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Flashcards in Unit 2 Deck (94):
1

Lambda genome of a virus

Very compact and hard to get the gene of interest

2

Organisms genome

Lots of junk DNA

3

Lab series 2 steps

1) restriction enzyme digests (lamba DNA digest and pUC19 digest)
2) ligation (ligate lambda DNA chunks with pUC19 plasmid)
3) electroporation (bacteria take up rDNA)
4) blue/white (select for cells with rDNA)
5) selection, expand white cells
6) electrophoresis to confirm rDNA
7) mutagenesis

4

Functional beta gal forms a

Tetramer

5

Mutated beta gal forms a

Can only form a dimer

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What have to be combined for functional beta gal protein to be made

Laczdeltam13 and ladzalpha

7

Cip reaction

Phosphate enzyme digested pUC19 (removes phosphate groups so just hydroxyl)

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White cells

PUC19 cut open and lambda DNA inserted, functional protein cannot be made

No laczdelta gene and no beta gal function

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Blue cells

Bacterial genome, mutated lacZ gene, and puc19 gene leads to functional beta gal protein

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Mutagenesis

Create a mutation in your assigned gene of interest using PCR

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What is an application of molecular cloning?

GMOs

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GMOs first

Papaya in Hawaii

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Explain GMOs in Hawaii

-ringspot virus was wiping out papaya crops
-strategy to control outbreak
(Infect papaya plants with weakened ringspot virus)
-new strategy
(Papaya plant that expresses ringspot viral protein, can’t get infected by natural virus)
GMO- viral coat protein inserted into papaya genome

14

Tobacco and corn gmo example

-plants being killed by borer bug
-bt bacteria kills borer bug

GMO- bt toxin protein (cry gene) cloned into tobacco and corn

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Examples of GMOs

Papayas *cp
Corn/tobacco *bt
Round up *gmo crops resistant to glyphosphate herbicide

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DNA characteristics

1) semi conservative
2) semi discontinuous
3) DNA replication requires/uses RNA primers
4) occurs bidirectionally

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Explain semi conservative

-distinguish old DNA from new
*old DNA (N15 containing DNA), grow E. coli in N15 media
*new DNA (N14 containing DNA) switch E. coli to N14 media
Separate DNA by weight (N15 vs N14)— gradient centrifugation

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DNA replication process could be

Semi conservative, conservative, and dispersive

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Semi discontinuous experiment hypothesis and conclusion

-lagging strand with short fragments
Okazaki fragments

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Semi discontinuous experimental methods

Gradient centrifugation
-e coli cells without DNA ligase
-e coli cells in the middle of DNA rep (short pieces and long pieces of DNA, two bands)
-e coli cells that completed DNA rep

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DNA replication requires/uses rna primers hypothesis

DNA polymerase required short RNA primer to initiate synthesis

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DNase-

digests DNA but not RNA but can’t digest complementary so short RNA primers that don’t get digested

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Proteins involved in DNA replication

-DNA polymerase
-helicase
-single stranded binding
-beta sliding clamp
-clamp loader
-Tao
-primase

24

DNA polymerase

Enzyme that synthesizes new strand of DNA
-makes a phosphodiester bond
-synthesizes phosphodiester bond only if bass pairing is correct

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Helicase

Breaks apart hydrogen bonds in dsDNA (aka DNA B, hexamer)

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SSB

Protects single stranded DNA from getting degraded by free radicals

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B sliding clamp

Keeps DNA polymerase and DNA associated with each other

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Clamp loader

Protein that loads the clamp onto the DNA

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Tao

Keeps everyone held together

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Primase

Synthesizes short RNA primers, doesn’t need B/OH

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Initiation

The start of DNA replication (how do all the proteins find each other and the DNA)

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Origin of replication

(Ori c) special sequence in E. coli genome where dna replication initiates

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Elongation

Once DNA polymerase is binding dna and starts synthesizing new dna

-lagging strand synthesis is complicated
-lagging Okazaki fragments get ligated together

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Termination

Messy process, 2 replicaron forks will meet, low fidelity of replication (no mistakes or segments don’t get replicated)

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Termination in E. coli genome

Ter sites where termination happens
No protein coding genes in this region (ok if termination is messy)
Tus protein binds each Ter site

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Regulation in relation to the cell cycle

DNA replication occurs in S phase
-important dna replication only happens once per cell cycle

Lots of regulation, extra copies of genome mess up mitosis

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1st mechanism of regulation

DnaA protein

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DnaA protein binds to either

DatA or 9-mer DNA sequences

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DnaA protein mechanism

Binds to 9-mer sites and unwinds 13 mer sites
DnaA gets sequestered after replication is initiated

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Sequestered definition

Gets stuck binding to a different dna seq

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DnaA prefers to bind

DatA vs 9 mer in oric

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How many proteins is ideal and are needed to initiate replication once but not twice?

374 at least DnaA
350 bind datA and 4 bind oric

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How many proteins needed to bind to oric to initiate?

