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323 Cards in this Set
- Front
- Back
3 Components of a nucleotide
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phosphate, sugar, nitrogenous base
|
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features of ribonucleotide sugar
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A pentose (5 carbon) sugar with carbon atoms numbered 1'C to 5'C
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difference between RNA and DNA sugar
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RNA has an OH group attached to the 2'C, DNA has only H
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which bases are purines
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adenine and guanine
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which bases are pyrimidines
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cytosine, thymine (and uracil)
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purines and pyrimidines, which are 2 ring strucures
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purines
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where do sugar and base join and what type of bond
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1'C of the sugar by a N-glycoside linkage
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where do sugar and phosphate join and what type of bond
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5'C of the sugar by an ester linkage
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how many phosphates on a nucleotide
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1-3
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what is the structure of a nucleoside
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nucleotide without phosphate groups, only sugar and base.
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where do nucleotides join and what type of bond
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through sugar and phosphate
groups by phosphodiester bonds at the 3'OH to the alpha phosphate group |
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what type of reaction joins nucleotides
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condensation reaction
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direction of nucleotide joining
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5' to 3'
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what are needed for nucleotides to join
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free 5' phosphate group or a free 3'OH
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what will a DNAchain with 5' polarity have
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a free 5' phosphate
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what will a DNA chain with 3' polarity have
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a free 3' OH
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what are bonds joining DNA strands
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hydrogen bonds between anti-parallel chains
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how do A and T bond between DNA chains
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2 H bonds
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how do C and G bond between DNA chains
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3 H bonds
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4 things required for DNA synthesis
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template strand, primer sequence, free, dNTP's, DNA polymerase
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features of a DNA primer
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upto 20 nucleotides, complementary to template, free 3'OH
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what happens as a dNTP is attached to growing DNA chain
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complementary dNTP arrives, DNA polymerase catalyses 3'OH nucleophilic attack of alpha phosphate on incoming dNTP, phosphodiester bond formed and extra pyrophosphate released
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preparation steps for DNA synthesis
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unwinding, separation, primer binds
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direction of DNA synthesis
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5' to 3'
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what consequence does ribose sugar rather than 2'-deoxyribose sugar have in RNA
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2' to 5' bonds can form as well as 3' to 5', succeptible to alkaline hydrolysis at high pH = less stable
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what structures tend to form when RNA forms double strands
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hairpin loops within a single strand
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hierarchical structure of chromosome
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nucleosome of 8 histone proteins with DNA wound round.
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in eukaryotes where does protein biosynthesis occur
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cytoplasm
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stages in protein biosynthesis
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transcription to mRNA, goes to cytoplasm, translated to protein
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transcription requires
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RNA polymerase, free NTP's, NO PRIMER, template DNA, promoter, terminator.
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where does translation occur
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ribosomes
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what happens at translation
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ribosome binds 5' end of mRNA and moves along until AUG start codon, tRNA attached to AA binds to AUG, tRNA leaves and next codon is 'read' and polypeptide chain forms until stop codon when it is released.
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direction of RNA synthesis in transcription
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5' to 3'
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how many total codons and what do they code
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64, 3 stop, 61 encode 20 AA's
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what is RNA processing
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pre-RNA has 5' cap and 3' poly-A tail added, introns removed and exons spliced to make mRNA
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where does RNA processing occur
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nucleus
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6 points where genes can be regulated
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• Initiation of transcription
• Elongation of transcription • mRNA modification • mRNA stability and transport • Translation • Protein stabilit |
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at what stage does most gene regulation occur
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control of transcription
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what does RNApol1 do
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transcribes rRNA
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what does RNApol2 do
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transcribes mRNA
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what does RNApol3 do
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transcribes tRNA
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how does RNA polymerase bind to promoter
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accessory proteins, the general transcription factors.
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what binding would repress RNA polymerase transcription
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The binding of a transcriptional repressor to a silencer site would block RNA polymerase binding
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what binding would enhance RNA polymerase transcription
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binding of a transcriptional activator to an enhancer site
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what may regulate enhancers and silencers of transcription
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intra- and extra-cellular signals.
hormones, growth factors, stress signals (e.g. increased temperature), or nutritional signals (e.g. glucose levels or a lack of a particular amino acid). |
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2 important domains of transcriptional activators
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DNA binding domain, activation domain
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what bonds are cut by endonucleases
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phosphodiester bonds
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what are free on the cut ends of DNA after digestion with a restriction enzyme
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3'OH and 5'P
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how may DNA be treated to protect from endonuclease digestion
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methylation at recognition site
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types of restriction enzyme used in recombinant DNA tech and features of it.
