Genetics and Molecular Biology

who termed the word nuclein

fredrich miescher

what did frederick meischer do

isolated genetic material from white blood cells, noticed it had an acidic nature and called it nuclein

who determined the components of DNA

Phoebus Levene

what was phoebus levine wrong and right about

wrong: 4 nucleotides per DNA molecule, DNA does not store genetic code

Right:components (adenine, guanine, thymine, cytosine, desoxyriboe phosphate)

what did frederick griffith do

studied pneumoniae with R(harmless) and S(deadly) strains, if you injected a mouse with heat killed S strain it would live, but if you injected a mouse with heat killed S strain + harmless R strain it would die, did not determine cause

what did Avery, Macleod and McCarty do

they determined why the mouse died, they mixed the live R and heat treated s mix with either protease or DNAse, found that the mouse lived in the DNAse mix because the R DNA had been mutated by the dead S strain causing it to become deadly but when the DNase killed the DNA then it became ineffective

what did erwin chargoff do

examined the abundance of nucleobases and found that adenine and thymine were the same and guanine and cytosine were the same

he did not realise the importance of his discoveries but did share them with watson and crick

what did hershey and chase do

concluded that DNA was the genetic material and not protein

what are watson and crick famous for

they described DNA as a double helix with sugars and phosphates at the center and nucleotides facing the outside, they were proven wrong, but after rosalind franklins discovery, they flipped their model inside out which turned out to be right

what did rosalind franklin do

originally discovered proof of the double helixbut it was then stole and given to watson and crick

did rosalind franklin ever get a nobel prize

no she died too soon and they’re only given to living people, she prolly died because of the exposure to radiation she suffered during her research

describe the physical structure of DNA

backbone made of sugar and phosphate groups

hydrogen bonds between nucleobases (A and T) (C and G)

overall it is a double stranded helix

what is the base structure of a nucleotide

sugar (desoxyribose for DNA and ribose for RNA)

phosphate group

nitrogenous base

what is the difference between purines and pyrimidines

purines (Adenine and guanine) have 2 rings

pyrimidines (cytosine and thymine) have 1 ring

what are the chemical differences between DNA and RNA

they are similar except RNA uses a ribose instead of a desoxyribose as the sugar

RNA uses the pyrimidine base uracil instead of thymine

what is the nomenclature of an adenine in DNA compared to RNA

DNA=Deoxyadenosine 5’-monophosphate (dAMP)

RNA=Adenosine 5’-monophosphate (AMP)

what is Chargaffs rule

A=T. C=G. Purines/Pyrimidies=1 in DNA

how far apart are each base pair on a strand of DNA

0.34 nm

what are the names of the 2 grooves on a DNA strand

major and minor

what is more conserved in DNA the structure or the sequence

structure which is what allows the sequence to change

what part of DNA is soluble in water

the sugar and phosophate background, the bases are insoluble

what is melting of DNA

speration of 2 strands of DNA

is DNA seperation reversible

yes

what does the reversable seperation of DNA molecules allow for

allows artificial hybrid molecules to form (strands from different sources)

what are the 4 ways to melt DNA

increase temperature, reduce salt concentration, increase pH, solvents can also change the melting temperature

how is DNA melting measured

by measuring absorbance, as the DNA duplex seperates, absorbance increases

what is hyperchromi shift

as DNA duplex seperates, absorbance increases

what is Tm

melting temperature, an indication of the stability of hybridized DNA molecules

what does Tm say about the stability of a DNA helix

the higher the Tm, the more stable a DNA helix

what are the factors that affect a DNA strands melting temperature

G-C content

salt concentration of solution (ionic strength of buffer in M)

length of DNA

how does the G-C content affect melting temperature

the higher the G-C content, the higher the melting temperature

because more energy is required to break the 3 hydrogen bonds between G and C than the 2 between A and T

how does salt concentration affect melting temperature

the higher the salt concentration, the higher the melting temp

because, negative charges are shielded by salt ions which stabilize the helix and increase the energy needed to break it

what are things melting temperature is used for

-use to classify organisms by DNA

rare gene mutations can be detected as mutated sequences melt at different temperatures than normal ranges

-DNA melting serves an important role in many molecular biology techniqueswh

what were the 3 initially proposed models for DNA replication and which one was correct

conservative replication

Dispersive replication

semi-conservative replication (Correct)

what is the conservative replication model for DNA replication

the entire double stranded DNA molecule serves as a template for the new strand of DNA

what is the dispersive replication model for DNA replication

each strand of DNA is broken into fragment and proportion of new DNA increases at each replication

what is the semi conservative model for DNA replication

it is the correct model and it is an intermediate between the other 2 models,

the 2 nucleotide strands unwind and each serves as a template for a new DNA molecule

how was the semi-conservative model for replication confirmed

mixed original (heavy) DNA with light DNA, the first replication was 100% mixed between the 2 (so 50% light and 50% heavy), the second replication was 50% mixed and 50% light DNA, in subsequent replications the mixed band became thinner while the light band became thicker

in semi-conservative replication, what does each daughter DNA molecule consist of

one parental strand and one newly synthesized strand

what are the 4 requirements for DNA synthesis

one template strand of single stranded DNA (ssDNA)

