Micro/Genome 411 Lecture 4 DELETION MAPPING I. Genetic mapping A. Review of mapping (from last lecture) B. "Spot cross" assays C. Deletion mapping 1. Sample problem: Mapping with known deletions 2. Sample problem: Mapping with unknown mutations II. Investigating the nature of the genetic code A. Background B. T4 rII frameshift mutants 1. Creating an initial frameshift mutant (FC0) 2. Isolating FC0 "suppressor" mutants 3. "Suppressor of suppressor" mutants C. Test crosses of frameshift mutants D. Inferring the general nature of the genetic code Spot pairs of rII mutants on the non-permissive host (strain K?): rII-1 + rII-2rII-2 + rII-3rII-1 + rII-3 Complete lysis No plaques Individual plaques Incubate The "spot cross" assay of Benzer: Lecture 4: Deletion Mapping Micro/Genome 411 1 2 3 4 5 6 7 AB 1 2 3 4 5 6 7 + + ø + + ø + ø + = recombination ø = no recombination 123456789 1ØfrCfrØfrCØØ 2 ØCfrfrfrCfrfr 3 ØCCCfrCfr 4 ØfrfrCfrfr 5 ØfrCfrØ 6 ØCfrØ 7 ØCØ 8 Øfr 9 Ø Results of spot test crosses of T4 rII mutants on E. coli K?: C = confluent lysis of the spot fr = a few individual plaques seen within the spot Ø = no plaques seen Factual knowledge / conceptual base concerning the genetic code: ?DNA consists of a string (sequence) of nucleotide bases. ?Protein consists of a string (sequence) of amino acids. ?The sequence of amino acids in a protein is genetically determined Inference: The sequence of DNA bases is a linear code that correspondingly translates as a sequence of amino acids. Added insight: The code is probably NOT overlapping. ?Mutant proteins are known to vary by a single amino acid. DNA mutations are to change single nucleotides. Single base pair changes should affect multiple amino acids if the code is overlapping. Non-overlapping code Overlapping code General questions about the organization or nature of the genetic code: ?Is the code "punctuated" or "processive"? ?How many nucleotides encode an amino acid? ?Is the code redundant? ?Do all possible codons encode amino acids? Lecture 4: Deletion Mapping Micro/Genome 411 Acridine dyes as mutagens ?Create random mutations that map as single base changes (point mutations). ?Single base substitutions commonly have "leaky" phenotypes. ?Acridine-induced mutations are almost never "leaky". Conclusion: Acridine-induced mutations are random, single base mutations that are NOT simple substitutions. Wild-type sequence CATCATCATCATCATCAT CATGCATCATCTCATCAT Addition ("+1") frameshift mutation CATGCATCATCATCATCA Deletion ("-1") frameshift suppressor mutation Suppressor mutation: "A mutation elsewhere in the DNA that alleviates the [phenotypic] effects of another [prior] mutation." FC0 E. coli K? Plaques = Wild-type revertants Suppressor mutants Wild-type FC0 revertants FC0 suppressor mutants Wild-type T4Wild-type T4 Isolating FC0 frameshift suppressor mutations Cross progeny phage with wild-type T4 (on E. coli B): Procedure: ?Mutagenize T4 with acridine dye (proflavin). ?Plate on E. coli B. ?Pick "r" mutant plaques and screen to identify rII mutants. ?Map the rII mutations. ?HOPE that one maps to the B1 region of the rIIB gene. Lecture 4: Deletion Mapping Micro/Genome 411 Anticipated ability of rII frameshift mutant crosses to replicate on E. coli K? FC0 (+1) 1° suppressor mutations (-1) 2° suppressor mutations (+1) 3° suppressor mutations (-1) FC0 (+1) ? 1° suppressor mutations (-1) 2° suppressor mutations (+1) 3° suppressor mutations (-1) Wild-type sequence CATCATCATCATCATCAT "+1" frameshift mutation CATGCATCATCATCATCA Second "+1" frameshift mutation CATGCAGTCATCATCATC Third "+1" frameshift mutation CATGCAGTGCATCATCAT Kendall Gray Microsoft Word - Lecture 4 handout.doc
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