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type 2, recognises 4-6 bp palindrome
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what restriction enzyme will tend to make more cuts, a 4 bp recognition sequence one or a 6 bp
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4, more chance of this sequence occuring
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3 types of ends produced by restriction enzyme digestion
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sticky (5' or 3' overhang) and blunt
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how are restriction fragments separated
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gel electrophoresis
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what causes restriction fragments to move and separate
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charge (overall negative charge due to P groups) causes travel towards +ve pole and separation by molecular size
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features of an agarose gel
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They allow the separation of DNA fragments typically between ~100bp and 20kb, large pores, organic
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features of a polyacrymalide gel
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smaller, more homogeneous pores, They allow the separation of shorter DNA fragments (1-1000bp range), good resolution (1bp discrimination)
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3 determinants of separtion on gel by electrophoresis
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charge, size, shape
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what factor influences restriction fragment separation and why
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size, charge and shape are constant due to double helix shape
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what is necessary to estimating restriction fragment sizes
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DNA size ladder
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what is used to stain restriction fragments separated on gel
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ethidium bromide (fluoresces in UV light)
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what is used to join DNA fragments
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DNA ligase
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what does DNA ligase do
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reforms DNA breaks by reforming the phosphodiester bond
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necessities of DNA fragments for ligation
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identical overhangs (or blunt ends) free 3'OH and 5'P. must be dsDNA
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what is recombinant DNA
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cut and ligated DNA from different sources
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2 types of DNA cloning
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cell-based and cell-free
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cell based DNA cloning
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recombinant DNA cloned, usually in e Coli
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cell free DNA cloning
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PCR
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3 features of a cloning vector
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origin of replication, selectable marker, cloning site
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origin of replication
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in a cloning vector- allows vector to replicate independent of host
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selectable marker of vector
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allows survival of only transformed vectors, most common is B lactam resistant e coli infected by ampicillin resistant vectors, B lactamase cleaves these drugs
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onecloning site on vector
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a single site for inserting DNA using specific restriction enzyme, only on recognition site
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4 types of vectors
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1. Bacterial plasmids
2. Bacteriophage vectors 3. Cosmids 4. Artificial chromosomes |
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features of plasmids
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circular dsDNA, have an origin of replication, multiple cloning sites for different restriction enzymes, recombinant marker -typically a insertional inactivation (lac Z causes untransformed vectors to produce blue ecoli colonies)
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2 methods of transformation
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electroporation- bacteria washed, plasmid added and pulses of electricity creates small pores. chemical- at low temp CaCl2 added to bacteria, plasmid added, short 42 degree heat shock, membrane transiently disrupted
|
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most commonly used phage vector
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lambda phage
|
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how does cloning with lambda replacement vector happen
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15kb section of phage genome removed (stuffer fragment), desired dna ligated into phage, recombinant phage mixed with ecoli and plated together with uninfected ecoli, cell lysis and infection cascade, plaque formed.
|
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how are untransformed phages identified/eliminated
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without stuffer fragment phage genome is too small to survive so only those with an insert of 12-20 kb survive.
|
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features of cosmid vectors
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upto 45kb inserts, act as plasmids but infected in a phage head, collonies not plaques form.
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yeast artificial chromosomes
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hybrid of plasmid and yeast dna can accept 1mb inserts.
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artificial chromosome types
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yeast and bacterial
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bacterial artificial chromosomes insert size
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up to 300 kb
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max plasmid insert
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10kb
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max phage insert
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20kb
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max cosmid insert
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45kb
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max BAC insert
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300kb
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max YAC insert
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1MB
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4 possible ligation products in non-directional cloning with a plasmid vector
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untransformed vector, uninserted circularised insert, transformed plasmid with insert in one of 2 directions.
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why might direction of insert insertion into a plasmid be important
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if a protein product expressed by insert is desired then 5' end of insert must be next to promoter
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directional cloning
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2 restriction enzymes with non-complementary sticky ends, a small section of plasmid is excised but restriction sites should be close together to minimise this
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why might direction cloning be impossible and how would desired ligations be achieved in this situation
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lack of suitable restriction sites, alkaline phosphatase treatment of linearised plasmid suppresses self-ligation by removing 5'P, this prevents DNA ligase from joining ends, untreated insert is still inserted and the missing P nicks are later repaired by bacterial repair systems
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2 enzymes involved in blunt end ligation and their purpose
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5'-overhanging ends filled in by Klenow fragment of E.coli DNA polymerase I, 3'-overhanging ends trimmed by 3'→5' exonuclease activity of T4 DNA polymerase
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T4 DNA polymerase does what
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3'→5' exonuclease activity, trims 3' overhang
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Klenow fragment of E.coli DNA polymerase I does what
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fills in 5' overhang
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5 requirements for PCR
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excess of dNTP's, template DNA, primers to bind 3' end and provide free 3' OH, heat stable DNA polymerase, magnesium ions
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3 steps of PCR and temperatures
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1. DNA denatured at 95°C (unwinds and H bonds break)
2. primers annealed at 55°C 3. primer extension at 72°C, 5' to 3' |
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where is PCR carried out
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a thermocycler
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what will a PCR control involve
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a parallel run with no template
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after PCR is finished what happens to product
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gel electrophoresis separation versus a DNA ladder
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2 principle limitations of PCR
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only fragments up to 2kb, sequence of flanking region must be known to design primers
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differences in first 2 cycles of PCR
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original template will produce complementary strands with overhang, therafter only this will occur once with every cycle, defined fragments will have a primer at each end and will exponentially produce defined product.
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determinants of good primer design for PCR
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1. length 18-24 nucleotides
2. base composition, lots of G and C, more stable due to extra H bonds, avoid long runs of single nucleotides 3. primer melting temps, make both primers melt at similar temps, affected by length and base composition 4. primer binding sequence but not occur elsewhere in DNA 5.primers that do not form secondary structures or self anneal (no complementary ends) |
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how to stop low temperature non-specific low temperature primer extension by Taq polymerase
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hot start, Ab conjugated DNA polymerase that is inactivated
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what are the consequences of Taq polymerase lacking exonuclease activity
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no proof reading, introduction of error that will be propagated to differing amounts depending on which cycle they are introduced in.
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low Mg2+ in PCR will cause
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low polymerase activity
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too high Mg2+ in PCR causes
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non-specific annealing, multiple PCR prducts
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3 ways manipulation of PCR can be used to insert suitable restriction sequences for directional cloning
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1. attaching a restriction site sequence to the 5' ends of the PCR primers, only the 3' end may anneal but the restriction sequence will be copied into PCR product.
2. TA cloning, Taq polymerase tends to attach an extra single nucleotide to the 3' end on PCR product (terminal transerase activity creates a tiny sticky end, most commonly A), linearised vector will bind if it has a 3' T overhang, this can be created by treatment of linearised vector with Taq polymerase and dTTP's. 3. Pfu DNA polymerase produces blunt ended DNA that may be inserted to blunt ended vector or TA cloning can be used. 2&3 are non-directional |
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detection of genetic alterations involving a deletion, insertion or rearrangement by PCR
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using primers to flank the regionPCR amoplification id performed and compared to a control by electrophoresis separation, different length in disease, 2 different lengths in diseased sample if heterozygous.
