radiation causes adjacent thymine to pair up together

1) tumour-suppressor genes slows cell division by producing proteins that stop cells from dividing or cause cells to self-destruct (apoptosis) - if gene is mutated, the protein isn't produced so the cells divide uncontrollably into a tumour

2) proto-oncogenes stimulate cell division by producing proteins that make cells divide - if gene is mutated, the gene becomes overactive, causing the cells to divide uncontrollably into a tumour

tumours can be benign (not cancerous, grow slower, can cause blockages and put pressure on organs) or malignant (cancerous, grow rapidly and metastasise and invade other cells by spreading in bloodstream or lymphatic system)

appearance: irregular shape, larger and darker nucleus, non-functioning proteins, different antigens, grow rapidly

3) abnormal methylation 

- hypermethylation of tumour-suppressor gene means genes for proteins are not transcribed so they're not made, thus cells divide uncontrollably

- hypomethylation of proto-oncogenes cause them to act as oncogenes which increases the production of proteins that encourage cell division which stimulates cells to divide uncontrollably

4) increased oestrogen (breast cancer)

- oestrogen can stimulate certain breast cells to divide and replicate 

- increased cell division increases chance of mutation

- if cell mutates and becomes cancerous, division is stimulated due to oestrogen so tumours form quickly

unspecialised cells that can divide to become new specialised cells

1) totipotent - can mature into any body cell and are only present in mammals in the first few cell divisions of an embryo

2) pluripotent - can mature into any body cell (apart from the placenta) and from from totipotent cells 

3) multipotent - adult mammal cell, can differentiate into few types of cell e.g. red and white blood cells from the blood marrow

4) unipotent - adult mammal cell, can only differentiate into one type of cell e.g. cardiomyocytes are replaced by small supply of unipotent stem cells in the heart

5) induced pluripotent stem cells (iPS cells) - created in lab by reprogramming specialised adult body cells so that they become pluripotent i.e. cells made to express transcriptional factors associated with pluripotency by infecting them with specially-modified virus which has genes coding for transcription factors within its DNA which are passed onto cell DNA so that it produces these transcription factors

stem cell therapies e.g. bone marrow transplant replaces fautly bone marrow that produce abnormal blood cells - stem cells in transplanted bone marrow divide and specialise to produce healthy blood cells (successfully treated leukaemia and lymphoma, sickle-cell anemia and SCID)

can treat other diseases e.g.: spinal cord injuries, heart disease, bladder conditions, respiratory diseases, organ transplants, etc.

methods: 

- adult stem cells - multipotent stem cells obtained from bone marrow, little risk but some discomfort

- embyonic stem cells - pluripotent stem cells obtained from embyros grown in vitro in a lab and are destroyed afterwards

- iPS stem cells

pros: save lives or improve quality of life, more ethical is adult stem cells / cons: murder of embryo 

transcription of genes is controlled by transcription factors:

- transcription factors move from cytoplasm to nucleus and bind to promotor region on DNA of target gene

- control rate of transcription e.g. activators stimulate/increase rate of transcription by helping RNA polymerase to bind to start of gene and activate transcription

- repressors inhibit/decrease rate of transcription by blocking RNA polymerase from binding, thus stopping transcription

oestrogen (steroid) can initate transcription

- binds to transcription factor called oestrogen receptor, forming oestrogen-oestrogen receptor complex

- complex moves from cyptoplasm to nucleus and binds to promotor region on DNA of target gene and helps RNA polymerase to bind to gene

RNAi - small non-coding double-stranded RNA molecules that stop mRNA from target genes being translated into proteins

1) siRNA 

- associates with several proteins and unwinds after mRNA has been transcribed and leaves the cyptoplasm

- single strand of siRNA binds to complementary base sequence sections on mRNA

- proteins associated with siRNA cut the mRNA fragments so it can no longer be translated which move into a processing body that degrades it

2) miRNA

- less specific than siRNA so targets more than one mRNA molecule

- associates with proteins and binds to mRNA in cytoplasm 

- miRNA-protein comlex physically blocks translation of mRNA 

epigenome or "chemical markers" determine whether gene is swtiched on/off by altering how easy it is for enzymes/transcription factors to transcribe DNA

1) increased methylation switches genes off

- methyl group attached to CpG site on DNA which is where a cytosine and guanine base are next to each other 

