Exam 3

restriction enzymes

enzyme that cuts DNA at a specific, palindromic sequence of nucleotides

gel electrophoresis separates DNA fragments based on

size

4 steps in gel electrophoresis

1. load DNA into gel
2. DNA migrates to "+" end of pole
3. stain w/ ethidium bromide
4. expose to UV light
- smaller molecules will move faster toward the + pole

steps for blotting

1. gel electrophoresis
2. transfer the results on the gel to a membrane
3. hybridize with sequence specific radioactive probe
4. remove unbound probe (washing)
5. expose X-ray to membrane (probe will be bound to complementary sequence)

Southern blotting detects

genomic DNA

Northern blotting detects

RNA

a higher intensity band in a Northern blot is indicative of

more RNA (more probe binding)

different band sizes in Northern blots can indicate

- size of introns (by comparing to genomic locus)
- alternative splicing

the size of fragments in Southern blots is determined by

the location of the restriction enzyme recognition sites

southern blot is \_____ for all tissues of an organism

the same (bc the genome is the same)

Northern and Western blots allow you to compare

tissues, developmental stages, organisms

Western blotting steps

1. primary antibody is designed to recognize a specific protein
2. secondary antibody binds the primary antibody and amplifies signal
3. enzyme on secondary Ab converts a substrate into a detectable product

higher intensity band on a western blot is indicative of

more protein (more Ab binding)

size of band in western blotting is determined by

the length of protein
- can indicate alternative splicing or protein cleavage

Western blotting detects

proteins

all 3 blotting techniques can determine

size

Northern and Western blotting can determine \_____, but Southern cannot

abundance (Southern simply tells you presence or absence)

DNA cloning steps

1. cut foreign DNA and vector using restriction enzymes
2. transform bacteria
3. plate on Amp and X-Gal

DNA cloning amplification

1. force bacteria to take up plasmid DNA by transformation
2. rely on bacterial replication to make copies of plasmid
3. grow large cultures and isolate plasmid DNA

components needed for PCR

template, pair of primers, dNTPs, Taq polymerase, and buffer

does PCR require knowledge of the DNA sequence?

only in the region of interest (for primers)

PCR does NOT need

restriction enzymes or host cells

PCR requires \_____ amounts of starting material

small

PCR primers must:

- be complementary oligonucleotides
- pair with different strands
- have 3' ends pointing at one another

3 basic steps of PCR

denaturation, annealing, extension

PCR denaturation

reaction heated to denature DNA into single strands

PCR primer annealing

reaction temp decreased to allow primers to hybridize with complementary DNA

PCR primer extension

Taq polymerase synthesizes DNA by extending primers

amplification of the DNA region of interest in PCR will occur \_____, while the extended DNA will only grow \______

exponentially, linearly (there will be more of just the DNA of interest in the end)

RT-PCR (reverse transcriptase-polymerase chain reaction) steps

1. oligo dT anneals to polyA tail
2. reverse transcriptase copies RNA to DNA
3. generates double stranded cDNA
4. amplify by PCR

how is DNA and cDNA different

cDNA is copied from mature mRNA and thus only contains exons whereas genomic DNA contains both introns and exons

molecular "library"

combinations of cloned fragments inserted into plasmids for further use

ddNTPs

DNA chain-terminating subunits \-- lack 3' OH

Sanger sequencing requires

DNA polymerase, DNA primers, dNTPs, ddNTPs

RT-PCR requires

reverse transcriptase, oligo dT

constitutively expressed genes

genes that are expressed continuously regardless of biological conditions

regulated genes

genes whose expression changes in response to biological conditions

inducible genes

normally off, turn ON under specific conditions

repressible genes

normally on, turn OFF in response to environmental change

in negative control, repressor proteins will

bind DNA and prevent transcription

repressor with inducer

- when bound to DNA, no transcription occurs
- when inducer molecule binds to the repressor, the repressor unbinds from DNA and transcription occurs

repressor with corepressor

- bound to DNA with the repressor/corepressor complex and no transcription occurs
- when corepressor unbinds repressor, the repressor unbinds the DNA and transcription occurs

in positive control, activator proteins will

bind DNA and initiate transcription

activator with co-activator

- transcription will not occur without the activator bound, and the activator will not bind without co-activator
- when the co-activator binds the activator, the activator binds the DNA and initiates transcription

activator with inhibitor

- when inhibitor is bound to activator, the activator will not bind the DNA and transcription will not occur
- when inhibitor unbinds the activator, the activator will bind DNA and initiate transcription

allosteric effectors

bind reversibly and change the confirmation of target protein

lac+

can grow in the presence of lactose and absence of glucose

lac-

cannot grow in presence of lactose without glucose

β-galactosidase (β-Gal)

catalyzes the cleavage of lactose into galactose and glucose, some lactose converted into allolactase (inducer)

operator (O)

