MCDB 101 B MID 2

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  1. Gene Conversion
    • 1. allele converted to another = DNA Difference
    • 2. always coverts to the other allele in the cross NOT a mutation
    • 3. Often correlatin with nearby recombination event
    • 4. Double stranded breaks occur in meitotic prophase??

    • *conserved in humans
    • usually in the Heteroduplex DNA during recombinations
  2. Double stranded Gap Repair Model of Recombination
    Over view
    • After a ds break, exonuclease chews down the 3` overhangs
    • -3` overhang can be recombination ( to compensate for the chewed up dna sequence)
    • -strand invasion by the homolog ( where you get the gene conversion)!
  3. Step 1 of Ds break repair model of Recombination
    1. Spo-11 exonuclease creates a DS break
  4. Step 2
    • Exonuclease chews back the 5` end creating a 3` overhang
    • - 5` to 3` exonuclease
    • *recall rec A : in prokaryotes promotes strand invasion and creation of D loops
  5. Step 3
    • strand invasion of Homolog by 3` overhang
    • -by DMC-1 : has recA like activity
    • forms D-loops ~ to T loops junction
  6. Step 4 : DNA synthesis ( D loop extension)
    • The 3' OH: serves as a primer
    • Template is the other strand of the homolog that is not involved in Recombination

    Also, there is a dispaced DNA strand can act like a template for the bottom strand

    And gets ligated ( joined together) with 2 holliday junctions that have Heteroduplex DNA
  7. Step 5: branch migration ( movement of Holliday junction up and down chromosome)
    Ruv AB like activity catalyzes branch migration
  8. RNA Pol
    • RNA POL I: insensity to alpha amanitan
    • POL II: moderate
    • On rna Pol II
    • - has ser #5
    • - and ser #2
    • - DOES NOT BIND to DNA on its own
  10. CTD
    on RNA pol II
  11. RNA pol II . Promoters
    • where the Tfactors bind and recruit RNA POL
    • *RNA POL does not bind to DNA on its own, must be recruited
  12. TF II D
    • primary T factor that binds to promoter site has subunits that consist of T.ata B.inding P.roteins and TAF
    • TBP -associated factors
  13. Pre Initiation Complex
    TF IID , TBP, TAF's + RNA POL II = PIC ; read to transcribe
  14. TFII H
    • Helicase that binds after the PIC has assembled
    • -opens up the ds DNA to reveal DNA template
    • Kinase phosphorylates CTD
  15. Enhancers
    • DNA elements that can be located far.. upstream or downstream of the promoter ( serve as additional binding sites) for additional T factors ( activators)
    • -Required for high levels of transcription
    • - usually has a dyad symmetry ( must bind dimers)
    • -D loops are important for the enhancers to be able to
  16. T factors and enhancers: recognition site
    • Enhancer sitse : usually have dyad symmetry promotes strong binding
    • Jun TF

    • Jun:
    • has a leucine zipper: a dimerization domain +
    • DNA binding domain
    • and could have an

    • **Leucines interact together
    • Activators have an activation domain ( the part that inteact with T factors)
  17. Zn finger T factor
  18. steroid hormone receptors
    in the absence of hormone, the receptor stays in cytoplas of the cell, and if it gets into the nucleus doesnt bind to DNA to activate transcription
  19. Mediator complex
    • Coactivators
    • do not bind DNA themselves , they mediate between DNA binding proteins
    • ie activators bound in enchancers and T factors bound at promot
  20. Activators
    • binds to enchacer
    • the D loop brings it around
    • interact with promoter region and inc basal transcription
  21. Repressor
    binds to the enhancer region, and can decrease transcription bc it does not have an activation region. Has a dimerization domain and DNA binding domain
  22. Quenching
    • type I: binds to the enhancer (DNA) and prevents the activator from binding to the enhancer
    • TYpe II: Activator binds to the enchancer but repressor blocks the activation domain

    TYPE III: some can reduce transcription via Histone De acetylases . They remove the acetyl group and increase histone + charge to increase interaction = dna less accessible
  23. Reporter Construct
    • How to analyze the control elements
    • a construct is:
    • you take potential enhancer DNA and put it next to a BASAL promoter that is driving a Reporter that you can visualize the activity of

    ex. LAC Z
  24. Max Network
    • ex. of Complex gene regulation network
    • Max binds as a dimer to the enhancer

    • can be
    • Max-Max: has DNA binding domain and Dimerization domain. Max is ON in ALL cells, NO activation domain ; can act as an REPRESSOR
    • Myc: has DNA binding dommain, Dimerization domain + * Activation domain ; can act like an Activator!

    Mad: '' "' = region that binds to Histone De acetylase. NO activation domain ; can act like a REPRESSOR
  25. Helix - Loop- helix
    family of DNA binding protein that binds as homodimers/ heterodimers

    ex. Max
  26. Insulator
    DNA element, which prevents the action of enhancers ( transcriptional activators) going beyond themselves and works only on the genes they should be.
  27. SPT-6/ SPT 6 mut
    Moves/ displaces the hexamer of Histome

    in a mutant, FACT removes dimer and then RNA POL plows through and knocks off the hexamer .. NO SPT6 to put back the hexamer
  28. FACT
    acetylases/ deacetylases and removes/ adds back the dimer of histon
  29. RNA processing
    • of Primary mRNA transcript
    • usually done by snRNPs ( small nuclear RNA + protein ) complex