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detection of point mutation by PCR
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allele-specific PCR, requires the mutation to be known, 2 PCR's run each with a primer specific for mutated or wild type forms (3' ends terminate at point mutation), only correctly base paired primer will extend, a second pair of primers that flank the mutated region are used as a control to ensure the PCR process is working.
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example of allele specific PCR detection
|
K-ras oncogene mutations in colorectal cancer detected in faecal DNA, specific point mutations known to occur early at codon 12 and 13, useful diagnostic tool
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diagnostic uses of PCR
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genetic mutations separated by length v's control, point mutations detected by allele specific PCR, viral or bacterial detection of infection eg TB faster than culture or Ab
|
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what is the starting template material for RT PCR
|
a single stranded piece of RNA
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2 stages of RT PCR
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1. RT reaction. primer 1 complementary to 3' end of desired mRNA, reverse transcriptase extends this to a complementary cDNA strand
2. PCR. in cycle 1 primer 2 extends sscDNA to ds. thereafter primer 1 and 2 extend both strands |
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2 clinical/research uses of RT PCR
|
diagnostic eg HPV specific mRNA products & in research to determine the transcription of specific genes under
different experimental conditions |
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basic principal of real time PCR
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new DNA produced in each cycle is detected as it is produced, number of cycle until cycle threshold - Ct is reached is udes to calculate either the absolute or relative quantity of starting mRNA/cDNA
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how is measurement done in real time PCR - 2 methods
|
special thermocycler that can detect fluorescent product
1. SYBR Green fluorescent dye will fluoresce only when bound to dsDNA. 2. fluorescent reporter probes e.g. TaqMan probes, probe is designed to bind specifically to the target DNA sequence between the two PCR primers, it has a 5' fluorescent reporter dye and a 3' quencher. At annealing stage it binds to ssDNA and at extension stage the e 5'→3' exonuclease activity of Taq DNA polymerase chops off the quencher so fluorescence occurs. |
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how is PCR used in DNA sequencing
|
dideoxy chain termination
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what is needed to perform dideoxy chain termination PCR
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a high fidelity DNA polymerase, only one primer, dNTP's and labeled ddNTP's, template.
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how do dNTP and ddNTP's differ
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ddNTP is 2' 3' di-deoxyribose and has a 3' H rather than OH
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how is the product of dideoxy chain termination PCR read
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run on polyacrymalide gel (gives one nucleotide resolution), each possible length of temple fragment will have been copied with a fluorescing ddNTP at the end which is scanned and is complemtary to the initial template.
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2 types of DNA library
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1. genomic DNA library composed of fragemtns of DNA inserted into plasmid or phage λ that constitute the entire genome including regulatory regions, introns and noncoding areas.
2. cDNA library, cDNA fragments corresponding to all mRNA present in a cell at sampling this is both tissue- and developmental stage- specific |
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4 steps in constructing a genome library
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1. Isolation of genomic DNA, usually from WBC
2. Fragmentation by partial digestion to produce random, ovelapping similar sized fragments. 3. isolation and ligation, gel separation and extraction then vector ligation 4. Plating out, vectors infect usually ecoli, with plasmid, cosmid and BAC vectors each colony represent a fragment with phage it is a plaque. |
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what does a genome library look like
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A genomic library consists of a large number of agar plates containing colonies or
plaques, each of which carries a different insert of genomic DNA |
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what factors determine how many colonies are needed to represent a genome in a genome library
|
size of genome and size of vector insert
|
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Construction of a cDNA Library
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1. isolation of mRNA, using
oligo-dT's to isolate polyadenylated mRNA 2. ss cDNA synthesis using reverse transcriptase and oligo-dT primer to bind poly A tail, treatment with alkali NaOH to destroy RNA but leave DNA. 3. ds cDNA synthesis, to allow one primer to bind all cDNA present Homo-polymer tailing is done by incubating with terminal transferase and one dNTP (eg C) which adds a poly C tail to 3' end without a primer. A oligo-dG primer can then be used to bind all. 4. ligation into vector 5. plating out. |
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genome library screening by nucleic acid hybridisation
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1. colony lift onto nitrocellulose filter
2.treated to lyse the bacteria 3.alkali (NaOH) to denature the dsDNA 4. hybridised with a radioactively-labeled probe 5. autoradiography detects colony that binds to desired probe. |
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how to screen a genome library using a probe from another species
|
a homologous probe is totally specific and will be used at higher temperature and lower salt concentration (high stringency) a heterologous probe needs lower temperature and higher salt concentrations in hybridisation and washes but the sequence homology should be sufficient to still get a match between eg mouse and human
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to screen acDNA library if the protein product is known but not the DNA sequence
|
back-translation from AA sequence of the protein to DNA (several codons encode most AA's so must use all possible) this is a degenerate probe -all possible
nucleotide sequences. choose a section of at least 6 AA's preferably with methionine and tryptophan (only one codon each) and use as a probe. Run at high stringency to eliminate partial matches. |
|
immuno screening a cDNA library
|
antibody raised to cDNA protein product, λ vectors used so nitrocellulose filter placed on plaque, ecoli lysed in plaque so proteins in plaque transfer to filter, incubated with primary antibody, washed, secondary labeled eg anti-rabbit ab applied and read.
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what is southern blotting used for
|
analyse complex mixtures of DNA fragments
|
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procedures of southern blotting
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1. genomic DNA digested to fragments by restriction enzyme
2. separated by gel electrophoresis 3. NaOH to denature the dsDNA fragments on gel. 4. ssDNA fragments blotted onto nitrocellulose membrane (blotting solution drawn up blotting paper by cappilary action, through gel and carries fragments onto membrane to make transfer of gel) 3. fragemnts baked or UV onto membrane 4. membrane hybridized with a labeled probe complementary to area of interest 5. exposed to X-ray film to visualise |
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what information can be obtained from a southern blot
|
• The size of the fragment that binds to the probe.
