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T7 genetics - Coggle Diagram
T7 genetics
DNA
origins
hershey and chase: determined that DNA was the carrier of genetic information.
they grew virsues in radioactive sulphur or phosphorous as sulphur only appeared in proteins and phosphorous only appeared in DNA
then they took the viruses and placed them in a centrifuge, the spinning causes all the heavy stuff to sink to the bottom, therefore the nucleus (contatining the genetic info) should also sink to the bottom.
as a result they found that only those viruses grown in phosphate had a radioavitve pellet (sink to bottom) and those grown in sulphur were only radioactive in the supernatant (liquid)
franklin rosevelt and x-ray crystallography: used to deduce the structure of DNA:
- took DNA and laid them vertically within a glass tube
- shot x rays at it and prokected the diffractions on photographic film
from this they deduced that:
- must be double helix, double stranded
- phospahte outside, nucleotide inside
when building the model they found that there were equal number of purines and purimidines , as well as AT was double bonded and CG was triple bonded, leading the conclusion that during DNA replication:
- the molecules run by complentary base pairing
- the direction is bidirectional
DNA replication
there are a few important enzymes that are involved in dna replication:
- helicase - the unwinding enzyme that splits the helix into 2 strands, occurs at specific regions (called origins of replication) and creates replication fork
- DNA gyrase - relieves torsional strain on the DNA as it is uncoiling by negative supercoiling
- SSB proteinS (single stranded binding) - bonds to DNA strands after they have been seprated to stop re-annealling, and protect the nucleotides from being idgested by nucleases will depart once DNA polymerase III arrives
DNA primase - adds RNA primers to singal the begining of replication
DNA polymerase III: extends chain by adding nucleotide (does this as free nucleotides exist as triphosphates, DNA pol III cleaves the 2 phosphates and uses the energy to generate a phosphodiester bond) that are lined up agaisnt DNA strand via complementary base pairing, bonds to 3' end of primer and moves in a 5->3"
-- leads to a lagging strand, away from fork
-- and leading strand, toward the fork
if lagging strand, need 2 more enzymes:
- DNA polymerase I, replaces the primers with nucleotide bases
- DNA ligase - joins the okazaki fragments of DNA together - by creating phosphodiester bonds
okazaki fragments : sequences of dna created during dna replication on the lagging strand
as dna strands are anti-parrallel the DNA pol II must move in opposite directions, therefore on the lagging strand the Pol would need to always return and discontinoulsy make fragements
origin of replication: sequences where dna replication is inititated within a genome,
- in bacteria as they are circular DNA, they have only 1 origin
- as eukaryotes are linear and have more dna in general, they will have multiple points
- DNA synthesis may occur bi-directionally, which forms a replication bubble
-- the replication bubble will eventually fuse as the two forks meet, however its ability to go both ways drasticlaly decreases the time for DNA replication (i.e. withough replication for 1 chrom would take 1 month)
telomeres: regions of repetitive dna on the ends of chromosomes that help prevent chromosomal deterioration.
- as with each replication, the terminal RNA primer is unable to be replaced by DNA pol I at the extreme ends of the chromosome, therefore it will inevitably get shorter
- this is also why the shortening of telomeres is associated with ageing (usulaly 40-60 replications - hayflick limit) as cells may stop dividing when too much telomere dna is lost
- however telomere can be lengthed by an enzyme called telomerase, yet too much = cancer, so cancer researchers are finding inhibition drugs for telomerase to treat cancer
DNA sequencing: a method used to present the DNA seqeucnce of something, and is done through using dideoxynucleotides (ddNTP)
- ddNTP are unable to form phosphodiester bonds due to a lack of the hydroxyl group, this means that they will terminate the dna replication seqeucen when meeting is complementary pair, i.e. if using ddGTP, then the place where it terminates must have a cytosine
appication:
- need 4 PCR set ups, each tube contating the DNA, free nucleotides and 1 type of ddNTP,
- then do PCR to generate a billion copies and place solution in side a gel electrophoresis machine
- as each PCR contains a difference ddNTP, the overall gel electrophoresis should be enough to piece together what the DNA sequece looks like
noncoding dna: only 1.5% of the human genome consists of coding genes, the rest, before thought to be useless are actually pretty functional (e.g. STRs in dna profiling) not exaclty non-coding DNA but coding DNA vs repetitive dna
nucleosomes: are when dna is wrapped around an ocatmer of histones to help with condensing DNA during supercoiling:
- dna is wrapped around an octamer of histones
- histone tails link to form a chromatasome
- chromatasome coil to form a solenoid
- solenoid coils to form a 30nm fibre
- the fibre then folds to form chromatin
- chromatin supercoils to from chromosomes
histones are used to be bound to dna, as the negative dna is attracted to the positive on the amino acids
- histones also have N-terminal tails which extrude outwards from the nucleosome and during condesation, they link up with each other to form a chromatasome
supercoiling: the additional twist of DNA strands which create strain on that strand
- there are two types of supercoiling: positive (oerwound) and negative (underwound), most DNA is underwound
- supercoiling works to be more efficient when packing dna into the body
- nucleosomes help with super coiling and during dna replication, positive supercoiling occurs, putting strain, therefore dna gyrase will negativly coil the dna strand in order to reduce the strand
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