all 4 desoxyribonucleoside 5’-triphosphate

DNA polymerase and other enzymes

free 3’ -OH group

what does DNA polymerase do

catalyses phosphodiester bonds, extends an existing DNA strand

can DNA polymerase make a a new strand of DNA

can only extend an existing chain

what is the absolute requirement in order for DNA polymerase to work

a pre-existing 3’ -OH

in which direction does DNA polymerase elongate a chain

5’ → 3’

in what direction does DNA polymerase read a template strand

3’ → 5’

where does DNA synthesis occur

at the replication fork

where does replication begin

at a specific nucleotide sequence called the origin of replication

within what does DNA synthesis take place

replication bubble

which DNA strand is synthesized first at the replication fork

both DNA strands are synthesized simultaneously

what is a replicon

any DNA molecule or region of DNA that replicates from a single origin of replication

if synthesis is always 5’→3’ how are both antiparallel strands copied simultaneously at the replication form

replication is semi-discontinuous

in the leading strand, synthesis is continous and in the direction of the fork

in the laging strand synthesis is discontinous and occurs in the opposite direction of the fork

what is the difference between synthesis in the leading strand vs the lagging strand

leading strand has continuous synthesis and occurs in the direction of the fork

lagging strand has discontinuous synthesis and occurs in the opposite direction of the fork

what is the lagging strand

strand that runs 5’→3’ towards the fork

what is the leading strand

strand that runs 3’→5’ towards the fork

what are the modes of replication of circular genomes

theta replication (bacteria)

rolling circle replication (viruses)

how many modes of replication are there for linear eukaryotic genomes

1 (linear replication)

describe theta replication

single replicon

bidirectional replication-2 replication forks within a bubble

replication is semi discontinuous at both forks

describe rolling circle replication

no replication bubble

one replication fork-uncoupling of the 2 strands of the DNA molecule

replication is continuous

what si the difference between theta replication and rolling replication

theta replication produces a single replicon while rolling circle replication produces 0 replicons, theta replication is semi discontinuous (2 forks) while rolling circle replication is continuous

how many replicons, origins of replication and replicaion bubbles are present in linear replication

multiple

describe initiation in prokaryote replication

-initiator proteins bind to origin of replication (Oric)

-a short section of DNA is unwound and proteins bind to the single stranded DNA

-single stranded binding proteins keep DNA strands seperate

-helicase binds to laging strand template and breaks hydrogen bonds

-DNA gyrase travels ahead of the replication fork and alleviates supercoiling caused by unwinding

what keeps DNA strands seperate in unwinding

single strand binding proteins

what does helicase do

binds to the lagging strand template and breaks hydrogen bonds, it cannot itself innitiate unwinding

what does DNA gyrase do

travels ahead of th replication fork and alleviates supercoiling caused by unwinding

describe the events of chain elongation

-a short stretch of RNA nucleotides (RNA primer)is synthesized by primase

-this RNA primer provides the free 3’ -OH for DNA polymerase to use

-Lagging strand synthesis is discontinuous so requires a new primer at the begining of each “okazaki fragment”

-RNA primer is later removed and replaced with DNA nucleotides

what does primase do

synthesizes RNA primer which provides the 3’ -OH required by DNA polymerase

which strand requires new RNA primers at the begining of each okazaki fragment and why

lagging strand, because synthesis is discontinuous

what replaces RNA primer once it’S removed in elongation

DNA nucleotides

why is RNA primer used in elongation

it provides a free 3’ -OH necessary by DNA polymerase and it’S production does not require a 3’ end

what element in the unwinding phase of replication uses ATP

DNA gyrase

what forms a complex that moves along the template of the lagging strand

primase and helicase

how many polymerases does E.coli have

5

what are the 2 DNA polymerases of E.Coli that we need to know

Pol 3 and Pol 1

what does E.coli polymerase 3 do

it is the principle replication enzyme that adds new nucleotides to the 3’ end of a growing DNA strand

what does E.Coli polymerase 1 do

removes and replaces RNA primers with DNA

what is exonuclease activity

the ability of an enzyme to remove nucleotides one by one from the end of a DNA or RNA strand by breaking phosphodiester bonds

which E.Coli DNA polymerases posess exonuclease activity

Polymerase 1 and polymerase 3 at least

what does DNA ligase do

seals the nick in the sugar backbone after DNA polymerase 1 removes and replaces RNA primers with DNA by catalysing the phosphodiester bond without addition of nucleotides

when does termination occur

when 2 replication forks meet or when specific sequences are reached

what differentiates replication of eukaryotic DNA from replication of prokaryotic DNA

-much greater genome size

-requires multiple origins of replication

-linear chromosomes cause a problem at termination stage of replication

-nucleosome must be dissassembled and reassembled during DNA replication

-more closely tied to the cell cycle while in prokaryotes it is continuous

when does replication begin in eukaryotes

at the end of the G1 phase

how does replication begin in eukaryotes

at the end of G1 phase, initiator proteins called ORC loads helicase onto the double stranded DNAs origin of replication

when is helicase activated in eukaryotic replication

in the S phase