DNA sequence to which Lac repressor binds

Lac repressor gene (lac I)

encodes lac repressor which is a diffusible protein, constitutively expressed

cis mutation

mutation will only affect gene expression on the same DNA molecule

trans mutation

affects gene expression whether its on the same or different DNA molecule

lacZ mutant

recessive

operator mutations are

cis-dominant, if Oc is present, the repressor will not be able to bind and LacZ will be expressed regardless of inducer presence

repressor mutations are

trans-acting, the mutation can act on any DNA molecule in the cell

I- (repressor mutation)

no active repressor --\> get lacZ expression regardless of inducer presence (as long as lacZ+)

repressor super mutant (Is)

- trans-acting
- always binding the operator and repressing expression even if the inducer is present

I+ (wild type)

- repressor binds operator when inducer is absent (no transcription)
- no repressor when inducer present (transcription occurs)

in the presence of glucose there is \_____ cAMP and \_____ lac mRNA

low cAMP, very little lac mRNA

in the absence of glucose there is \_____ cAMP and \_____ lac mRNA

high cAMP, abundant lac mRN

when tryptophan is absent, transcription is

high (no repressor)

when tryptophan is present, transcription is

low (repressor bound)

attenuation

control of gene expression by premature termination of transcription

trp attenuation utilizes

simultaneous transcription and translation

1-2 stem loop

pause stem loop

2-3 stem loop

antitermination stem loop

3-4 stem loop

termination stem loop

attenuation under high trp levels

- leader translated
- ribosome occupies regions 1 and 2
- 3-4 stem loop forms
- transcription is terminated

attenuation under low trp levels

- ribosome stalls at trp codons
- regions 2-3 pair (antitermination)
- regions 3-4 cannot pair
- termination continues

sigma factors control

clusters of unlinked genes

how do sigma factors coordinate expression

bacteria can switch sigma factors during heat-stress conditions and activate different genes to be expressed

promoters and promoter proximal elements (PPEs)

regulate the amount of gene transcription / generally located close to the gene of interest

enhancers

stimulate transcription

silencers

repressor proteins that may bind to DNA sequences and inhibit the start of transcription

promoters and PPEs are always located

upstream and near the transcriptional start site

enhancers and silencers can be located

upstream, downstream, or intronic and can be up to \>100kb away from promoter

insulators

block the effects of enhancers (can redirect enhancer activity to another gene, prevent transcription, or prevent enhancer from acting long distance)

function of the Gal pathway (in yeast)

when galactose is the only sugar available, wild type yeast induce transcription of the enzyme producing genes

coordinated control of the GAL system...

each gene is regulated by GAL4-UAS

when galactose is present (in yeast)

GAL 7, 10, 1 and 2 are expressed

when galactose is absent (in yeast)

GAL 7, 10, 1 and 2 are NOT expressed

regulation of GAL when galactose is absent

- Gal80 (represser) is bound to Gal40
- no transcription

regulation of GAL when galactose is present

- galactose and Gal3 bind Gal80
- Gal80 unbinds from Gal4
- Gal4 recruits RNA pol and begins transcription

DNA binding domain (DBD)

determines what genes are regulated

activation domain (AD)

recruits transcription machinery

steroid hormone receptors without hormone

receptor anchored in cytoplasm

steroid hormone receptors with hormone

receptor translocates to nucleus, able to regulate target gene expression

Myc/Max heterodimer

activator

Max/Max homodimer

repressor

Myc features

- activation domain
- DNA binding domain (DBD)
- dimerization domain

Max features

- NO activation domain
- DBD
- dimerization domain

in resting cells what does Myc/Max expression look like

- only Max expressed
- no transcription

in proliferating cells what does Myc/Max expression look like

- Myc and Max expressed
- transcription activated

ways of modifying chromatin for RNA pol II binding

- post-translational histone modification
- chromatin remodeling

the centromere is a location with a lot of

heterochromatin (inactive/silencing)

histone methyltransferase (HMT)

adds a methyl group to histone tails (causes it to become condensed/inaccessible)

histone demethylase (HMDT)

removes methyl groups from histone tails (causes it to become relaxed/accessible)

chromatin writers

adds markers to histone tails

chromatin erasers

removes markers from histone tails