• The copy number of a particular genomic sequence (i.e. the number of copies that are present). For example, a large genomic sequence may be absent or amplified (present in greater number) in cancer cells. • Deletions, insertions or large scale rearrangements (e.g., a translocation) within the genomic fragment of interest. • The presence of specific ‘forei |
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how can a DNA probe be labeled
|
5' P removed from probe by alkaline phosphatase and radioactive phosphate added by polynucleotide kinase
|
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function of northern blotting
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analyse complex mixtures of RNA.
|
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type of probe used in northern blotting
|
cDNA
|
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northern blotting proceedures
|
1. total cellular RNA removed
2. separated by electrophoresis with formaldehyde to prevent secondary structure formation 3. stained with ethidium bromide (intact RNA sample will have sharp bands that correspond to the two large rRNA molecules) 4. transferred and fixed to nitrocellulose membrane as in Southern blot. 5. probed with labeled 6. xray visualisation cDNA |
|
information from northern blot
|
1. The size of a specific mRNA molecule (transcript).
2. The relative abundance of a particular mRNA 3. Expression (transcription) patterns between tissues or developmental stages |
|
function of Western blotting
|
analyse complex mixtures of protein.
|
|
probe type used in western blotting
|
Ab
|
|
steps in Western blotting
|
1. protein extracted
2. sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) separation 3. transferred onto a nitrocellulose membrane by electro-blotting 4. Ab probe 5. secondary Ab 6. visualisation |
|
information from western blot
|
size of protein, If a protein has been modified e.g. phosphorylated (will alter size, or detection by Ab to phosphorylated form), relatie protein abundance, expression pattern between tissues or developmental stages.
|
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what is the transcriptome
|
all the mRNA molecules (transcripts) of a particular cell population under specific conditions.
|
|
oligonucleotide
|
a short sequence od bp's commonly synthetic in origin
|
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What is a Microarray?
|
an ordered arrangement of DNA molecules immobilised usually on a glass slide. cDNAs, PCR fragments or oligonucleotides. each spot contains multiple copies of a short stretch of DNA
|
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preparation for using a microarray
|
mRNA reverse transcribed to ss cDNA using reverse transcriptase, an oligo-dT primer, and fluorescently-labelled dNTPs
|
|
how is microarray analysis quantitative
|
each spot contains millions of copies and will not be saturated by hybridisation with probe so degree of fluorescence indicates relative abundance. This is relative quantity.
|
|
what is an expression profile
|
the results of a microarray analysis which shows the mRNA (DNA expression levels) of thousands of genes in a cell population
|
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comparative microarray analysis
|
comparison of expression profile of 2 cell populations eg cancerous and not or different developmental stages
|
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how are the expression profiles of 2 cell populations compared on one microarray
|
One of the mRNA populations is reverse transcribed into green fluorescing cDNAs by incorporating dNTPs labelled with the green fluorescent marker the other red. equal amounts hybridised and washed off then imaged. spots will be either unstained, red, green or yellow (both stain)
|
|
what broad uses may microarray analysis have clinically
|
diagnosis (versus known disease profiles), prognosis (versus known staging examples), effectiveness of therapy (retesting)
|
|
Serial Analysis of Gene Expression (SAGE)
|
cDNA tags unique for each gene, ligated, cloned, sequenced
|
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creating SAGE
|
mRNA removed and RT into cDNA, tag created for each cDNA, tags ligated, put in vector, cloned and sequenced, software analysis of sequence counts number of times a tag occurs
|
|
use of SAGE
|
usually comparison of 2 cell populations
|
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size of a tag for SAGE
|
10-14 bases
|
|
meaning of SAGE results
|
The number of times a particular tag is identified corresponds to the abundance of its parental cDNA in the starting mRNA. 2 cell populations comparable.
|
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what is the principal difference between SAGE and microarray
|
Sage is based on sequencing, microarray is based on hybridisation. Therefore SAGE can detect previously unknown genes, microarray can only detect what is know.
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what are the limitations of mRNA analysis
|
although most gene control is done at the level of transcriptional control so mRNA well represents cell status proteins that require post translational modifications or genes regulated post translationally are not discriminated
|
|
proteomics
|
is the large-scale study of proteins, particularly their structures, functions and interactions.
|
|
number of estimated human genes
|
30000
|
|
number of estimated human proteins
|
500000
|
|
by what means are more proteins produced than genes exist
|
alternative promoters, polyadenylation sites, alternative splicing, post-translational phosphorylation or glycosylation
|
|
2-dimensional polyacrylamide gel electrophoresis (2D PAGE)
|
Proteins from a particular cell sample can be extracted and separated into more than 1,000 discrete protein spots by two-dimensional polyacrylamide gel electrophoresis
|
|
2D PAGE method
|
Proteins are separated by net charge in one dimension and by size in the second dimension.
|
|
problems with 2D PAGE
|
not sensitive enough to detect rare proteins, and many proteins will not be resolved on the gel
|
|
how are spots generated by 2D PAGE analysed
|
mass spectrometry
|
|
protein identification following 2D PAGE
|
1. protease digestion to produce peptides (trypsin)
2. peptides identified by mass spectrometry giving a unique peptide mass fingerprint identification only possible if protein is previously known |
|
how does mass spectrometry detect post-translational modifications
|
phosphorylation etc will distinctively alter mass in a detectable way.
|
|
method for analysing protein-protein interactions
|
Yeast 2-hybrid analysis (Y2H)
|
|
what is learned by finding protein protein interactions
|
may indicate function especially regulatory networks or signal transduction cascades
|
|
what is the DNA binding domain
|
region of a protein that recognizes and binds to a specific recognition sequence in DNA
|
|
what is the activation domain
|
protein region that interacts with other proteins to activate transcription of the target gene.
|
|
basic Y2H set up
|
1. yeast transcriptional activator, GAL4 binds to a GAL4-recognition sequence with its DBD just in front of its target gene promoter and activates transcription with its AD.
