Eukaryotic Genome

• Protein coding
• Tandemly repeated genes
• Repeating
• Spacer

• Solitary: represented only once
• Duplicated/Diverged (gene families): similiar proteins/sequences with similiar fxn very close together on genome

• rRNA
• tRNA
• Histones

• Simple: satellite DNAs
• Moderate: Mobile elements + Tandem Repeats + duplicated genes

• Satellite: @ centromere, repeates <500 bp tandem Millions in length (kinetochore)
• MiniSatellite: Highly polymorphic, 15-100 bp <3000 repeats, "fingerprinting"
• MicroSatellite: 1-4 bp repeated 50-100 times, "backward slipage"
• *sequences are highly conserved, number of repeates is highly variable*

• Cut from genome
• Put back into genome
• Replicative + non-replicative

• DNA mediated - non-replicative (inverted repeats target sites)
• RNA Mediated - replicative (direct repeats target site)

• Autonomous: has Ac gene ( encodes transposase)
• Non- Autonomous: Lacks Ac gene (Ds - dissociation)

• Transposase
• Makes blunt cut on IS + recognizes target sequence and makes sticky end/staggered cut + Ligates IS to 5' end of target

• Viral: LTRs present (250-600bp), follows retrovirus life cycle, all virus genes (-) envelope, Less common in mammals, DNA made in cytosol
• Non-viral: Lack LTR, DNA made in nucleus

Read in sense direction = 3' LTR (DNA) --> promoter, 5' LTR (pre-mRNA trx) --> signals host enzymes to process pre-mRNA

• LINES: 6 kb, 15% of genome
• SINES: 300 bp, 10% of genome

• Target site - Promoter region - ORF1-ORF2- direct repeat target site
• ORF1: codes for RNA binding protein assists in nuclear export
• ORF2: codes multifunctional protein - revertranscriptase+endonuclease

• Alu1: site for specific restriction enzyme, SINES have them, and they are located all over genome = possible insertion sites (always located in areas that do not dirupt gene expression)
• Transpose like LINES
• NF-1: Nurofibromatosis type 1- Alu insertion in intron which causes deletion of downstream exon during splicing = ORF shift

• spontaneous mutation
• gene duplication
• exon shuffling
• enhancer shuffling
• Evolution

• Linear DNA
• 10nm fiber/Beads on-a-string: Nucleosome - linker DNA - Nucleosome
• 30nm fiber: Condensed nucleosomes
• Loops of 30nm fiber: 30 nm Fiber looped on scaffold protein
• Higher order: compacted scaffold DNA

• 5 types of histones
• octamer of histones: 2(H2A, H2B, H3, H4)
• 147 bp surrounding histone complex (all mammals)

• Repress trx by preventing Pol from attatching or functioning
• Activate trx by recruiting pol or stimulating activity

• cis: are DNA chararacteristics and features (enhancers, promoters/promoter proximal, chromatin state)
• Trans: Elements that bind DNA (TF [activators/repressors], co-activators/repressors, HATs/HDACs, Mediator complexes)

• Short sequences near start site - recruit RNA pol initiation factors
• TATA box: -34-28 bp us
• Initiators: some genes
• CpG islands: present in low trx rate genes
• Promoter proximal elements: Promoters within 200 bp

• Promoter deletion analysis: way to find regions of DNA with promoters, promoter of interest has 5' deletions, fused to reporter gene, recombinant techniques.
• Linker Scanning Mutation Analysis: Methods for mapping enhancer regions, scramble regions and place back in. link to reporter gene
• Site-directed mutagenesis (aka Deng and Nicoloff Procedure): 1) Denature plasmid and anneal primer with point mutation, 1b) add polymerase. 2) Use Dpn 1 to digest methylated parent strand. 3) Transform with new plasmid (mutated)

dsDNA - restriction enzyme digest - gel - membrane transfer - probe- wash - analyze

• Specific tissues - isolate mRNA - linearize with formaldehyde - gel - membrane transfer - probe with complementary DNA - analyze
• *use control gene for load control*

• RNase mapping assay: isolate mRNA from cell of interest 2) hybridize with probe for gene of interest 3) digest in high salt (keeps dsRNA [ss tag ends digested]) 4) gel
• RT-PCR: use olig-dT Primer (run PCR) gel [non quantitative]
• qRT-PCR: product analysed after each round - normalize to HK gene (taqman vs sybr GRn)

• TF binds DNA
• co-TF binds TF

• Used to examine regulatory elements
• 1) DNA fragment is radio labeled and 2) incubated with cell/nuclear extract 3) run on gel (higher up band have more TF attached)

use column chromatography to create fraction of cell/nuclear extract. Run gel with DNA sequence in every well and different fractions in each well. High band fraction has TF for that sequence

1) Take putative TF, 2) incubate 5' radiolabeled (1 end only!) sequence with known control element, 3) add small amount DNase 1 = 1 cut in every DNA, 4) run on gel, gap in gel = strand length that has protein bound to it

• Modulatory: DNA binding site, Activation domain, Flexible protein portion
• *can have more than one activation site *
• *Activation sites are universal - DNA binding domain is specific*
• *modules never overlap*
• *modules can occur in any order*

• Used to determine domains
• N and C terminus deletions + internal Deletions
• look at different to see which bound DNA and which activate trx

• Fish for proteins that interact with bait domains - method for screening for protein-protein interations between TF
• Fish domain: bound to activation domain comes from cDNA library
• Bait domain: known protein bound to DNA binding domain
• Need 3 selections: Bait vector, Fish Vector, activation of GI selection (select for bait that caught fish) - use 3 different antibiotics

• Tells you the cDNA in the fish vector interacts with the protein of interest. You do not however, know if the protein is a TF.
• Clone newly found gene into vector (no need for selection - reporter will show you)
• Clone another vector with "protein-x" binding site and reporter gene