1. deamination in light of RNA editing
    conversion of C base to U: take the amino group off hte C and replace with a double bonded O; converts easily

    example: in the ApoB gene in intestine v. liver (intestine has the deaminated version)

  2. mRNA will never just diffuse across nuclear envelope:
    has to be actively transported through a nuclear pore

    in the pore, you can see the filaments have formed a basket
  3. FG nucleoporins
    • proteins that contribute to the nucelar pore; NUPs, line the central channel
    • have FG domains; hydrophilic sequence interspersed between short breaks of hydrophobic (phenylalanine and glycine)
    • are loosely associated based on (DYNAMIC nature) hydrophobic interactions
  4. transporters
    moves molecules (ex. mRNA) from nucleus to cytoplasm through nuclear pore; have a surface structure with hydrophobic residues on their surface; dynamic net of FG nucleoporins in the central channel of nuclear pore can associate with hydrophobic regions of transporter
  5. NXF1
    =/same as TAP; get mRNA through the pore and associate with the mRNA via REF (RNA export factor)
  6. NPL3 protein
    • can be modified by phosphorylation (by kinase)
    • -once mRNA has been completely transcribed, Glc7 (a phosphatase) that specifially removed phosphate group from NPL3 (only does it when tx is complete with polyA tail)
    • -NXF1/Nxt1 can now bind and transport out of nucleus into cytoplasm
    • -now in cytoplasm, kinase Sky1 adds phosphate group back to NPL3 === no longer associates with mRNA
    • -transporter gets sent back to nucleus as does phosphorylated NPL3

    basically in its phosphorylated state, NPL3 doesn't associate with mRNA, but once it is dephosphorylated it attached to transporter and it can now move

    • -Glc7 dephosphorylates in nucleus
    • -Sky1 phosphorylates in cytoplasm
  7. Rev
    HIV protein that promotes the transport of viral (HIV) mRNA into the cytoplasm for translation; rev inihibitors are potential anti-HIV drugs
  8. fractal globule
    Image Upload 1
    different sections/'colors' of chromosome are compacted close to sections that are similar to each other; comes apart easily, not just tangled together

    teh bottom one is how the chromosome is actually organized
  9. in order to label all of chromosome 5, your probe would be....:
    • microdissection of chromosomes: know from karyotype you can tell the difference between two chromosomes
    • -can go into nucleus, disect out a chromosome, label it using chromosome paint, can use the entire chromosome as a probe
    • to do FISH; CAN'T be an antibody, because there's no protein that hybridizes to the entire chromosome
  10. silenced chromatin:
    • heterochromatin associated with nuclear envelope
    • -but there are also active genes (euchromatin) near the periphery; more specifically, the nuclear PORE. super convinient to be close to the pore because after Tx it can be easily exported
  11. LBR
    • lamin B receptor; sits/is associated within the nuclear envelope
    • ALSO associates with HP1 (ick, heterochromatin)
  12. Adapt
    stands for Adaptor; protein bringing relevant things (proteins, euchromatin) to the pore
  13. interface between euchromatin and heterochromatin:
    barrier; boundary element; insulator
  14. connection of all this to a disease:
    • background:
    • H3K4me3 (lysine 4 methyl 3): an active mark, associated with euchromatin

    if it spreads inapropriately, then you have activation of genes that shouldn't be activated (Cancerous/leukemia)
  15. Image Upload 2
    process of Tx gene to mRNA is efficient, mRNA is fairly stable (doesn't decay at a high rate), mRNA is efficiently translated into protein, protein is fairly stable and remains around for an intermediate period of time

    as the protein builds up, the mRNA will begin to decay; equilibrium is reached
  16. Image Upload 3
    mrNA produced inefficiently

    less mRNA, but you get hte same amount of protein because you're producing so much PER mRNA

    protein decay rate is higher, but you get the same equilibrium
  17. Image Upload 4
    long lived protein that doesn't go away; don't want protein level to flucctuate

    • ex. ribosomal proteins; best way to achieve this is via a long lived protein
    • (because Transcription tends to happen in bursts)
  18. decapping pathway (deadenylation-independent)
    decapping enzyme REMOVES 5' cap; other specific enzyme (RNA exonuclease) recognizes broken mRNA and chomps mRNA from it's 5' to 3' end (5' to 3' exonucleolytic decay)
  19. deadenylation-dependent pathways
    • starts at the 3' end
    • deadenylase REMOVES polyA tail; once it gets below some length, relationship between PABPs and polyA tail (aka mRNA) destabilizes
    • -3' - 5' end attachment (reinitiation of translation) is affected
    • -once association is broken, you also tend to get decapping (via decapping enzyme)
    • -also the degredation of the 3' end makes it a substrate for exosome