1. GAL4 positioned beside HIS3 gene (only activation by GAL4 will produce histidine thus allowing yeast to grow on histidine free medium. 3. hybrids of 2 proteins to be tested attached to either GAL4 DBD or GAL4 AD are created by recombinant plasmids 4. when vectors inserted into yeast products produced and GAL4 DBD binds to GAL4 recognition sequence but AD will only attach if 2 proteins interact. |
|
creating hybrid proteins for Y2H
|
inserting the cDNA of the DBD and the cDNA of protein X into a vector so that it can express a hybrid protein then the same with protein Y and cDNA of GAL4 AD
|
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how to use Y2H analysis to screen a cDNA library for protein interactions
|
protein of interest is hybridised to GAL4 DBD in bait plasmid. All cDNA in library hybridised with GAL4 AD cloned into prey plasmids and translated to produce hybrid protein protein. Bait and prey transformed into yeast and selectable marker used to identify only yeast containing both. Yeat growing on histidine-free medium indicated protein-protein interaction has occurred. Prey plasmid then isolated and sequenced to identify.
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|
Gene knock-outs
|
genetically engineered mice with a specific gene removed, the effect can then be observed
|
|
Gene knock-down advantages
|
1. can study essential genes
2. can be applied to most eukaryotes including cultures 3. can be a more accurate model of disease |
|
what is RNA interference (RNAi).
|
small non-coding dsRNA target complementary mRNA transcripts for destruction so regulate gene expression.
|
|
how does RNA interference (RNAi) work. 4 steps
|
1. dsRNA is chopped up by the enzyme Dicer into shorter fragments known as short interfering RNAs
2. incorporated into a multi-protein complex - RNA-induced silencing complex (RISC). 3. ds siRNA then becomes single stranded and one of the strands (the one complementary to the mRNA) guides the RISC to the target mRNA 4. The ss siRNA binds to the mRNA and directs the RISC to degrade the mRNA |
|
how are genes knocked down
|
siRNA oligos introduced to bind and destroy specific mRNA. This is done in culture.
|
|
Recombinant protein production
|
an expression vector used to produce recombinant protein
|
|
uses of recombinant proteins
|
1. industrial production eg insulin
2. analysis of protein function, can introduce protein with known mutations or overexpress. |
|
where are recombinant proteins made
|
ecoli or yeast, insect or mammal cells
|
|
what would cause a recombinant protein to need to be made in eukaryotic cells
|
1. requires post-translationally glycosylated (ecoli cannot do this)
2. prokaryotic proteins may be misfolded 3. if protein is toxic to bacteria |
|
2 types of gene therapy
|
1. germ cell, changes may be inherited, illegal in humans
2. somatic |
|
challenges of gene therapy
|
1. must be delivered to specific target cells
2. must be inserted in the DNA so that it will be expressed (transcribed and translated) permanently and stably at a level which is of benefit |
|
2 somatic cell gene therapy strategies
|
1 ex vivo
2 in vivo |
|
ex vivo somatic gene therapy
|
patients cells removed, altered and reintroduced. must be able to be cultured (eg stem cells in BM), good for targeting
|
|
in vivo somatic gene therapy
|
gene delivered direct into body, very hard to target, only option for lung epithelial cells, which have been targeted for therapy of cystic fibrosis
|
|
targeting in vivo somatic gene therapy
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viral vectors
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problems with viral vectors
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targeted by immune system, reversion to virulence
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types of viral vector and features
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1. retroviruses: RNA genome is converted into dsDNA by the retroviral enzyme reverse transcriptase and stably integrates into host. can only infect dividing cells,only small gene inserts due to stability problems,Random integration of virus carries the risk of inactivating genes at the site of insertion.
2. Adenoviruses, DNA, can infect dividing and non-dividing cells, larger inserts, do not integrate with host genome so repeat delivery required. |
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Non-viral gene therapy
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liposomes
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what are liposomes and what is their function in potential therapy
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tiny lipid bubles, may be able to deliver DNA for gene therapy, fuse with cell membranes, not target of immune system, large DNA inserted, no infection risk, modifying bubble may allow targeting, most lost prior to arrival at target, weak and transient action
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cancer gene therapy
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introduce several genes into tumour cells. aim to kill cells, slow growth, cause expression of a protein to attract immune response, sensitise to chemo and radio therapy. Also target immune cells to induce sensitivity and action against tumour cells.
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external oncogenic stimuli
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smoking, radiation, sunlight and various chemicals, viruses, baceria
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3 basic stages of cancer progression
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1. initiation of a first genetic change
2. promotion, clonal expansion and accumulation of further mutations 3. progression, uncontrolled proliferation, switch from benign to cancerous |
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example of a tissue, mutation type and source of radiation damage
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ionising radiation eg Xray, DNA strand breaks, Production of ROS, causes Base damage, Deletions, Transitions, Transversions. Skin cancer
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example of a tissue, mutation type and source of chemical damage
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Polycyclic Aromatic hydrocarbons from Combustion of organic
compounds cause DNA adducts, Transversions, Frameshifts & Point mutations causing Lung cancer |
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example of a tissue, mutation type and source of viral damage
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Human Papilloma virus a DNA tumour virus causes Inappropriate gene expression & Cervical cancer
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internal oncogenic stimuli
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spontaneous mutation
1.DNA replication errors eg frameshift insertion/deletion, point mutations 2.chemical modification of bases eg deamination or alkylation 3. mutation by metabolism products. reactive oxygen species (superoxide, hydroxyl radical, and hydrogen peroxide), these can also be external stimuli. Antioxidants eg vitamin C and E are defense |
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what is a missense mutation
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frameshift causing wrong AA to be made
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nonsense mutation
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frameshift resulting in stop codon
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what is a transition mutation
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a point mutation when a pyrimidine is replaced by a pyrimidine or a purine by a purine e.g. G-C becomes A-T
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what is a transversion mutation
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a point mutation which occur when a purine replaced by a pyrimidine or vice versa (e.g. A-T becomes T-A or C-G)
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what is deamination
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amine group on a base is replaced by carbonyl group, this can alter the base type eg C turned to U
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what can remove U bases from DNA
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U-DNA-glycosidase
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partial internal partial external ongogenic stimuli
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metabolism products of drugs etc ingested Xenobiotics
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how are Heterocyclic amines (HCAs) linked to cancer
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bbq meat
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action of UV on DNA
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cross-links between adjacent pyrimidines, most frequent are cyclobutane pyrimidine dimers (CPDs), usually formed between adjacent Thymine bases, pull bases closer distorting DNA and increasing errors at subsequent replication
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what are (6-4) photoproducts
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Dimers formed by a single covalent bond between the 6 position of one pyrimidine and the 4 position of the adjacent pyrimidine on the 3' side, distorts DNA leads to errors
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what are cyclobutane pyrimidine dimers
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cross links usually between adjacent thymine bases, distrots DNA leads to errors
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5 types of DNA repair
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•Repair by Alkyl Transferase (AT)
•Base Excision Repair (BER) •Nucleotide Excision Repair (NER) •DNA mismatch repair •Recombinational Repair |
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what is a DNA adduct
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DNA covalently bonded to carcinogen
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what is alkyl transferase
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a form of DNA repair where alkylated bases are repaired by transferring the alkyl group to a cysteine residue in this this enzyme
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base excision repair. 5 steps
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repairs oxidative damage, often spontaneous
1. N-glycosidase removes damaged base 2. endonuclease cuts 5' end of abasic site 3. DNA polymerase B cuts 3' abasic end 4. DNA polymerase B fills site 5. DNA ligase seals ends |
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nucleotide excision repair corrects what type of damage
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complicated, multi-protein process to repair DNA lesions eg UV CPDs and 6-4 photoproducts.