- increased methylation changes DNA structure so transcriptional machinery can't interact with the gene so it is switched off

2) decreased acetylation switches genes off

- histones can be epigenetically marked by addition/removal of acetyl group

- histones acetyled = chromatin less condensed so transcriptional machinery has access to DNA

- histones deacetyled via histone deacetylase = highly wound around histones so cannot be accessed 

- adavances in technology allow effective gene sequencing (e.g. pyrosequencing)

- automated, cost-effective gene sequencing allows scientists to sequence genomes of a variety of organisms by looking at DNA fragements

- also allows scientists to seqeunce proteome

- harder to sequence complex organisms as contain large sections of non-coding DNA and regulatory genes 

- however, Human Genome Project has found over 30,000 human proteins in human proteome

recombinant DNA technology - transferring fragment of DNA from one organism to another (transgenic organism)

1) using reverse transcriptase - mRNA is isolated from cells then mixed with free DNA nucleotides and reverse transcriptase which uses mRNA as template to synthesise a strand of cDNA

2) using restriction endonuclease enzymes - palindromic sequences recognised by restriction endonucleases (complementary to active site) which cut DNA at these places. DNA then incubated with restriction endonuclease which cuts the DNA fragment out via hydrolysis. Cut can leave sticky ends which can be used to bind (anneal) the fragment to another piece of DNA that has sticky ends with complementary sequences

3) gene machine - required sequence is designed and synthesised from scratch by adding nucleotides in correct order step-by-step with "protecting groups" (to make sure they join properly) to produce oligonucleotides (20 nucleotides long) which are broken off and connected to other DNA fragments  

1) DNA fragment inserted into vector (e.g. plasmid or bacteriophage) - vector DNA cut using same restriction endonuclease used to isolate DNA fragment on target gene (so sticky ends of vector are complementary to sticky ends of DNA fragment) - vector DNA and DNA fragment are mixed together with DNA ligase which joins the sticky ends of DNA fragment and vector DNA together via ligation = recombinant DNA

2) vector transfers DNA fragment to host cells - if vector is plasmid, host cells must be placed in ice-cold calcium chloride solution to make their cells more permeable, then vector is added and the mixture is heat-shocked which encouraged the cells to take in the plasmids. If bacteriophage, it will infect host becterium by injecting its DNA into it and the phage DNA integrates into the bacterial DNA 

3) identifying transformed host cells - around 5% will take up vector DNA, so marker genes inserted into vectors at same time as gene so transformed host cells will contain gene and marker gene. Host cells grown on agar plates where transformed cells will produce colonies where all cells contain cloned gene and marker gene. Marker can be resistant to antibiotics so grown with specific antibiotic (identified using inhibition zone?) or be fluorescent and show under UV light

polymerase chain reaction

- mixture set up with DNA sample, free nucleotides, primers (short pieces of DNA complementary to fragment), and DNA polymerase

- mixture heated to  95 degrees celcius to break H bonds and cooled to 50/60 degrees celcius so primers can bind (anneal) to strands

- mixture heated to 72 degrees celcius so DNA polymerase can line up free DNA nucleotides alongside each template strand (specific base pairing means new complementary strands are forrmed)

- two new copies of fragment of DNA are formed - 1 cycle of PCR

- each cycle repeats same procedure but copies are doubled each time 

1) agriculture - transformed to give higher yields, be more nutritious, have pest resistance 

pros: reduces risk of famine and malnutrition e.g. drought-resistant crops for drought-prone areas / pest resistant so fewer pesticides needed which reduces costs and problems associated with pesticides i.e. leaching / transformed crops can be used to produce useful pharmaceutical products e.g. vaccines which makes drugs more readily available to people where vaccination storage is not available

cons: produces monocultures which reduces biodiversity and increases risk of whole crop falling vulnerable to one genetic disease which could wipe it out entirely / transformed crops could interbreed with wild plants producing superweeds that are resistant to herbicides / farmers cannot sell crops as organic as GM seeds that have been blown over may contaminate fields 

2) industry - use of biological catalysts (e.g. enzymes) which can be produced from transformed organisms 

pros: produced in large quantities for less money, reducing costs

cons: anti-globalisation activists opppose large biotechnology companies as they control some forms of genetic engineering and as this technology increases these companies become bigger and powerful which forces smaller companies out of business as they can't compete / without proper labelling, some people won't have a choice about whether to consume food made from genetically engineered organisms / some consumer markets e.g. EU won't import GM foods which can cause economic loss to producers