  20. exosome
    • major 3' to 5' exonuclease
    • -exists in nucleuse and cytoplasm
    • -in nucleus, more used for improperly constructed mRNA (ex. introns still present)
    • -in cytoplasm, takes action when the polyA tail has been reduced to a threshold number of residues
  21. endonucleolytic pathway
    • cut mRNA's in the middle: leave one half w/out 5' cap and one without polyA tail
    • -fragments created get sent to exosome (eats @ 3' end) and exonucleases (eats from 5' capless end)
  22. miRNAs
    Image Upload 5
    • short single-stranded RNAs, 21-23 nucleotides in length; have complementary binding sites in 3' UTR's of transcripts (MEANING THAT THE COMPLEMENTARY REGION OF THE miRNA IS IN ITS 5' END; don't get confused)
    • -main effect: inhibit translation of mRNA
  23. peripheral blood stem cells
    • capable of creating the different types of blood cells (multipotent but not only found in the bone marrow)
    • can also return and repopulate the bone marrow if needed

    a decrease in CXCR4 protein causes the INCREASE of circulation of circulating peripheral blood stem cells....might be useful!
  24. how miRNA's are made:
    • -uses RNApol II; starts transcription as usual, produces a pri-miRNA transcript (Can be a really long sequence)
    • -one essential feature is that it's self-complementary and it forms a hairpin

    -nucleus: this hairpin is recognized in the nucleus by DROSHA (nucelase that cuts the hairpin to its 70 nt length) and DGCR8 (aka Pasha)

    -now leaves nucleus and enters cytoplasm via Exportin 5 (special transporter)

    -Cytoplasm: associates with 2 different proteins (TRBP) and Dicer (also is a nuclease; cuts off the top of the loop leaving mostly just base pairing with overhang on the 3' END)

    NOW: only one strand (usually) is loaded into a protein complex called RISC
  25. argonaute
    protein in RISC complex (RNA induced silencing complex); the one that actually binds the miRNA
  26. usually requires at least:
    TWO RISC complexes sitting on a 3' UTR of an mRNA to inhibit translation
  27. how does the RISC complex stop translation?
    doesn't immediately suggest translation inhibition: aka complexes bind to 3' UTR, translation starts at the 5' end....

    • key is P bodies: places in CYTOPLASM where NO ribosomes are around; have a high concentration of RNA decapping and exonuclease enzymes
    • -RISC bound mRNA's are found here
    • -if the only mechanism is bringing mRNA's to P bodies, that might be a good inhibition mechanism
  28. short interfering RNA (siRNA)
    • similar to miRNA
    • siRNA: 1) cause target RNA to be cleaved --- directly degraded 2) siRNA are PERFECTLY paired with targets 3) common in plants
    • -predicting targets of siRNA is easier (than miRNA); just need complementary sequence

    miRNA 1) prevents translation 2) are IMperfectly paired with targets

    *ARGONAUT with siRNA acts as the enzyme that cleaves; knows perfect base-pairing has occured
  29. hairpin construct
    Image Upload 6
    a gene that forms siRNA; meaning that one end when trancribed is complementary to the subsequent end of the gene

    used for in vivo contruction of siRNA
  30. hierarchical genome sequencing
    Collins/HGP/NIH: used BAC vectors that held between 100-200 kb of DNA

    basically just seems like you insert as many fragments into BAC's, and then align their sequences using markers; if the markers line up (like 2 or more) you can order all of them together
  31. whole-genome sequencing

    just RANDOMLY (shotgun) sequenced random fragments and lined them up?
  32. cytogenetic map
    Image Upload 7
    binds to AT-rich regions of genome; Giemsa staining creates bands in chromosome
  33. FISH (Flourescent In Situ Hybridization)
    Image Upload 8
    localizes the presence of specific DNA sequences on chromosomes; uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity
  34. radiation hybrid map
    method to determine not only the distance between chromosome markers but also their order on the chromosome

    • 1) chromosomes are separated from one another and broken into several fragments using high doses of x-rays
    • -the farther apart 2 DNA markers are from each other, the more likely a given dose of x-ray will break the chromosome between them, therefore placing them on two different fragments
    • 2) order is determined by estimating frequency of breakage
  35. DNA markers
    short, repetitive DNA sequences, most often located in noncoding regions of the genome, that have proven valuable for localizing human disease genes in the genome
  36. to find an exon:
    need to identify an ORF without a stop codon

    ALSO: GT and AG on either side of an intron (identifiers)
  37. Image Upload 9
    gene identifiers; can define introns and exons
  38. EST sequence
    expressed sequence tag;

    short sub-sequence of cDNA sequence used to identify gene transcripts, and are instrumental in gene discovery and gene sequence determination

    EST results from one-shot sequencing of a cloned mRNA (i.e. several hundred base pairs of sequence starting from an end of a cDNA). The cDNAs used for EST generation are typically individual clones from a cDNA library
  39. synteny:
    when two genomes in different organisms are syntenic, their genes are in the same place/order on a specific chromosome
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