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5 basic steps in nucleotide excision repair
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1. Detection of DNA damage is carried out by XPA, XPC, RPA
2. helix opened and TF-IIH (Transcription Factor IIH) macromolecule extends single strand 3.The DNA is cleaved on either side of the lesion by XPF & XPG 4. gap is filled in by DNA Polymerase ε, PCNA (Proliferation Control Nuclear Antigen) 5. DNA ligase seals chain. |
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NER damage detection
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XPA, XPC, RPA (Replication Protein A)
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NER strand separation
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TF-IIH (Transcription Factor IIH). This composes of 6 subunits including XPB, XPD, ERCC
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NER DNA cleavage
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XPF & XPG
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NER gap filling and sealing
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DNA Polymerase ε, PCNA (Proliferation Control Nuclear Antigen) and the pieces ligated by DNA ligase
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2 types of NER
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transcription coupled repair (TCR) and global genomic repair (GGR)
TCR ensures that transcribed strand damage is repaired first. |
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differences in proteins used in TCR NER and GGR NER
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TCR uses CS-A and CS-B to recognise damage not XPC
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cockayne syndrome is caused by
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CS-A or CS-B defects result in functional GGR NER but defective TCR NER
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DNA MISMATCH REPAIR (MMR), 3 steps
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wrong base has been inserted
1. MSH2:MSH6 heterodimer binds to mismatched base 2. MLH1:PMS1 heterodimer binds MSH2:MSH6, exonuclease cleaves strand 3. gap filled by DNA polymerase and DNA ligase |
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recombinational repair
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a mutated section of dna (eg pyrimidine dimer) is skipped in replication and the gap is filled using the normal strand as a template. This is not repair but a way to tolerate damage.
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what is the mutator phenotype
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inactivity or deficiency of genes involved in repairing damaged DNA making the cell more error-prone and lead to increased mutation rate in other genes. These cells then outcompete neighbours.
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4 main stages of cell cycle
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G1, S, G2, M
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what happens at G1
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Growth and preparation of the chromosomes for replication
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what happens at S
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(synthesis) Replication of DNA
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what happens at G2
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Preparation for mitosis
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what happens at M
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(mitosis) Division of cell into 2 daughter cells
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what is the function of cyclin dependant kinases
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catalyse the phosphorylation of other proteins, thereby activating them. This regulated the cell cycle
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what are the principal regulators of G1
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cyclin D & E
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how is a a CDK activated and produce its action
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binds with cyclin, activated by phosphorylation, this activates a transcription factor by removing an inhibitor. This induces gene transcription for cell cycle progression and also next CDK and cyclin genes
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RB example of CDK system
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p16 INK4a inhibits cyclin D1/CDK4 complex, if uninhibited D1/CDK4 hyperphosphorylates pRB in the pRB/E2F complex, this dissociates activating the transcription factor E2F which stimulates transcription of various growth genes eg cdc2, myc. This activates other transcription factors eg Fos, jun in a cascade.
