Detailed Notes for the 10/13/05 Lecture

  1. Even Phage DNA can recombine - Benzer (1961) and T4 phage genetics

    1. Points to remember

      1. Got lots of T4 mutants at rII because mutations were not lethal

        1. Wild-type plaques are small and ragged on E. coli B

        2. RII mutations give large, round plaques on E. coli B

      2. Two types of mutants

        1. Revertible - point mutations

        2. Nonrevertible - deletions (better criteria to follow)

    1. Mutations within a cistron can recombine

      1. RII mutants cannot grow on E. coli K(λ), but rII+ can - conditional lethal

      2. Multiple infection on K(λ) yields rare recombinant phage

      3. Note: recombinant rare; complementation frequent

      4. This gives rise to the cis trans test

      5. Some mutations can recombine with others (point mutations); some cannot (deletions)

    2. Fine structure mapping

      1. Looked at recombination with a set of deletions

      2. Use finer and finer regions

      3. He could see recombination to the single nucleotide level

        1. Could detect recombinants at a level of 0.0001%, but didn't

        2. Either zero (same site) - note: two different mutations at the same site.

        3. Or 0.01% - smallest amount of recombination = 0.02% (only look at one recombination class)

        4. T4 has about 1500 mu, so 0.02/1500 = 1.3 x 10-5 genome

        5. T4 has 2 X 105 nucleotides, so ~2 nucleotides separate

        6. Actually can look at single nucleotide.

        7. So genes are not units of recombination

      4. Distribution of mutations

        1. Wide variation

        2. Not what one expects from Poisson

        3. Hot spots

  1. Using phage to map bacterial genes

    1. Integration can be at one site (lambda) or many (mu)

    2. Excision (removal from the chromosome) can be precise or imprecise

      1. Imprecise excision (in which adjacent DNA is taken up the phage) leads to specialized transduction

      2. Example is λ(gal)

    3. Some phages allow for host DNA to be put into their protein coats

      1. P1 (free - large plasmid)

      2. P22 (lysogen)

      3. Can use this to map genes (generalized transduction)

        1. RF = recombination frequency = recombinant phage/total phage = a+b-/(a+b- + a+b+)

        2. NOTE: this is not recombination, but co-packaging

        3. Can do fine structure mapping - in fact this is how the map of E. coli was made

  1. Tetrad analysis and mapping in fungi – mapping with a centrosome and with identified products of meiosis

    1. Ordered versus nonordered tetrads and octads

    2. Detecting linkage with nonordered tetrads as with yeast

      1. Types of tetrads : PD, NPD, T

      2. If non-linked PD = NPD

      3. If linked PD>>NPD

        1. NCO

            ab/ab/++/++ PD

        2. SCO

            ab/a+/+b/++ T

        3. DCO

            ab/ab/++/++ PD

            a+/ab/+b/++ T

            a+/a+/+b/+b NPD

            ab/a+/++/+b T

      4. Calculating RF

        1. DCO = 4 NPD

        2. SCO = T - 2 NPD (from the DCO)

        3. M = SCO + 2 DCO = T + 6 NPD

        4. RF = 2 T + 3 NPD

    3. What is tetrad analysis used for now

      1. Not for mapping genes - other ways are available

      2. Look for segregation patterns

  2. Modeling recombination

    1. Phenomena to be explained

      1. Unusual (nonMendelian) octads

        1. Chromatid Conversion (2:6) ++++++mm

        2. Half-chromatid conversion (3:5, 3:1:1:3) +++mmmmm

      2. Polarity - gradient of gene conversion events

      3. Half of all conversion events are associated with recombination

      4. Co-conversion

    2. (Robin) Holliday model

      1. Break (Same position for both homologues)

      2. Ligate

      3. Branch Migration

      4. Resolution - break and reform (same or different strands)

        1. Same - no recombination

        2. Different - recombination

      5. Can explain the four observations with gene conversion