    *most processing machinery ( ie snRNPs) bind to RNA pol's CTD
  30. 5' Capping Enzyme Complex
    • 5' Tri phosphate at the end of the primary transcripts
    • 5' Capping enzyme adds a 7 methyl G to the phophate 5' end

    so that it is linked 5' to 5'

    * only caps mRNA
  31. Why is mRNA's the only one's that are capped
    • after phosphorylation on the Ser #5 of CTD
    • - capping enzyme complex assembles
    • -as the transcript emerges
    • - capping enyzme complex assembles ONLY ON THE RNA POL II CTD
  32. 3' Poly Adenylation
    type of RNA processing

    • -protein complex assemble on RNA POL II CTD
    • - complex recognizes a sequece/signal AAUAAA on mRNA then ~25 bp downstream will Cleave the mRNA and then add hundreds of A's
  33. Poly A tail Importance
    1. mRNA stability - no 5' cap mRNA is VERY vulnerable to 5' exonucleases2. very important for Translation initiation
  34. 5' Cap
    1. mRNA stability - no 5' cap mRNA is VERY vulnerable to 5' exonucleases2. very important for Translation initiation
  35. Transcription Termination Model 1
    • there is an exonuclease that is still chewing on the transcript of the RNA pol ( even after the mRNA has been cleaved by the Poly A complex)
    • - when the exonuclease reaches RNA pol II transcription stops
  36. RNA POL recycling
    Phosphotase : de phos. the ser #4 and ser#5
  37. Splicing
    why the mRNA that is translated is so much smaller than the primary transcript

    EXONs are in the mRNA, Introns are spliced OUT of the mRNA

    carried out by the Spliceosome
  38. Spliceosome
    • attaches to the CTD of RNA POL II
    • -need to precisely remove as you transcribe you remove introns

    • -made of snRNA( small nuclear RNA)
    • * although you can can take spliceosomes + mRNA without RNA pol and you will still get introns spliced out
  39. snRNA
    • part of the Spliceosome
    • some are semi-homolgous to the splicing signals
  40. Splicing signals
    • Splicing donor : first 2 nucleotdies = GU ( in dna GT)
    • Branch site
    • Splice Acceptor : usually many CAG

    these are semi- homologous to the spliceosome thats how it recognizes it
  41. Splicing mechanism
    Cut occurs at Splice donor site

    • takes the G ( from GU conserved area) and links it to the
    • to the internal A residue in the intron
    • 5' to 2' link
    • = forms lariet structure

    • cut at the DS splice acceptor ( freeing the molecule intron)
    • and join the exon to the DS exon.

    EJC: is a marker of SUCCESSFUL splicing
  42. EJC
    • deposited UPSTREAM of a successful splice
    • - is very important for getting the mRNA out of the nucleus and into the cytoplasm
    • -and very important for quality control in translation
  43. Gene isoforms
    • why there are very few genes, but we are so complex
    • due to:

    • :alternative promoters on the same gene ( one promoter active in one type of cell/ and other promoter in other cells)
    • :alternative splicing
    • :alternative poly A site
  44. Mechanism of repressor action
    Repressor binds to the enhancer, blocks the binding of an activator = no activation of basal factors at promoter

    repressors bind to the activation domain of an activator preventing it from interacting with basal factors
  45. SWI/ SWF?
    uses ATP to alter chromatin struture ( or chromatin remodellling) at the 5 ' ends of genes. This allows initiator factors access to promoter and enhancer regions
  46. Spt-6
    atlers nucleosomes ahead of pol II ( along with fact ) and replaces nucleosomes after pol II has transcribed through. More specifically . spt6 replaces/ removes the hexasome after FAT has removed/ replaced the H2A/b dimer
  47. eIF-4E
    in translation, EUKARYOTIC initiation factor that binds the 5' CAP of the exported mRNA
  48. Drosha
    A nuclease that process the primary transcript of miR-?? 375 by cleaving off the 5' and 3'ends
  49. Poly A polymerase
    Adds the poly A tail to the 3' end of the primary transcript
  50. Transcription elongation ( details)
    • 1. NELF: is released from POL II followign phos. of ser # 2's on teh CTD
    • 2. FACT and spt6 work to remove nucleosomes ahead of Pol II ( acetylase histone tails, fACT removes H2A/B dimer, spt6 removes the hexamer)
    • 3. Pol II transcribes through
    • 4. FACT and spt6 work backwords to replace nucleosome to protect HDAC
  51. mRNA export
    • Adaptor proteins bind to the EJC
    • adaptor proteins ( tMRNA) bind nuclear pore complex facilitating export of the mRNA in to the cytoplasm
  52. Translation initiation
    • 1. CBP: binds to the 5'Cap
    • 2. eIF-4A : helicase unwinds any mRNA in a ds form
    • 3. eIF 4 G: makes the PABP bind to the CBP ( bends DNA)
    • 4. eIF's interact to form mRNA cap to tail initiation structure
    • 5. small subnit ( 40s) scans until finds a good AUG in context.
  53. chi squared
    (Observed - expected)^2 / expected

    reject null hypothesis if 0.5>

    cannot reject if 0.1 < more
  54. Interference ( recombination)
    1- (Obs/ exp = I
  55. Chi squared hypothesis for Recombination
    1. genes are NOT linked

    • --> groups Parentals = Recombinants
    • dF = 1
  56. how to determine the recombination frequence if no testcross is available???
    Hope its X linked --> then look at ONLY BOYS
  57. Fungi / yeast ascus
    the outcome of a single meisosis is seen in ONE ascus
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MCDB 101 B MID 2
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