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if erros occur in the cell cycle what are used to halt progression, examples of these
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cyclin dependant kinase inhibitors, P21 family
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key cell cycle checkpoints
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•before the cell enters S phase (a G1 checkpoint)
•during S phase •after DNA replication (a G2 checkpoint). |
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proto-oncogene
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a normal gene that can become oncogenic if mutated
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5 ways a proto-oncogene may become and oncogene
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•Retroviruses
•Protein-Protein interaction (from DNA viruses) •Chromosome translocation •Gene amplification •Point mutation |
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2 modes of retrovirus activation of proto-oncogene to oncogene
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transduction- host genome gene is incorporated into virus and produced (acute transforming viruses)
transfection - the virus inserts its genome into the host genome (non-acute transforming viruses) |
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what are long terminal repeats (LTR)and what is their role in oncogenic activation
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retroviral genomic elements that enhance gene expression. If a host gene is transfected into a retrovirus it may upregulated by viral LTR and if viral RNA is converted to DNA and inserted into host genome the viral LTR may act on host genes
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DNA viruses in oncogene activation
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viral proteins may bind host proteins preventing action eg HBV E7 protein binds host Rb (tumour suppressor) so preventing it from inhibiting E2F transcription factor
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chromosome translocations in oncogene activation, 2 modes of action
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1. increase normal product, if a proto-oncogene is translocated to be influenced by a strong promoter
2.expression of combined product, chimeric protein |
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example of chromosome translocation increasing normal product to induce oncogene
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burkitts lymphoma, c-myc proto-oncogene on C8 is translocated to C14 and influenced by Ig gene enhancer
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example of chimeric protein causing oncogenic action
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CML, Philadelphia gene, c-abl on C9 combines with BCR on C22. The BCRabl chimeric product is a tyrosine kinase homologous to abl but with mutant qualities
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gene amplification and oncogen activation
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multiple copies of a proto-oncogene develop in chromosome due to defective start signalling in replication, copies are called double minute chromosomes. if all of these are transcribed then protein product will be greatly amplified
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double minute chromosomes
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multiple copies of a proto-oncogene that may lead to amplified transcription and so protein translation
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homogenous staining regions
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a chromosome region containing double minute chromosomes, gene copies that were amplified aberrantly
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example of gene amplification in oncogen activation
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HER-2 is over expressed in some breast cancers, this receptor protein-tyrosine kinase is overexpressed and binds epidermal growth factor, which stimulates cell proliferation. Target of Herceptin
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point mutations in oncogen activation
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single base pair mutation that alters protein enough to change function
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example of oncogenic point mutation
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RAS protein involved in signal transduction cascades, point mutated forms of the ras gene produce RAs protein that is not deactivated and constant stimulation drives cell cycle progression
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5 classes of oncogen
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•Secreted Growth factors
•Cell surface growth factor receptors •Components of intracellular signal transduction system •DNA binding gene regulatory proteins •Components of cell cycle : cyclins/CDKs/CDKIs |
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secreted growth factor oncogene example
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c-sis overexpression leads to overabundance of platelet derived growth factor -sarcomas and gliomas
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Cell surface growth factor receptor oncogene example
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erbB gene encodes EGF receptor, when mutated the usual phosphorylation does not inactivate so permanently on signalling for growth and division. Breast cancer
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Components of intracellular signal transduction system as oncogene- example
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ras, when mutated activated as normal by GTP binding but GTPase does not work on it so permanent activation, phosphorylation of downstream protein kinases inducing proliferation. Found in many cancers
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Transcription factors as oncogenes, example
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myc, fos, and jun TF's mutated forms mean growth factor stimulation is not required to induce transcription.
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Components of cell cycle: cyclins/CDKs/CDKIs as oncogenes, example
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D-type cyclins regulate progression through the early stages of G1. Cyclin D1 produced by oncogene PRAD-1
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features of apoptosis
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controlled programmed cell death, shrinkage, nuclear condensation, blebbing, fragments into vesicles, phagocytosed by macrophages
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2 important proteins in apoptosis
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bcl-2 and bax in balance. If bcl-2 levels become elevated it is encouraged to continue proliferating. Conversely, if bax levels are elevated the cell is encouraged to die
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example of apoptotic pathway mutation causing cancer
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follicular cell lymphoma, bcl-2 is over expressed due to translocation so is increased relative to bax and proliferation is promoted.
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example of oncogenic teamwork
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C-myc transcription factor and ras signalling protein, if bothmutated cancer is nearly 100% assured
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how can tailored changes be made to a genome, 2 stage process
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engineered endonucleases can cut dsDNA at desired point and DNA fragments containing th edesired change to the sequence are also inserted to the nucleus, these are then used as templates for filling in the gap by homology-directed repair (HDR)
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knudson model
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2 hit model of cancer progression, both copies of a gene must be knocked out or mutated
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classic 2 hit example
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retinoblastoma, an inherited recessive loss-of-function mutation to Rb on C13
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proportion of cancers with mutated p53
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60%
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features of p53
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TSG on C17, often deleted in cancers
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most common type of somatic mutation of p53
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point mutation
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inherited p53 mutation is commonly found as what disease
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Li-Fraumeni Syndrome, usually inherited one mutated allele and other then deleted. exhibit a wide range of tumours, including breast, brain, leukaemia and sarcomas.
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function of p53
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genomic guardian, the main player in the complex pathways that mediate cell cycle control and DNA repair
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effect of DNA damage on p53
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normal 20 minute half life is extended as it is stabilised so accumulates
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how is p53 normally controlled
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mdm2 binds p53 targeting it for ubiquitinylation and so destruction, also inhibits p53 activity as an activator of transcription.
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how does DNA damage lead to p53 build up
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amino terminus is phosphorylated so association with mdm2 does not happen
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how is p53 in an autoregulatory system and how is this mutated
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p53 activates the expression of its own regulator mdm2, mutated p53 this does not happen
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3 biological activities of p53
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1) Induction of G1 arrest and Suppression of Tumour cell growth
2) Induction of Apoptosis 3) Differentiation of B-cells |
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p21 function at G1 checkpoint
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cell cycle inhibitor which works by binding and inactivating the cyclin/CDK dimers that regulate the cell cycle
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GADD45 function at G1 checkpoint
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GADD45 protein inhibits DNA synthesis by binding PCNA and also promotes DNA excision repair. Over-expression of GADD45 suppresses cell growth.
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function of p53 in G1 arrest and tumour growth suppression
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1. cell cycle checkpoint protein. in response to damage activates the expression of several genes including GADD45, and p21 at G1 checkpoint.
2. Activated in response to over-expression of gene products from myc, ras, jun oncogenes to check unregulated proliferation |
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function of p53 in inducing apoptosis
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activation induces transcription of bax and inhibits bcl2 expression thus changing the ratio and inducing apoptosis
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function of p53 in B cell differentiation
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not fully known but aids development to mature form
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6 biochemical functions of p53
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1) Regulation of DNA synthesis
2) Sequence-Specific DNA binding 3) Transcriptional Activation 4) Transcriptional Repression 5) Promotion of DNA re-naturation and Strand Transfer 6) Translational Control |
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p53 in regulation of DNA synthesis
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binds DNA adjacent to origin of replication blocking attachment of initiation proteins, prevents helicase unwinding, binds and inactivates replication factor RPA.
