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What is base addition?
One or more nucleotides is added
What is base deletion?
One or more nucleotides is deleted
What is base substitution?
A nucleotide is replaced with another base
What is duplication?
One or more bases is repeated
What is inversion?
A sequence of bases is reversed
What is translocation?
A sequence of bases is moved from one location in the genome to another. This could be movement within the same chromosome or movement to a different chromosome
What is a frameshift?
When the mutation affects the whole of the rest of the code
What are mutagenic agents?
Things that increase the rate of mutations
Describe acting as a base
Chemicals called base analogs can substitute for a base during DNA replication, changing the base sequence in the new DNA
Describe altering bases
Some chemicals can delete, or alter, bases
Describe changing the structure of DNA
Some types of radiation can change the structure of DNA, which causes problems during DNA replication
What are stem cells?
Unspecialised cells that can develop into other types of cell
Describe totipotent stem cells
Cells that can mature into any type of body cell, occur only for a limited time in early mammalian embryos
Describe pluripotent stem cells
Cells that can mature into any type of body cell (except placental cells), found in mammalian embryos
Describe multipotent stem cells
Can divide to form a limited number of different cell types, found in mature mammals, e.g. bone marrow
Describe unipotent stem cells
Can only differentiate into one type of cell, found in mature mammals, e.g. cardiomyocytes
How do stem cells become specialised?
How do bone marrow transplants work?
Describe stem cell therapies of the future
Describe adult stem cells
Describe embryonic stem cells
Describe induced pluripotent stem cells (iPS cells)
What are the ethical considerations of using stem cells?
What are the benefits of stem cell therapy?
What are transcription factors and what do they do?
Transcription factors bind to promoters near the start of target genes, they control expression by controlling rate of transcription
What do activators do? Give an example
Stimulate or increase rate of transcription, e.g. help RNA polymerase to bind to target gene
What do repressors do? Give an example
Inhibit or decrease rate of transcription, e.g. bind to target gene, preventing RNA polymerase from binding
What is oestrogen?
Steroid hormone
How does oestrogen act as an activator?
What is RNAi and what does it do?
RNA interference, small lengths of non-coding RNA, RNAi stops mRNA from target genes being translated into proteins
What is siRNA?
Short interfering RNA
How does siRNA interfere with gene expression?
Double-stranded siRNA associates with several proteins and unwinds
One of the single strands is selected and the other strand is degraded
Single strand binds to target mRNA, base sequence of siRNA is complementary to base sequence in sections of target mRNA
Proteins associated with siRNA cutes mRNA into fragments so it can no longer be translated
Fragments move into a processing body, which contains 'tools' to degrade them
What is miRNA?
MicroRNA
Why is miRNA less specific than siRNA?
miRNA isn't fully complementary to target mRNA, s o it may target more than one mRNA molecule
How does miRNA interfere with gene expression in mammals?
When miRNA is first transcribed, it is a long, folded strand processed into a double strand, then 2 single strands by enzymes in cytoplasm
One strand associates with proteins and binds to target mRNA
miRNA-protein complex physically blocks translation of target mRNA
mRNA is moved to a processing body, where it can be stored or degraded. When it's stored, it can be returned and translated at another time
What are acquired mutations?
Mutations that occur in individual cells after fertilisation
What causes tumours?
When mutations occur in genes that control rate of mitosis, causing uncontrolled cell division
What are cancers?
Tumours that invade and destroy surrounding tissue
Name 2 genes that control cell division
Tumour suppressor genes and proto-oncogenes
What happens when tumour suppressor genes are functioning normally?
They slow cell division by producing proteins that stop cell division or cause apoptosis
What mutation occurs in tumour suppressor genes and what does it cause?
Hypermethylation means genes are not transcribed by proteins which slow cell division are not translated, so cells are able to divide uncontrollably
What happens when proton-oncogenes are functioning normally?
They stimulate cell division by producing proteins that make cells divide
What mutation occurs in proton-oncogenes and what does it cause?
Hypomethylation causes them to act as oncogenes, increasing production of proteins that encourage cell division, so cells are able to divide uncontrollably
What is hypermethylation?
When too many methyl groups added
What is hypomethylation?
When too few methyl groups are added
Describe the features of malignant tumours
Describe the features of benign tumours
Describe the features of tumour cells
Why does increased exposure to oestrogen over an extended period of time increase risk of breast cancer?
What is epigenetics?
Involves heritable changes in gene function without changes to the base sequence of DNA
Name 2 epigenetic mechanisms
Methylation of DNA and acetylation of histones
How does methylation of DNA alter gene expression?
How does acetylation of histones alter gene expression?
How can epigenetics be used to treat disease?
What is the problem with using epigenetics to treat disease?
These changes take place normally in a lot of cells, so drugs have to be as specific as possible
What is a genome?
Complete set of DNA in an organism
What is a proteome?
Complete set of proteins made by organism
What must be done before genomes can be sequenced?
DNA must be split into smaller fragments as gene sequencing methods only work on fragments
Why is it relatively easy to determine the proteome of simple organisms?
They don't have much non-coding DNA
Give an example of when it is useful to know the proteome of simple organisms?
Identifying antigens on bacteria to develop vaccines
Why is it more difficult to determine the proteome of more complex organisms?
They contain large sections of non-coding DNA and complex regulatory genes, which makes it more difficult to translate genome into proteome
How have sequencing methods become more specialised?
The techniques are often automated, more cost-effective and can be done on a large-scale, pyrosequencing can sequence 400 million bases in 10 hours
What does recombinant DNA technology involve?
Transferring a fragment of DNA from one organism to another
Why can transferred DNA be used to produce a protein in cells of recipient organism?
