Midsem test - topic 2 Flashcards
Polymorphism
Mutation carried by more than 1% of the population
Fluctuation test
- Luria and Delbruck
- Innoculated E.coli with bacteriophage to study mutations
- no poisson distribution so concluded that mutations are spontaneous and resistant mutants are pre-existing
Gene regulation
cellular transcription machinery controls whether or not genes are expressed and to what level
Structural mutations
alter protein/RNA structure or activity
Regulatory mutations
alter gene expression
Control at transcription
- gene copy number
- transcription efficiency = action of RNA pol
Control at translation
- post transcription control (mRNA modifications)
- mRNA stability (half life)
- mRNA translation - rate of initiation and translation
Control at folding
- protein stability
- post translation effects - modifications which repress or activate
What does transcriptional gene regulation involve
- whether or not gene is expressed
- level of gene expression
Promoter
specific sequence to aid gene recognition and start transcription, RNA pol binds here
Strong promoters
initiate transcription faster which has sequences that bind well
Weak promoters
bind RNA less well
Key sites for activator/repressor function
- DNA binding site = recognises specific DNA sequence
- allosteric site = effector binds here causing conformational change which sets binding site to functional or non-functional
Lac operon - NO LACTOSE
repressor binds to operator - no transcription
Lac operon - LACTOSE PRESENT
lactose binds to repressor so it cannot bind to operator, transcription occurs
Oc mutations
- constitutive mutation in operator
- repressor cannot bind to operator
- operon always on
- cis acting
cis acting
effect restricted to only the chromosome where the mutation is located
I mutation
defective repressor protein which cant bind to operon
operon always on
trans acting
Trans acting
effects multiple genes
cAMP in lac operon
- in high presence when low glucose
- cAMP forms complex with CAP which activates transcription of lac operon
- complex bends DNA to improve RNA polymerase recognition of binding site
high glucose, no lactose, no cAMP
no transcription
high glucose, high lactose, no cAMP
little transcription
low glucose, high lactose, high cAMP
high transcription
Negative control
inactivates repressor
Positive control
activator required for expression
Arabinose operon
- both negative and positive control
- positive control = AraC binds to araI to initiate when arabinose is present, CAP-cAMP complex
- negative control = AraC binds to AraO and AraI, creates a DNA loop so transcription cannot occur, arabinose is absent
Sigma factor
- RNA polymerase subunit
- when associated with RNA polymerase it allows RNA polymerase to bind to DNA sequence
- when transcription starts its released
- sigma 70 = recognises -35/-10
Consensus sequence
comprises the most commonly encountered nucleotides found at a specific location in DNA or RNA, made by comparing sequences via sequence alignments
Multicellular organisms
start as pluripotent stem cells and differentiate into all cell types in body
Pluripotent
cells that can differentiate into any cell type
Similarities between eukaryote and prokaryote gene expression
- promoter sequences vary to specific level of transcription initation
- use activator or repressor proteins
Differences of eukaryotes to prokaryotes
- have intron RNA that needs to be removed before translation
- in eukaryotes transcription and translation occur separately due to nucleus
- default state of eukaryote DNA is off due to the nucleosome position blocking promoter
Enhancers
where activators/repressors bind in eukaryotes, distant from gene
Transcription factors
- bind specific DNA sequences
- help RNA polymerase bind the promoter
- interact with mediator protein complex to promote transcription to determine whether RNA polymerase binds
TAD
chromosomes organised into 3D territories into loops
GAL system - NO GALACTOSE
- repressor Gal80 binds to Gal4
- Gal4 cannot activate transcription
GAL system - GALACTOSE
- Gal 3 binds galactose
- conformational change allows increased binding of gal 3 to gal 80
- gal 3 binds to gal 80
- gal 4 can bind to mediator and activate gene expression
- TFIID can bind
- DNA loops
Chromatin
complex of proteins, RNA and DNA that forms chromosomes within nuclei
Euchromatin
open and accessible
Heterochromatin
highly condensed and inaccessible
Constitutive heterochromatin
part of gene is permanently heterchromatin as its gene poor
Facultative heterochromatin
heterochromatin can change to euchromatin
Histone acetylation
associated with active transcription
4 major chromatin states
- active euchromatin
- inactive euchromatin
- facultative heterochromatin
- constitutive heterochromatin
Position effect variegation
- when a gene normally in euchromatin is juxtaposed with heterochromatin by rearrangement or transposition
- if gene is integrated beside heterochromatin it can spread to gene and silence it
- in drosophila white colour gene is inverted which allows heterochromatin to spread giving patches of red and white
DNA methylation
- cytosine methylated to methylated cytosine
- represses gene expression
- can be inherited - CG bases
- long term repression method
small RNA regulation
- DICER cuts long DSRNA into short dsRNAS
- RISC binds small RNA and denatures it
- incorporates one strand
- IF siRNA = RISC finds complementary mRNA and degrades it
- IF mIRNA = RISC pairs imperfectly with mRNA which leads to inhibition of translation