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Sequence-Specific DNA binding by p53
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most p53 mutations occur in the DNA binding domains
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p53 Transcriptional Activation
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N-terminal domain of p53 is a strong transcriptional activator of genes with a p53-binding motif on their promoter. Rb1, GADD45, EGF, creatine kinase, mdm2 and p53
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p53 Transcriptional Repression
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repress the transcription of growth promoting genes- c-fos, c-jun and c-myc. normal levels do not suppress but when elevated by DNA damage it does.
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p53 Promotion of DNA re-naturation and Strand Transfer
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catalyze re-naturation of complementary strands of ss DNA
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p53 Translational Control
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associate with the cellular translation machinery eg TGF-β mediates down-regulation of CDK4 synthesis at the translational level and TGF-β controlled by p53
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functional p53 response to DNA damage detection
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stabilised, lengthened half life acts to halt cell cycle and induce DNA repair at G1 or G2 checkpoint, if repaired then cycle continues, if not then apoptosis initiated
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how may p53 be involved in failure of chemotherapy and radiotherapy
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chemotherapy and radiotherapy aim to cause just enough cellular damage to activate the in-built cellular mechanism for over-expression of p53 and so apoptosis, this will not work if p53 itself is mutated.
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p53 as therapy
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in cancers where p53 is mutated chemo and radio therapy will be less effective so adenovirus vector introduction of unmutated p53 is being trialed
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are oncogenes or TSG associated with hereditary cancers
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TSG, think RB
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what is the distinction between a benign tumour and a malignancy
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is it confined within a capsule
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at what stage are oncogenic genetic alterations likely to occur and why
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mitogenesis (the induction of mitosis), short time i not allow sufficient time for repair. Also ss DNA is more sensitive to damage
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how can epigenetics affect tumour development
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DNA methylation, transcriptional activation or translational control may all control gene expression eg DNA methylation silences genes (perhaps TSG), if this process is disturbed TSG may be overly silenced or correct silencing of proto-oncogenes may not occur
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example od a compound that is thought o affect epigenetics
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folic acid, B12
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what happens in the promotion phase of tumour development
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clonal expansion of originally mutated cell, further mutations develop (especially if original mutation confers competitive advantage) replication becomes less accurate.
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what natural process may act to stall growth or induce apoptosis in a growing tumour mass
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central hypoxia induces p53
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features of malignant cells
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incompletely differentiated, rapid growth, invasive, loss of capsule, prone to metastasis
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what cell feature makes cells a target of chemotherapy drugs and what is the problem with this
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rapid division, means that chemo drugs also affect normally rapid dividing cells eg hair and GIT
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stages of metastasis
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1. detachment
2. release of angiogenic factors 3. dissemination 4. establish new foci |
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Detachment
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first stage of metastasis development. loss of CAM's eg e-cadherin. normally proliferation requires adherence to ECM, this is Anchorage dependance.
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what is anchorage dependance
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cells must normally be correctly adhered to the ECM to proliferate
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2 types of cellular adherance must be lost for detachment
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1. intercellular adhesion
2. adhesion with cell and ECM |
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how do cell normally attach to basement membrane
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integrins
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how may a tumour cell break adherance to basement membrane yet still be able to adhere again at a new loci
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tumour cell produces lamanin and lamanin receptor with lamanin already bound.Lamanin has heparin and collagen binding sites on it allowing ECM binding. To move Type IV collagenase is released by tumour cell breaking lamanin-collagen bond
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what is anchorage independence
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normally detached cells have inactivated regulatory protein Cyclin E-CDK2 which stalls growth, in tumour cells oncogenes relay false signals to the nucleus maintaining Cyclin E-CDK2 activity
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what is Cyclin E-CDK2
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a growth regulatory protein that normally stalls growth by inactivating in detached cells. This action is maintained in metastasising tumour cells, anchorage independence. Normal function involves dissociating Rb protein from transcription factor that initiates transcription of cell cycle proteins
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how do tumour cell in the centre of a mass obtain oxygen
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release of angiogenic factors
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how is angiogenesis normally controlled
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p53 controls transcription of TSP-1 angiogenic inhibitor thrombospondin 1, loss of P53 removes this control, seen in Li-fraumeni
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4 modes of tumour dissemination
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blood, lymph, locally, across bod cavities
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haematogenous metastasis
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tumour releases matrix metalloproteases MMP that break down ECM and basement membranes travel in blood, aggregate platelets and lymphocytes, attach to wall and after further protease breakdown extravasation
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what is intravasation
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tumour breakdown of blood vessel wall to enter
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what is extravasation
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tumour release of proteases to break down vessel wall ECM of blood vessels to leave circulation
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how do tumour cell survive in the blood
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must attach to the lining of the blood vessels and aggregate platelets and lymphocytes to protect from killer T cells and increase adhesion. this aggregate then travels and re-adheres elsewhere
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what is a carcinoma and how does it usually disseminate
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epithelial cancer usually with lymphatic metastasis
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what is a sarcoma and how does it usually disseminate
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connective tissue cancer usually with haemaotologenous metastasis
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why are the lungs common points for metastasis
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the first point of contact for vasculature leaving many tissues is the lungs
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what factors are thought to underly the preference of some tumour types for certain tissues for metastasis, example
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the concentrations of growth factors and hormones and surface receptors eg prostate cancer and bone marrow,
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tumour staging
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TNM method
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what do TNM stand for and how is it scored
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T- primary tumour site
N - extent of lymph node involvement M - extent of metastasis each scored 1-4 |
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what are metastatic factors and an example
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patterns of gene expression within tumour cells that are predictive of metastatic spread eg HER-2 in breast cancer, many p53 mutations lead to agressive and invasive cancers
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XERODERMA PIGMENTOSUM
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severe sunlight sensitivity, 2000x skin cancer risk. UV-induced lesions in DNA are inefficiently removed or not at all
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XERODERMA PIGMENTOSUM genetic cause
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nucleotide excision repair (NER) pathway defect, 7different types - A-G
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cockayne syndrome
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inability of cells to carry out transcription-coupled repair (TCR). so although general NER is functional errors occur specifically at transcribed regions
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