Cellular Control + Genome manipulation Flashcards
what is gene expression ?
gene is transcribed and used to make a functional protein (switched on)
which stages is gene expression controlled ?
transcriptional, post-transcriptional, post-translational
what are transcription factors ?
proteins that bind to DNA and switch on/off genes by increasing/decreasing rate of transcription
state the name of transcription factors that switch genes on
activators
state the name of transcription factors that switch genes
repressors
how is gene expression controlled at transcriptional level?
shape of transcription factor altered (caused by certain molecules) – changes binding ability – changes rate of transcription
where do transcription factors bind in eukaryotes ?
specific DNA sites near start of target gene
where do transcription factors bind in prokaryotes ?
operon
what is an operon ?
section of DNA containing structural genes, control elements and (sometimes) regulatory gene
what are the structural genes in an operon ?
gene that codes for useful proteins (enzymes..)
what are the control elements in an operon ?
promoter - DNA seq. before structural genes that RNA pol. binds to
operator - DNA seq. that transcription factors bind to
what is the regulatory gene in an operon ?
codes for activator or repressor
what is the lac operon ?
E. coli gene that produces enzymes to respire lactose when not enough glucose
what are the three structural genes of the lac operon ?
lacZ / lacY / lacA
Describe lac operon when no lactose present
- regulator gene produces lac repressor
- lac rep. binds to operator site
- blocks RNA pol. from binding to promoter
Describe lac operon when lactose present
- lactose binds to repressor
- repressor changes shape so can’t bind to operator site
- RNA pol. can transcribe structural genes
what are introns ?
DNA sections that don’t code for amino acids
what are extrons ?
DNA sections that do code for amino acids
what happens at post-transcriptional level?
pre-mRNA is spliced to form mature mRNA
what is splicing ?
removal of introns
what happens at post-translational level?
cAMP produced from binding of molecules to membrane – cAMP activates protein by changing 3D structure
describe how cAMP activates Protein kinase A (PKA)
- cAMP changes 3D structure
- causes subunits to unbind from one another
- PKA now active
what are hox genes ?
genes which code for proteins that control body plan development
what are homeobox sequences ?
highly conserved (little change between organisms) regions of hox genes
How do hox genes control development?
- homeobox seq. codes for homeodomain
- homeodomain binds to DNA
- protein enabled to work as transcription factor
what is apoptosis ?
programmed cell death
describe apoptosis process
- enzymes break down components
- cell shrinks and breaks into fragments
- fragments are engulfed and digested by phagocytes
give example of how gene regulates apoptosis from internal stimuli
DNA damage can trigger apoptosis
give example of how gene regulates apoptosis from external stimuli
attack by pathogen
what are the three terms for changing DNA seq. ?
substitution / deletion / insertion
state the three ways a mutation can be neutral
- mutation doesn’t change amino acid
- mutation changes amino acid, but is chemically similar so functions similarly
- codes amino acid not involved w/ function
what does PCR do?
amplify fragment of DNA
what does PCR stand for ?
polymerase chain reaction
what are the reactants for PCR?
DNA sample, free nucleotides, primers, DNA polymerase
what are primers?
short pieces of DNA complementary to start of DNA fragment
outline the process of PCR (3)
- heat mixture - break H bonds
- cool mixture - primers anneal
- heat a bit - DNA pol lines up free nucleotides (com base pair)
Two copies of DNA formed
what is special about enzyme used in PCR?
adapted bacterial DNA pol. that can withstand higher temp – can carry out multiple cycles
what does electrophoresis do ?
uses electrical current to separate DNA / RNA fragments and proteins depending on size
Outline process of gel electrophoresis
- Gel box w/ agrose gel and buffer, wells closest to cathode
- loading dye added to DNA samples, samples put into wells
- DNA frags move to +ve charged anode (small travel faster)
- finally stain w/ staining solution, rinse
what charge is cathode ?
negative
why is loading dye needed for electrophoresis ?
helps samples sink in wells and makes easier to see
what do you use in electrophoresis for putting samples in wells ?
micro pipette
what are size of DNA fragments measured in?
bases (b)
what must you do for protein electrophoresis ?
denature so that all are same charge
what are palindromic sequence of nucleotides ?
sequences of anti parallel base pairs that read the same in opposite directions
what are restriction enzymes ?
enzymes that recognise and cut specific palindromic (recognition) sequences
why are restriction enzymes specific ?
shape of recognition seq. is complementary to active site
Outline process of using restriction enzyme to cut DNA fragments (3)
- DNA sample incubated w/ specific RE
- RE digest specific recognition seq. via hydrolysis
- sticky ends anneal DNA frags together (com.bas.pair)
what is DNA profiling ?
looking at the repeats of the non-coding sequences in the genome
how is DNA profiling used in forensic science ?
