lectures 10-21 Flashcards
genome sizes are stated as….
bp per haploid genome
benefits from HGP?
personalised medicine
the central dogma
dna makes rna makes protein
dna and rna replication transfers info
DNA replication, transcription, translation, RNA replication, reverse transcription
reverse transcription
info at RNA level is copied to DNA
RNA viruses
retroviruses
mostly in RNA level
insert RNA into our DNA
why did Mendel work with peas?
large numbers of offspring short generation time self-fertilisation and cross-fertilisation possible cheap convenient
the first law of inheritance: the law of segregatin
2 coexisting alleles of an individual for each trait, segregate during gamete formation so that each gamete gets only 1 of the 2 alleles
what acts like Mendelian factors?
sex chromosomes
what did T. H. Morgan propose?
X chromosomes carried genes other than sex determinants (eye-colour in fruit flies)
sex-linked
what did Walter Sutton propose?
chromosome theory of inheritance: sex was determined by chromosome based inheritance
alkaptonuria disease
secrete homogenistic acid into urine which goes black following exposure to air
what did Garrod propose?
a gene for a metabolic step was defective in albinism and alkaptonuria
Life cycle of Neurospora crassa (haploid organism)
haploid ascospores (4A, 4a), ascus germination growth of conidiospores (reproduces by vegetative haploid spores) germinating conidium vegetative mycelium of A and a
cells of opposite mating type (A and a) fuse to form binucleate heterokaryon (2 diff nuclei) nuclear fusion meiosis, mitosis to form diploid nucleus fruiting body separate to haploid ascospores
Beadle and Tatum’s question
what did they do?
is there a separate gene for each step?
made arginine auxotrophs of Neurospora crassa
mutated arginine biosynthesis
X-RAY MUTATION in A and a, mate them to form fruiting body, contained mutants in arginine biosynthesis
MATE wild and mutated type of both A and a types
DISSECT individual microscopic ascospores to individual test tubes, grow colonies
IDENTIFY MUTANTS by transferring to minimal medium so failure to grow identified nutritional requirements
IDENTIFY NUTRITIONAL REQUIREMENT by diff minimal mediums so mutation in AA biosynthesis
IDENTIFY ARGININE AUXOTROPHS by testing all AAs
auxotroph
mutant that requires a particular additional nutrient
prototroph
normal strain which does not require that nutritional supplement
evidence for multiple steps of pathway
if auxotrophs came from different asci, probably have different mutations
if defective in different parts of arginine pathway
mated, then mutants might complement each other so heterokaryon would grow in absence of arginine
provides evidence of multiple steps
how does complementation work
heterokaryon contains both nuclei so between them they can perform all steps in complementation
defects complement each other
Beadle and Tatum’s results
each step of metabolism requires individual gene
What did Friedrich Miescher find?
discovered nucleic acids
sticky substance in pus
like protein but rich in phosphorus with no detectable sulphur
Griffith’s experiment on the transforming principle/factor
Avery-MacLeod-McCarthy follow up experiment
R rough colonies are non-pathogenic
S smooth colonies are pathogenic
dead S cells don’t cause pneumonia, no cells left
mixture of dead S and living R caused pneumonia so S cells transformed the R cells into pathogens
living S killed by heat, with enzyme treatment, with living R, only DNase destroyed the transforming principle
also purified DNA from S cells added to R cells transformed R cells to S cells, therefore hereditary material
T2 ‘phage research
phage infects E.coli with attachment mediated by base plate and fibres
remain attached but heads appear empty
labelled their DNA radioactively infect, centrifuge some bacteria were now radio-labelled grow bacteria in fresh medium phages were radiolabelled - confirms DNA is genetic material
labelled phage protein, not transferred to E.coli so not genetic material
what sugar is used in DNA?
pentose deoxyribose (5 carbon)
purine bases
pyrimidine bases
adenine, guanine (2 rings)
cytosine, thymine (1 ring)
which bond joins a nitrogenous base to deoxyribose sugar?
glycosidic bond
between sugar C-1’ and N-9 (purine) or N-1 (pyrimidine)
what other bond is in DNA?
ester bonds are formed between sugar C-5’ and phosphate
phosphate in DNA makes it …
negative
DNA has a 5’ and 3’ end so it is…
polar
phosphodiester bond in DNA
links 3’ C of 1 nucleotide to 5’ C of next so has polarity
5’ end
3’ end
phosphate group
hydroxyl group
what was Levene’s model?
4 nucleotides in tetranucleotide blocks with bases pointing out
so DNA simple and not repetitive so not genetic material
also meant that each base present in equal amounts (not true)
x-ray crystallography
cross is typical of helical structure
stretched out DNA and left hydrated, produced cross pattern because alligned helices effectively form a diffraction grid which produces a cross pattern
closer spots on diffraction pattern means larger actual distance
calculated distances in DNA
why was Linus Pauling’s DNA model wrong?
triple helix means that sugar-phosphate backbone faced inwards but negative charges would repel each other
B-DNA
Z-DNA
right-handed clockwise (if slid down it)
left-handed anti-clockwise
6 key features of Watson-Crick model of DNA
right-handed
anti-parallel strands (for bases to match)
bases inwards, sugar-phosphate outwards
complementary base pairing
base pair distance (0.34nm apart)
major and minor grooves (backbones not equally spaced, important with how proteins interact w/ DNA)
how many hydrogen bond when A pairs with T?