3) medicine - drugs and vaccines produced by transformed organisms (e.g. human insulin is made from transformed microorgansism using a cloned insulin gene to treat diabetes I using in vivo cloning)

pros: made quickly, cheaply and in large quantites and can be used in gene therapy to treat diseases. method:

- if defective genes caused by two mutated recessive alleles, add working dominant allele

- if defective genes caused by mutated dominant, silence gene by adding DNA in middle of allele so it stops working

- allele inserted into cell using vector

- two types: somatic (altering alleles in body cells which doesn't affect sex cells) or germ line (altering alleles in sex cells so every cell of offspring won't suffer from disease)

cons: companies who own genetic engineering technologies may limit use at risk of losing lives / technology may be used unethically to make "designer babies"

DNA probes = used to locate specific alleles on genes (to look for mutatation of disease). They are short strands of DNA that have specific base sequence that's complementary to base sequence on target allele which means it can bind (hybridise) to target allele if it's present in a sample of DNA 

- DNA probe made by sequencing allele that's being screened then using PCR to produce multiple complementary copies of part of the allele

- sample of DNA cut into fragments using restriction endonuclease and seperated using gel electrophoresis (DNA placed into well in slab of gel, covered in buffer solution that conduct electricity - electrical current passed through gel, DNA fragments -ve so move towards +ve electrode at end of gel - small DNA fragments travel faster and further so fragments separate according to size)

- separated DNA fragments then transferred to nylon membrane and incubated with fluorescently labelled DNA probe - if allele is present, DNA probe will bind (hybridise) to it

- membrane exposed to UV light - if gene is present, fluorescent band shows 

DNA microarray (screens lots of genes at same time) 

- glass slide with microscopic spots of different DNA probes attached to it in rows

- sample of fluorescently labelled human DNA washed over array

- if labelled human DNA contains any DNA sequences that match to any of the probes, it will stick to it

- array is washed to remove any unstuck DNA

- UV light shone on array so any labelled DNA attached to a probe will fluoresce which means that the person's DNA contains that specific allele

helps to:

- identify inherited conditions 

- determine how a patient will repsond to specific drugs

- identify health risks (e.g. genetic predispositions due to inheriting certain mutated alleles that could lead to diabetes, depression etc.)

- used in genetic counselling whereby doctors can advise patients and relatives about the risks of genetic disorders and can suggests what to do/not to do in order to deal with it and what procedures they can undergo to combat it

- used in personalised medicine (different people respond to same drugs in different ways, so personalised medicines can be tailored to an individual's DNA by using a patient's genetic information to predict how they will respond to different drugs)

genomes contain non-coding variable number tandem repeats (VNTRs) which are base sequences that don't code for proteins and repeat next to each other over and over - these differ from person to person so are compared between individuals = genetic fingerprinting

- sample of DNA obtained from blood/saliva/skin/hair/faeces

- PCR used to make many copies of areas of DNA that contain VNTRs 

- primers used to bind (anneal) to either side of these repeats so whole repeat is amplified

- length of DNA fragments correspond to number of repeats person has at each specific position

- fluorescent tag added so DNA fragments can be viewed under UV light, then undergoes gel electrophoresis

- DNA fragments viewed as bands under UV light = genetic fingerprinting

- comparison e.g. fingerprints with band at same location suggests same no. of nucleotides and thus same no. of VNTRs = match 

determines genetic relationships - more bands on genetic fingerprint that match, more closely related (inherit VNTRs from parents so used for paternity testing)

determines genetic variability within a population - the greater number of brands that don't match, the greater the genetic difference between people (comparing no. of repeats at several places in the genome to find out how genetically varied the population is)

used in forensic science to link a suspect to a crime scene - DNA of suspect isolated, replicated using PCR, run on an electrophoresis gel and comapred to DNA found at crime scene

used in medical diagnosis - can be used to diagnose genetic disorders and cancer or unknown mutation (because it identifies broader, altered genetic pattern)

used in animal and plant breeding - genetic fingerprinting an be used to identfiy how closely-related individuals are - the more closely related, the more similar their genetic fingerprint will be - the least related individuals will be bred together. this prevents interbreeding (which causes increased risk of genetic disorders which can lead to health, productivity and reproductive problems)