- different ppl have different no. of repeats
- PCR + gel electrophoresis can be used to compare DNA profiles of suspects
how is DNA profiling used in medical diagnosis ?
compare DNA profile of faulty region of parents’ DNA and embryos DNA (e.g. cystic fibrosis)
state the meaning of recombinant DNA
DNA formed from joining genetic material from different sources
what is a transformed organism ?
genetically modified organism
what is a transgenic organism ?
organism with gene from another species
outline the process of genetic engineering (3)
- DNA frag and vector DNA cut w/ same RE (so that sticky ends compl.)
- mixed w/ DNA ligase
- vector w/ recombinant DNA now transfers gene into bacterial cell
what are the two types of vectors used in genetic engineering ?
plasmids and bacteriophages
how does plasmid vector transfer gene to bacterial DNA ?
electroporation - electrical field created to increase bacterial cell membrane permeability
how does bacteriophages vector transfer gene to bacterial DNA ?
- bacteriophage injects its DNA into bacterium
- phage DNA integrates into bacterial DNA
what does the genetically modified soy bean gene do and where does it come from ?
- codes for protein toxic to insects
- comes from the bacteria Bt
how are soy beans genetically modified ? (5)
- RE cuts gene from Bt
- inserted into plasmid
- plasmid put back into bacteria
- plant cells infected by transformed bacteria
- gene gets inserted into plant cell’s DNA
what is good about GM plants ?
reduce amount of chemical pesticides used, which are harmful to environment
what is bad about GM plants ?
encourages mono culture of plant, decreases biodiversity so more susceptible to disease
what is pharming ?
pharmaceuticals being produced from genetically modified organisms
what is good about pharming ?
drugs made in large quantities so more available to people
what is bad about pharming ?
manipulating animals genes could cause harmful side-effects to the animal
what are the negatives for using GM pathogens ?
- GM pathogen could revert back to original form and cause outbreak of disease
- could be used as weapons for biowarfare
what is good about companies owning GM organisms / products ?
scientists will compete, so new products are developed faster
what is bad about companies owning GM organisms / products ?
poorer countries may not be able to pay the high prices that have been set
what is gene therapy ?
altering alleles to cure genetic disorders
what to do w/ gene therapy if disease cause by two recessive alleles ?
add a working dominant allele
what to do w/ gene therapy if disease cause by a dominant allele ?
‘silence’ dominant allele by adding DNA in middle of allele so it doesn’t work
what is somatic therapy ?
- gene therapy by altering the alleles in body cells
- offspring not affected
what is germ line therapy ?
- gene therapy by altering the alleles in sex cells
- offspring affected
what are the positives of gene therapy ?
- prolong lives
- give better quality of life
- carrier might be able to conceive without the worry of passing on disorder
what are the negatives of gene therapy ?
- expensive process (could be used for other health services)
- immune response may be triggered against vectors
- inserted allele may get over expressed, producing too much of the protein
what is chain-termination method ?
method of sequencing DNA
what is in the mixture of the chain-termination method ?
- single stranded DNA template
- DNA primer
- DNA polymerase
- free nucleotides
- fluorescently tagged modified nucleotide
what is different about a modified nucleotide ?
no more bases are added once its added to DNA strand
outline process of chain termination method (5)
- 4 separate tubes w/ mixture, modified nucleotides are different
- PCR to produce many strands (different lengths)
- DNA frags separated by electrophoresis
- visualised with UV light
- complementary base seq. read from bottom to top
why can’t whole genomes be sequenced w/ chain term method ?
only works on small fragments (up to 750bp long)
Outline the process of sequencing whole genomes
- genome cut into fragments w/ RE
- Frags inserted into different bacterial artificial chromosomes (BACs)
- BACs inserted into diff bacteria which divide
- DNA extracted from colonies and cut w/ RE
- Each DNA piece sequences w/ chain-term
- pieces put back in order of BAC seq.
- frags from all BACs put back in order
what are some improvements in whole genome sequencing ?
- chain term has become automated and faster
- high throughput sequencing
- pyrosequencing
what is synthetic biology ?
creating biological molecules from scratch
what are some fields of synthetic biology ?
- building artificial biological systems
- redesigning biological systems
- designing new biological systems
how is gene sequencing useful for diseases like Marfan syndrome ?
- bioinformatics allows to compare all data and identify GENOTYPE-PHENOTYPE CORRELATIONS
- helps predict what symptoms likely to face
what is bioinformatics ?
using comp software to analyse, organise and store biological data
how is gene sequencing useful for health and disease within a population (epidemiology) ?
computerised comparisons between genomes can link particular mutations to diseases
how is gene sequencing useful for understanding evolutionary relationships ?
can compare how closely related species are by looking at genome similarity
which kingdoms have homeobox genes ?
animals plants fungi
state 3 roles of homeobox genes
- directing formation of limbs and organs
- regulate differentiation
- tumor suppressors (prevent cells dividing in an uncontrolled way)