Advantages of small RNA regulation
- easy to use - only have to make small RNA antisense to target gene
- transient - allows reduced expression of essential genes
- powerful and flexible - can introduce many small RNAs at once to decrease expression
Alternative splicing
- introns have to be spliced via spliceosome
- 5’ and 3’ splice sites are very short so spliceosome doesnt know which one is correct so leads to alternative splicing
- produces different proteins
Point mutations
- single nucleotide changes in sequence = substitution
- transitions = type of base is conserved
- transversions = type of base changes
INDELS
- insertions/deletions
Large scale mutations
- large duplications/deletions
- translocations = breakage and movement of DNA
- inversions = piece of DNA breaks out and rejoins in inverse orientation
- transposition = DNA moves via a transposon
Chromosome mutations
change in number of individual chromosomes due to non-disjunction = ANUEPLOIDY
Mechanisms of mutation
- errors during DNA replication and repair
- spontaneous chemical changes of bases that change their pairing
- induced chemical changes of bases from external mutagens
repair DNA polymerase vs normal DNA polymerase
repair DNA polymerase has lower accuracy so higher error rate
Spontaneous base chemical changes
- cytosine deamination to uracil, TRANSITION
- methylcytosine deamination to thymine, more prone to deamination than cytosine
Induced chemical changes of bases
- substitution of natural bases for base analogs which can act as mutagens if they have unusual base-pairing
- EMS acetylates bases which alters base pairing, Guanine will bind to thymine
- BP converted to a mutagen via CYP450 which binds irreversibly to guanine
- 5-bromouracil can pair with either adenine or guanine
- ionising radiation = induces breaks
effects of point mutations
- silent/synonymous mutation = change in AA sequence that doesnt change codon
- missense/nonsynonymous = change in AA causing a codon change
- conservative = nonsynonymous mutation which changes to a similar AA
- nonconservative = nonsynonymous which changes to dissimilar AA
- nonsense = premature stop codon
effects of INDELS
- can cause frameshift mutations if it occurs in coding region
effects of regulatory mutations
- affect level of gene expression so can alter transcription rate
- if in a trans regulatory gene it will affect gene expression of multiple genes
neutral mutation
has no effect on function
deleterious mutation
compromise function
beneficial mutation
improve function
Suppressor mutations
- suppress phenotype of another mutation
- tRNA mutations are example
- mutating tRNA anticodon to recognise mutated codon
Transposons in maize
- mutant causing a lack of colour gene but revered to WT colour at high frequency
- produced via transposition
- Ds jumped into c gene causing a frame shift
- movement of Ds relies on Ac
- when Ds moves out of C gene it restores function so purple reappears in patches
Inverted repeats
same sequence on opposite strands
direct repeats
- duplication of target site
- same sequence on same strand
- made by all transposons
2 classes of transposons in bacteria
- simple = single transposons with short inverted repeats at termini
- composite = a gene sandwiched between simple transposons and move as a block
transposases
- enzymes that catalyse movement of transposons
- bind transposon inverted repeats at termini
- causes cleavage and insertion into target DNA
class 2 transposons
- move via DNA intermediate
- DNA is copied/excised and moves to a new site
class 1 transposons
- retrotransposons
- move via RNA intermediate
- transposon transcribed then reverse transcribed back to DNA
- integrates into genome
- replicative = move by copy and paste
conservative transposon
move via cut and paste
autonomous transposon
encode transposase genes and catalyse their own transposition
non-autonomous transposon
rely on transposases from autonomous - Ds rely on Ac
effects of insertion of transposon
- cause mutations when inserted into genes or regulatory sequences
- white grapes = insertion into promoter, loss of colour
effects of transposon rearrangement
- recombo between transposons at different sites can cause duplications and deletions
- most occur in repair
- transposons on different chromosomes can cause translocations
- can cause problems with pairing in meiosis
LTR
- long terminal repeat retrotransposons
- retrovirus like
- no autonomous
- have promoter to allow for transcription
- have reverse transcriptase
- makes short target site duplications
LINES
- long interspersed nuclear elements
- have 5’ promoter and reverse transcriptase
- both autonomous and non autonomous
SINES
- small interspersed nuclear elements
- small, nonautonomous retrotransposons
- most abundance
- believed to use LINE transposases machinery so have a similar target side duplication
- almost all derived from tRNA genes that have mutated to transposons - EXCEPT Alu
- Alu = most abundant SINE, derived from small RNA
Transposon silence mechanisms
- heterochromatic chromatin with repressive histone modifications
- DNA methylation
- small RNAs with silencing mechanisms
- fungi target G/C to A/T mutations
conflict against transposon theory
transposon transcription is turned on in mammalian stem cells and early development
role of transposons
- help regulate global gene expression = LINES and SINES mediate different chromatin structures so different expression patterns
- regulating gene expression = placental development