4

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DnaA process

DatA gets replicated soon after initiation
-datA can bind 370 DnaA proteins
After initiation 740 DnaA proteins bind and 4 DnaA sequestered bind to the 2nd DatA sequence

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Range of DnaA proteins

Greater or equal to 374 but no more than 744

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2nd mechanism of regulation

Dam methylase sites

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How many minutes for dam methylase

1 min

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If DnaA mechanism fails, what is the backup

Seq A proteins binds hemimethylated DNA

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Mutation

When damaged dna gets replicated or transcribed d

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Damage

Change to dna chemical structure

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Types of damage

Endogenous or exogenous

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Endogenous

From inside the cell (spontaneous)

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Exogenous

From outside the cell (induced)

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Endogenous specific types of damage

Polymerase errors
ROS oxidative damage

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Exogenous types of damage

Radiation
Small chemicals
Bulky

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Polymerase errors repair mechanisms

Proofreading
Mismatch repair

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ROS oxidative damage repair mechanism

Base excision repair

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Radiation repair mechanism

Nucleotide excision repair

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Small chemicals repair mechanism

BER *base excision repair
Adaptive response

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Bulky

Nucleotide excision repair

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DNA polymerase mistakes

DNA polymerase active site should only fit proper base pairs, sometimes mistakes (wrong nucleotide) due to active site not catching

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Proofreading mechanism

Different region of the enzyme — sense mismatch and cut out

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Mismatch repair mechanism

Mut S, mut H, mut L

Hemimethylated state is important so cell can know which strand has the mistake

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Mut s

Homodimer, bind to the mismatch

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Mut h

Nuclease, cut mismatched dna

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Mut L

Bridge between Mut S and Mut H

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Steps of mismatch repair

1) mut L binds Mut H and Mut S
2) Mut H cut the new strand of dna unmethylated *can only do if binding Mut S/ Mut L
3) exonuclease (digest DNA starting at cut, up to where Mut S is binding)
4) DNA poly I resynthesizes the DNA

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Base excision repair

Interfere with base pairing and replication of transcription

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If not repaired due to BER , can result in

single nucleotide changes

70

Class of dna glycosylase proteins

- 5 hydroxycytosine dna glycosylase
- thymine glycol dna glycosylase

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5 hydroxycytosine dna glycosylase examples

-Damage
-5 hydroxycytosine dna glycosylase binds to damaged base, cut damaged base out by glycosylase
-repaired

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BER function and example

DNA glycosylase that recognize bases modified by alkyl groups
Ex) 3 me A dna glycosylase

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Adaptive response

6meG and 4meT, no corresponding dna glycosylase so no BER

If not repaired, misincorporation during rep or transcription

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Ada

Protein that recognizes and repairs 6meG and 4meT

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Ada functions

-transfers methyl group from 6meG and 4meT to sulfur atom in active site
-can only do this repair mechanism once (before dying, acts as transcription factor)

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Ada vs Ada-me

Ada-me has methyl group in its active site (acts as a Tf for Ada gene)

Can repair mechanism damage, doesn’t act as Tf

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Transcription factor

-protein involved in transcription (either enhance transcription or hinder)
-influence RNA polymerases ability to transcribe

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Ada is positive or negative feedback loop

Positive feedback loop turns off once methylation damage is repaired

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Nucleotide excision repair

-uv radiation (thymine dimers)
-bulky damage (chemotherapy agents, etbr, cigarette smoke)

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Proteins involved in nucleotide excision repair

UvrA, B, C, D

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UvrA

Recognizes damage, dimer

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UvrC

Cut the damaged DNA, nuclease activity

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UvrD

Removes damaged DNA, helicase activity

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Uv radiation and bulky damage

Capable of stalling dna poly

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Damage avoidance mechanisms

-if thymine dimers or bulky damage aren’t repaired (they stall DNAP) and signal for the cell to kill

86

Damage avoidance mechanisms E. coli cells

E. coli cells have mechanisms to bypass bulky damage or thymine dimers so that replication can be completed -survive

87

2 damage avoidance mechanisms

1) lesion bypass polymerase
2) recombination

88

Nucleotide excision repair mechanism steps

1) UvrA and B recognize damage
2) recruit UvrC and D to repair
3) UvrC will cut DNA while UvrD unwinds DNA
4) repaired by DNA poly I and ligase

89

Lesion bypass polymerase steps

-DNA poly III is stalled
-Lesion bypass polymerase enters
-DNA poly III falls
-Random nucleotides get incorporated
-DNA poly III enters
-LBP leaves
-Random nucleotides but no death
-NER can fix the damage (using random nucleotide template)
-Random nucleotides are used as a template for DNA poly I

90

Steps of recombination

1) DNA rep
2) DNA poly III fell off of DNA, reinitiated DNA replication
3) one DNA (has a gap and damage) uses other strand of DNA to fix the gap
4) recombination proteins pull damage strand into the other strand — base pairing
5) DNA poly synthesizes the damage strand with other strand as template
6) sealed by ligas and damage repaired by NER

91

Molecular cloning

Move a DNA sequence from one place to another while making copies

92

GMO example bt corn of molecular cloning

Bt-corn
*cry gene from bt bacteria cloned into corn genome
*isolated gene responsible for killing bug
(Toxin protein cry gene)
*put in corn through cloning

93

Steps of molecular cloning (general)

1) isolate gene from bacteria
2) insert gene into plasmid
3) move rDNA into host organism
4) select host organisms that contain rDNA

94

Plasmid

Circular piece of dsDNA has features/genes that we will use in subsequent steps