Eukaryotic Genome

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  1. Genomic DNA classifications
    • Protein coding
    • Tandemly repeated genes
    • Repeating
    • Spacer
  2. 2 Types of protein coding genes
    • Solitary: represented only once
    • Duplicated/Diverged (gene families): similiar proteins/sequences with similiar fxn very close together on genome
  3. 3 types of tandemly repeated genes
    • rRNA
    • tRNA
    • Histones
  4. 2 (3) Types of Repeated seqeunces
    • Simple: satellite DNAs
    • Moderate: Mobile elements + Tandem Repeats + duplicated genes
  5. 3 Types of simple sequences and properies of each
    • 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*
  6. 3 generic features of mobile elements
    • Cut from genome
    • Put back into genome
    • Replicative + non-replicative
  7. 2 classes of mobile elements
    • DNA mediated - non-replicative (inverted repeats target sites)
    • RNA Mediated - replicative (direct repeats target site)
  8. Types of DNa mediated Mobile elements
    • Autonomous: has Ac gene ( encodes transposase)
    • Non- Autonomous: Lacks Ac gene (Ds - dissociation)
  9. Ac gene codes for a _______ which ______.
    • Transposase
    • Makes blunt cut on IS + recognizes target sequence and makes sticky end/staggered cut + Ligates IS to 5' end of target
  10. Types of RNA mediated transpostion
    • 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
  11. LTR characteristics
    Read in sense direction = 3' LTR (DNA) --> promoter, 5' LTR (pre-mRNA trx) --> signals host enzymes to process pre-mRNA
  12. Types of non-viral retrotransposons
    • LINES: 6 kb, 15% of genome
    • SINES: 300 bp, 10% of genome
  13. LINE structure/mechanism
    • 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
  14. SINES
    • 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
  15. Importance of mobile elements (4 specific, 1 overall)
    • spontaneous mutation
    • gene duplication
    • exon shuffling
    • enhancer shuffling
    • Evolution
  16. DNA sturctures
    • 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
  17. Nucleosome structure
    • 5 types of histones
    • octamer of histones: 2(H2A, H2B, H3, H4)
    • 147 bp surrounding histone complex (all mammals)
  18. How do regulator genes function?
    • Repress trx by preventing Pol from attatching or functioning
    • Activate trx by recruiting pol or stimulating activity
  19. Cis vs Trans elements
    • 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)
  20. Regulatory elements are _____? Examples (4)?
    • 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
  21. Promter deletion analysis? is and performed.
    Linker scanning mutation analysis? is and performed
    Site-directed mutagenesis? alternate name, is, performed, important features (2)
    • 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)
  22. Souther blot steps
    dsDNA - restriction enzyme digest - gel - membrane transfer - probe- wash - analyze
  23. Northern Blot
    • Specific tissues - isolate mRNA - linearize with formaldehyde - gel - membrane transfer - probe with complementary DNA - analyze
    • *use control gene for load control*
  24. Methods for measuring Trx levels: (3)
    • 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)
  25. TF vs co-TF
    • TF binds DNA
    • co-TF binds TF
  26. explain Gel-Shift assay
    • 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)
  27. explain Electrophoretic mobility shift assay (EMSA)
    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
  28. explain DNase 1 footprinting
    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
  29. Describe TF structure
    • 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*
  30. Explain Mutational analysis of TFs
    • Used to determine domains
    • N and C terminus deletions + internal Deletions
    • look at different to see which bound DNA and which activate trx
  31. Explain Yeast two-Hybrid technique
    • 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
  32. What does Yeast-Two Hubrid technique tell you? How do you know if prot. is a TF?
    • 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
Card Set
Eukaryotic Genome
Eukaryotic Genome
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