C-G?
2
3
how is eukaryotic DNA organised?
DNA duplex (coiled in double helix) wrapped around histone proteins (nucleosome) chromatin fibre coiled chromatin fibre coiled coil metaphase chromatid (folded)
conservative model for DNA replication
parent strand transfers info to intermediate and this gets copied
parent helix is conserved and daughter is completely new
dispersive model for DNA replication
parent helix broken into fragments, dispersed, copied, assembled into 2 new helices
new and old DNA completely dispersed
caesium chloride equilibrium density gradient centrifugation
separates molecules on basis of densities
get gradient of CsCl
macromolecules will be where match CsCl density
Meselson and Stahl testing of models of DNA replication
used centrifugation to separate on densities: E.coli grown in light (N-14) and heavy (N-15) so when incorporated will label DNA
mixed together, separate, created dark bands with UV light
E.coli grown in heavy, then added light, so t=0 all would be heavy, as grow and divide would incorporate light
semi-conservative:heavy, intermediate, light and more intermediate, slowly goes to light and loses intermediate because more of new strands, loss of heavy and light (old and new)
this is what experiment showed
predicted bands for other models didn’t occur
what function does DNA polymerase I (Pol I) have other than 5’ to 3’ polymerising
5’ to 3’ exonuclease (break down)
3’ to 5’ exonuclease activity
so editing mistakes and proof-reading function
nucleotide incorporation into DNA
release of pyrophosphate (2 phosphates) from nucleotide and hydrolysis to phosphate
so 1 phosphate remains on nucleotide which binds to other nucleotide
DNA polymerase action during incorporation of nucleotides
conformational change in DNA polymerase, closes round DNA when correct nucleotide binds
testing DNA strand polarity
radio labelled phosphate on nucleotides about to be incorporated
phosphate is 5’ of new nucleotide that’s inserted, when DNAse breaks ester bonds so strand into pieces, labelled phosphate is now attached 3’ of nearest neighbour
so see how often bases near each other (phosphate go from 1 known radiolabelled base to next base)
proportions of bases can only match if strands are anti-parallel (GpA same amount as TpC so has be be opposite directions if correct bases are to match)
editing by DNA polymerase
incorrect nucleotide sticks out with unpaired 3’OH so DNA polymerase 3’ to 5’ exonuclease removes mismatch
enzyme has 2 active sites, 1 for polymerisation, 1 for editing
why is DNA replication asymmetrical?
because on lagging strand DNA replicated away from the fork because always 5’ to 3’ which means that as fork opens, needs to prime again so not continuous
what are the fragments on the lagging strand called?
Okazaki
why is there no 3’ to 5’ synthesis of DNA?
in 5’ to 3’, the E used to incorporate the next nucleotide is from when the high energy bond is broken when pyrophosphate is removed
if 3’ to 5’, when editing is required and the wrong nucleotide is removed, it will leave 5’ end with only 1 phosphate (because pyrophosphate was removed when nucleotide was added)
so there is no high E bond available for next time a nucleotide needs to be added
what evidence shows that the primer for Okazaki fragments is RNA not DNA?
DNAse cannot completely destroy Okazaki fragments, it left little pieces of RNA, therefore they are the primers
DNA primase
type of RNA polymerase that is rifampicin-sensitive
catalyses the synthesis of a short RNA segment called a primer to inititate DNA synthesis on the lagging strand
makes a primer but doesn’t need a primer itself
lagging strand synthesis
DNA primase makes RNA primers
DNA Pol III extends primer to make Okazaki fragments
old primers erased by 5’ - 3’ exonuclease Pol I activity
replaced with new DNA
gap sealed by DNA ligase
Okazaki fragment joining
DNA ligase releases pyrophosphate from ATP and attaches resulting AMP to 5’ phosphate of downstream fragment
AMP released and phosphodiester bond forms between upstream 3’ OH and downstream 5’ phosphate
Processivity
enzyme’s ability to catalyse consecutive reactions without releasing its substrate
Leading strand synthesis
DNA helicase unwinds helix, with ATP
DNA primase makes RNA primer
primed duplex captured by Pol III
clamp holder transfers 2 halves of β clamp to Pol III
(new clamp halves maintain clamp holder in state of readiness)
clamping converts Pol III to high processivity so can replicate long stretches of DNA
helicase continues to unwind and Pol replicates leading strand
lagging strand synthesis in terms of DNA Pol
DNA primers makes primers
primed duplex captured by Pol III, clamped
lagging strand into loop so that 5’ end the right way away from the fork (same way as leading strand)
until old Okazaki fragments pulled back to Pol III so triggers unclamping so goes back to low processivity (has to had low to release fragments easily)
DNA Pol I and DNA ligase repair gaps
process restarts by clamping new lagging strand prier
DNA Pol I doesn’t require high processivity because works short period
DNA polymerase structure
clamp loader with spare clamp halves
arms with Pol III core, β clamp on it
DNA primase and helicase on end of clamp loader
separately has DNA Pol I and DNA ligase, and single-stranded DNA binding protein
