Meeting 17

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Meeting 17
2011-11-10 13:03:09

5.2 (but not really)
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  1. Forward Genetics
    • 1. Mutagenize lots of individuals
    • 2. Screen for interesting phenotypes
    • 3. Clone the gene that carries themutation
    • 4. Deduce protein structure, function
    • (can be done in Yeast, worms, flies, zebrafish, human genetics)
  2. Reverse Genetics
    • 1. Start with a protein
    • 2. Deduce gene sequence
    • 3. Make a mutant in the gene
    • (can do in Mice, but now sequenced genomes and RNAi make this available to other species)
  3. mutations in certain areas such as ________ allow you to see phenotypically the results of DNA mutations
    • 1. Establishment of polarity
    • -Anterior-posterior
    • - Dorsal-ventral
    • 2. Specification of body plan
    • 3. Organization of particular structures in specific
    • positions (aka a head being anterior)
  4. why are model systems perfect for studying development?
    model systems: C. elegans, D. melanogaster, D. rerio (zebrafish), A. thaliana

    • -embryos are easily accessible/visible
    • -they have short generation times: worms: 2-3 days, flies: 10 days
    • -small genome: (ex. flies have 4 chromosomes) don’t want too many genes; and you also want an organism where the genes are conserved in the organism and in humans
  5. ways to induce mutations in the genome (3):
    1. chemical agents (EMS, ENU): EMS adds an alkyl group to G, which itself pairs with T during replication; in subsequent round of DNA replication, this changes GC's to AT's: results in (silent, missense, nonsense) point mutations in coding regions; this is done predominantly in MALES so that they can easily spread the mutation

    2. Radiation (X/gamma-rays): more serious defect because it causes breaks in the chromosome that can result in deletions/inversions/translocations

    3. inserting a transposon: useful when it disrupts a coding sequence; this is because you know the sequence of the inserted transposon and so therefore can find it and as a result use it to find the surrounding sequence it interupted
  6. CurlyO (CyO)
    -CyO is a dominant marker (therefore it takes the place of the general 'WT' when crossing heterozygous recessive mutants)

    -when homozygous, CyO is lethal (aka CyO/CyO)

    -but when heterozygous, CyO/+ flies have curly wings (this is how you know they're carrying the CyO gene and ONLY have one copy)
  7. which is balanced and which is unbalanced? why?
    1st: unbalanced; because it is */+, so for every crossing with either a +/+ or */+ organism, the * mutant gene will still eventually be canceled out by the + WT gene

    • 2nd: BALANCED; CyO/CyO flies (which is the analog to +/+ flies in an unbalanced cross) will DIE; therefore the * mutation will die out just as many times as the CyO mutation will

    ask if these two boxes are ACTUALLY showing the same thing
  8. however the benefits of a balanced cross can be voided IF crossing over occurs:
    -during meiosis, homologous chromosomes pair up and crossing over can occur

    -if this happens, then one chromosome will have both mutations and the other will be WT for both (this is BAD)

    -if the double mutant is mated with the double WT, both mutations will be lost in subsequent generations

    ·need to prevent recombination between two chromosomes carrying different mutations
  9. Inverted chromosomes PREVENT recombination:
    • ·during meiosis, chromosomes PAIR (aka synapse) on the basis of sequence similarity
    • ·if you invert one chromosome, then its homolog fails to form a synapse its nucleotide order does not match the order of its corresponding homolog (almost as if they aren't homologs at all)

    -chromosomes can’t recombine

    ·therefore CyO and * mutations remain present in future crosses
  10. balancer chromosome
    • in inverted homolog used to prevent crossing over (recombination) between homologous chromosomes during meiosis; the inversions are made by x-irradiation; allow populations of flies with heterozygous mutations to be maintained; have three important properties:
    • 1) they suppress recombination with their homologs
    • 2) carry dominant markers (ex. CyO)
    • 3) negatively affect reproductive fitness when carried homozygously......?
  11. crossover suppressor
    the make-up of balancer chromosomes is multiple chromosomal inversions so that synapsis between homologous chromosomes is disrupted; IF crossing over occurs (during meiosis) between balancer chromosome & balancer's homolog, each chromatid ends up lacking some genes and carrying two copies of others: progeny carrying these types of chromosomes die
  12. in this type of cross, the progeny you want to keep is:
    *367/CyO male crossed with CyO/Dominant female

    • ·you known that if the gene has the dominant marker, you don’t want it...
    • ·so CyO/NOT dominant is BY DEFAULT the progeny you want to keep; others have markers that rule out the presence of *367 or the CyO (curly wings) [both traits you want]

    -the next step from here is to cross these flies together (*367/CyO)

    • *367 = some recessive mutation
    • CyO = lethal dominant marker
    • Dominant = some other dominant marker (but not lethal) that's easily identifiable, like eye color
  13. if two mutations lead to the same phenotype, we can TEST whether they're actually on the same gene or not via COMPLEMENTATION:
    • there are two possible explanations for why two mutations lead to the same phenotype:
    • 1) both mutations occur in the SAME gene
    • 2) the mutations occur in different genes but affect the same PATHWAY
  14. describe complementation:
    • 1) cross two strains each containing a 'different' mutation
    • -1/4 of the progeny will DEFINATELY die because they'll be CyO/CyO
    • -1/2 of the progeny will have curly wings and either the *61 or *524 recessive mutation
    • -what happens to the REMAINING 1/4 of progeny determines whether the mutations are on the same gene:
    • -because you know the mutations are lethal (you just do), if the 1/4 remaining is DED, then *61 and *524 FAIL to complement each other meaning they are on the same gene (silly that you gave the same mutation two different names)
    • -BUT! if the remaining 1/4 of progeny (*61/*524) is alive, then you know the two mutations are on DIFFERENT genes and they complement each other

  15. probability = λk e/ k!
    • k = # of clones/mutations expected to be represented
    • λ = average # of ______ per each position

    • -so for the first question, λ was average clones per 1 genome, aka 1 clone/1genome
    • -for the second, λ = mutations per each gene, 3000mutations for every 1000 genes = 3