Because genetic code is universal and transcription and translation mechanisms are similar
What are transgenic organisms?
Organisms that contain transferred DNA
Why is mRNA often easier to obtain than DNA?
Because the cells that produce the protein coded for by target gene will contain complementary mRNA
How is reverse transcriptase used to make DNA fragments?
mRNA is isolated from cells
mRNA is mixed with free DNA nucleotides and reverse transcriptase
Reverse transcriptase uses mRNA as a template to synthesise new strands of complementary DNA (cDNA)
What are palindromic sequences of nucleotides?
Antiparallel base pairs
What do restriction endonucleases do?
Recognise specific palindromic sequences (recognition sequences) and cut (digest) DNA at these places
Why do different restriction endonucleases cut at different specific recognition sequences?
The shape of recognition sequence is complementary to enzyme's active site
How are restriction endonucleases used to make DNA fragments?
DNA sample is incubated with specific restriction endonuclease, which cuts DNA fragment out via hydrolysis
Sometimes the cut leaves sticky ends - small tails of unpaired bases at each end of fragment
Sticky ends can be used to bind (anneal) DNA fragment to another pieces of DNA that has sticky ends with complementary sequences
How is a 'gene machine' used to make DNA fragments?
Sequence that is required is designed (if one doesn't already exist)
The first nucleotide in the sequence is fixed to some sort of support, e.g. a bead
Nucleotides are added step by step in the correct order, in a cycle of processes that includes adding protecting groups
Short sections of DNA called oligonucleotides (roughly 20 nucleotides long) are produced
Once these are complete, they are broken off from support and protecting groups removed
Oligonucleotides can then be joined together to make longer DNA fragments
What are protecting groups?
The make sure that the nucleotides are joined at the right points, to prevent unwanted branching
Define gene cloning
Making lots of identical copies of a gene
Define in vivo cloning
Where gene copies are made within a living organism
Define in vitro cloning
Where gene copies are made outside of a living organism using the polymerase chain reaction
Describe the process of in vivo cloning
DNA fragment of interest from gene using same restriction endonuclease
Same enzyme used to cut vector DNA (plasmid), to ensure they are complementary
Fragment DNA inserted into vector DNA, aided by ligase (ligation)
Marker gene also added to vector DNA, to allow identification of transformed cells, e.g. antibiotic resistance or fluorescence
Vector inserted into host cell, e.g. bacteria
Host multiplies, producing many copies of cloned gene
To produce proteins, they must contain specific promoter and terminator regions
Name 2 ways of using marker genes
Antibiotic resistance marker genes or fluorescent marker genes
Describe the process of in vitro cloning (PCR)
Reaction mixture containing DNA sample, free nucleotides, primers and DNA polymerase is set up
Mixture heated to 95c to break H-bonds between strands and cooled to 50-65c so primers can bind (anneal) to strand
Mixture heated to 72c so DNA polymerase can line up free nucleotides alongside each template strand, new complementary strands formed
Two new copies of fragment of DNA are formed and one cycle of PCR is complete, cycle starts again
What does PCR stand for?
Polymerase chain reaction
What are primers?
Short pieces of DNA that are complementary to bases at start of fragment you want
What is the equation for finding number of double strands?
2^n (n = number of cycles)
What is the equation for finding number of single strands?
2 x 2^n (n = number of cycles)
How does genetic engineering work?
Using recombinant DNA technology
How are transformed plants produced?
Gene that codes for desirable protein is inserted into plasmid
Plasmid is added to bacterium (vector) and inserted into plant cells
If right promoter region has been added, transformed cells will produce desired protein
How are transformed animals produced?
Gene that codes for desirable protein inserted into an early embryo or into egg cells of female
Promoter regions that are only activated in specific cell types can be used to control exactly which cells the protein is produced in
If protein is only produced in certain cells, it can be harvested more easily, producing protein in wrong cells can damage the organism
What are the benefits of transformed organisms?
Agricultural crops can be transformed so they give higher yields or are more nutritious - reduces famine and malnutrition
Crops can be transformed to have resistance to pests or droughts
Enzymes can be produced from transformed organisms, so can be produced in large quantities for less money
Drugs, e.g. insulin, can be produced quickly, cheaply and in large quantities
What are the concerns about transformed organisms?
Monoculture makes whole crop vulnerable to disease, and reduces diversity
'Superweeds' that are resistant to herbicides
Contamination f crops from GM crops
Introduction of toxins into food industry
Forcing smaller companies out of business
'Unethical' designer babies
What are promoter regions?
Tell RNA polymerase where to start
What are terminator regions?
Tell RNA polymerase where to stop
How are transformed microorganisms produced?
In vivo cloning
How can gene therapy be used to treat a disease caused by 2 recessive alleles?
Add a dominant allele
How can gene therapy be used to treat a disease caused by a dominant allele?
You can 'silence' an allele by inserting a fragment of DNA into the middle of the allele
What are liposomes?
Spheres made of lipid
Describe somatic gene therapy
Describe germ line gene therapy
What are the ethical issues surrounding gene therapy?
What are DNA probes used for?
To locate specific alleles or to see if a person's DNA contains a mutated allele
What are DNA probes?
Short strands of DNA with complementary base sequence to target allele so it will hybridise to target allele, label attached so it can be detected
Describe the process used when DNA probes are used to detect genes
Sample of DNA digested into fragments using restriction enzymes and separated using electrophoresis
Separated DNA fragments transferred to nylon membrane and incubated with fluorescently labelled DNA probe, if allele is present, DNA probe will hybridise with it
Membrane exposed to UV light and if gene is present, there will be a fluorescent band