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z-Test #1
PCB 4524 Zervos, Antonis Fall '10
24
Biology
Undergraduate 4
09/26/2010

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Cards

Term
Central Dogma
Definition
DNA RNA  Protein
Retrovirus
Term
Transcription vs. Replication
Definition
Transcription is similar to DNA replication
 Makes a new complementary strand, but made of ribonucleotides
 RNA pol does not need a primer (de novo)
 Multiple RNA pol can work on the same strand
 RNA pol has lower fidelity
o Important difference
 Replication of the whole genome once
 Transcription of a part of the genome multiple times
Term
Types of RNA
Definition
mRNA (messenger RNA-codes for protein), rRNA (ribosomal RNA), tRNA (transfer RNA), snRNA (small nuclear RNA, splicing), snoRNA (used to process/modify rRNA)
Term
Eukaryotic vs. Prokaryotic - Gene
Definition
 Euk
• TATA Box
• Introns
• Monocistronic
 Prok
• -35 Box, -10 Box
• TATA Box
Polycistronic
Term
Eukaryotic vs. Prokaryotic - Transcriptional Regulation
Definition
 Euk
• more refined, development
• Differentiation = making a cell specialized
• Depends on the control of genes
 Prok
• main goal is to quickly change depending on the surrounding, Environmental changes (oxygen, food, temperature)
Term
Eukaryotic vs. Prokaryotic - Transcription
Definition
 Euk
• Has nuclear envelope
• Transcription separate from translation
• Pre-mRNA must be processed to mRNA
• Alternative splicing
• Monocistronic
 Prok
• No nuclei
• Coupled transcription and translation
• Polycistronic
Term
Eukaryotic vs. Prokaryotic - Transcription Initiation
Definition
 Euk
• General transcription factors (GTF)
o Finds the promoter
o Closedàopen complex
• All GTFs + RNA pol = pre-initiation complex
• TATA box is distorted by insertion of a β-sheet
 Prok
• σ factor
o Finds the promoter
o Closedàopen complex
Term
Eukaryotic vs. Prokaryotic - Promoters
Definition
 Euk
• Four different elements
o TATA Box
o BRE
o INR
o DPE
• TATA box
o Helps locate the start site
o Change in efficiency
• TATA-less have GC-boxes
o House-keeping genes
 Prok
• UP element, -10 and -35 boxes, extended -10, Discriminator
Term
DNase footprinting
Definition
• Protein bound to DNA protects it from digestion by DNase I
• Used to find the site in DNA which the protein binds to
• The DNA is radiolabeled (32P)
• Binding affinity can be estimated by protein concentration
Term
EMSA
Definition
• Electrophoretic Mobility Shift Assay (gel shift assay)
• Used to find where the protein binds specifically
• In a gel, protein-DNA complex is slower than free DNA (Shift)
o Found ‘higher’ in the nondenaturing gel
• Can be used in conjunction to mutagenesis
• DNA is radiolabeled for identification
• Addition of antibody (Super-shift)
• For specificity or disruption of DNA-protein interaction
Term
Yeast-two hybrid system
Definition
• A known protein is used as ‘bait’
• Hybridized with DNA Binding Domain (DBD)
• Found in the pGILDA vector
• Screened against a library, the ‘prey’ proteins
o Screening
 You start with a single known protein (bait)
 Screen against a million of possible interactors
 Potential interactors can grow in restrictive media
 These are then ‘patched’ in a second isolation step
 Blue colonies in galactose only have their plasmids removed
 Plasmids are then sequenced
 Y2HS must be followed by co-localization and co-IP
• Hybridized with Activating Domain (AD)
• Found in the pJG4-5 vector
• If the bait-prey interact, the DBD-AD are close enough
• Leads to activation of the reporter gene
• Can be seen, such as blue colonies
Term
RNA pol in Eukaryotes is made up of many different subunits
Definition
o (2) Alpha subunits: assist in enzyme assembly, promoter recognition
o (2) Beta subunits: catalytic center, Mg++ binging on center cleft, resemble pincer in claw shape model
o Omega Subunit: restore denatured RNA polymerase in vitro to its fully functional form
Term
RNA pol I
Definition
Large rRNA precursor
Term
RNA pol II
Definition
mRNA and snRNA
Term
RNA pol III
Definition
5S rRNA, U6 snRNA, tRNA
Term
Sigma Protein
Definition
responsible for promoter recognition in prok
Term
Epitope Tags
Definition
o Polyclonal antibody may disrupt protein-protein interaction
o Monoclonal antibody is good for endogenous co-IP
o Epitope tag is added to protein of interest
o Has an antibody against it that can be used for co-IP
Term
Regulatory Elements
Definition
o Upstream activation sequence (UAS)
 Increase expression, upstream, near
o Enhancers
 Increase expression, close or far, up or downstream
o Silencers & repressors
 Decrease expression, near or far, interfere with TF
o Boundary elements/insulators
 Keeps two closely located genes separately
Term
General Transcription Factors in Euk
Definition
TFIID, TFIIB, TFIIF, TFIIE, TFIIH, TFIID
Term
Mediator Complex
Definition
o Very complex
o Recruited along with TFIIA and TFIIB
o Contains many subunits
o Transcriptional coactivator
o Bridge between the enzymes and TF
Term
Microarray
Definition
o Put a large number (~100K) of cDNA sequences or synthetic DNA oligomers onto a glass slide (or other subtrate) in known locations on a grid.
o Target mRNA  cDNA via reverse transcriptase
o Label an DNA sample and hybridize
o Measure amounts of RNA bound to each square in the grid
o Make comparisons
 Cancerous vs. normal tissue
 Treated vs. untreated
 Time course
o Many applications in both basic and clinical research
Term
SNP's Disease Examples
Definition
• 10% of African Americans have polymorphic alleles of Glucose-6-phosphate dehydrogenase that lead to haemolytic anemia when they are given the anti-malarial drug primaquine.
• AZT and cytochrome P450
o AZT needs to be modified by human enzyme before it can block HIV replication.
o One enzyme that metabolizes a large number of this pre-drug is cytochrome P450.
o 10% of the Caucasian population is homozygous for alleles of the Cytochrome P450 gene CYP2D6 that do not metabolize the hypertension drug debrisoquine, which can lead to dangerous vascular hypotension.
• Succinylcholine Toxicity
o 0.04% of individuals are homozygous for alleles of psedocholineseterase that are unable to inactivate the muscle relaxant drug succinylcholine, leading to respiratory paralysis.
• Isoniazid Metabolism
o There are many polymorphic alleles of the N-acetlytransferase (NAT2) gene with reduced (or accelerated) ability to inactivate the drug isoniazid.
o Some individuals developed peripheral neuropathy in reaction to this drug
o Some alleles of the NAT2 gene are also associated with susceptibility to various forms of cancer
• ACE
Term
Trans-splicing
Definition
o Exons from different pre-mRNA
o RARE in mammals
o No lariat structure, forms a Y instead
Term
Exon Shuffling
Definition
o An exon shuffles to a new gene
 Not mRNA
o Can be found in an unrelated gene
o Gene disruption most likely in intron
o Splicing mechanism leads to a new gene
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