1 MUTATIONS/MUTANTS

2 MUTATIONS AND MUTANTS GENOTYPE AND PHENOTYPE GENOTYPE- nucleotide sequence of a gene - chromosome - genome wild-type, mutation (change in nucleotide sequence), mutant PHENOTYPE- characteristic one can observe wild-type and mutant Mutations in the lactose operon MUTANT SELECTION, ENRICHMENT, SCORING Streptomycin sensitivity and resistance Histidine auxotroph Replica plating MUTATIONS Substitutions, deletions, insertions, inversions, frameshifts Mutagens Nitrous acid - a chemical that reacts with DNA GENETIC CODE DOMINANCE Allele, heterozygous, phenotype determined by one allele when two different alleles are present Partial diploid (Fertility Factor, Hybrid Plasmid)

MUTATIONS AND MUTANTS 3 E. COLI 4.7 MEGA BASE PAIRS CIRCULAR CHROMOSOME ~ 5,300 GENES ~ 3 x 109 MOLECULAR WEIGHT HAPLOID EXAMPLE: TRYPTOPHAN OPERON trpA GENE GENOTYPE: WILD-TYPE trpA+ MUTANT trpA- GENE mRNA PROTEIN ACTIVE ENZYME INACTIVE ENZYME CATALYZES REACTION IN TRYPTOPHAN SYNTHESIS CANNOT CATALYZE ITS NORMAL REACTION

PHENOTYPE WILD-TYPE MUTANT 4 PHENOTYPE WILD-TYPE MUTANT GROWTH ON GLUCOSE GLUCOSE AND TRYPTOPHAN

[WILL NOT SPLIT LACTOSE] 5 LACTOSE OPERON WILD - TYPE MUTANT GENOTYPE: lacZ + lacZ - GENE PRODUCT: ACTIVE b-GALACTOSIDASE [HYDROLIZES LACTOSE] INACTIVE [WILL NOT SPLIT LACTOSE] PHENOTYPE: GROWTH ON GLUCOSE GROWTH ON LACTOSE

USE OF INDICATOR MEDIUM TO SCORE PHENOTYPE 6 USE OF INDICATOR MEDIUM TO SCORE PHENOTYPE lacZ + lacZ - MAC CONKEY'S INDICATOR MEDIUM AND LACTOSE WHITE COLONIES

14 SELECTION INHIBITION OF GROWTH OF ONE ORGANISM (OR GROUP OF ORGANISMS) ALLOWING GROWTH OF ANOTHER ORGANISM ENRICHMENT FAVORING GROWTH OF SOME ORGANISM SCORING TESTING THE PHENOTYPE OF INDIVIDUAL COLONIES OR CULTURES

MUTANT SELECTION 7 WILD-TYPE: STREPTOMYCIN-SENSITIVE RIBOSOME PROTEIN INHIBITOR- rpsL+ PLATE: RICH MEDIUM RICH + STREPTOMYCIN ~108 CELLS CONFLUENT

LET'S PLATE ~1010 CELLS 8 rpsL MUTANTS STREPTOMYCIN- RESISTANT ALTERED RIBOSOME PROTEIN STILL FUNCTIONS IN PROTEIN SYNTHESIS NO LONGER RECOGNIZES STREPTOMYCIN SPONTANEOUSLY OCCURRED ~10 rpsL-/ 1010 WILD TYPE WILD-TYPE MUTANT GENOTYPE: rpsL+ rpsL- PHENOTYPE: STR-SENSITIVE STR-RESISTANT

ISOLATE A HISTIDINE-REQUIRING MUTANT [AUXOTROPH] his- 9 WILD-TYPE his+ GROWS WITHOUT HISTIDINE [i.e., ON GLUCOSE] PROPORTION his-/ his+ WILD-TYPE POPULATION ~1/107 MUTAGENIZE AND GROW MUTAGEN SURVIVORS ~1/105 ENRICHMENT ~1/103 SCORE TO FIND 1/103: PLATE SO EVERYBODY GROWS [GLUCOSE AND HISTIDINE] TRANSFER COLONIES TO GLUCOSE ONLY [NO HISTIDINE]

SCORING-TESTING INDIVIDUAL COLONIES FOR GENOTYPE/PHENOTYPE 10 SCORING-TESTING INDIVIDUAL COLONIES FOR GENOTYPE/PHENOTYPE OR, HOW TO FIND 1 his- MUTANT CELL (OR COLONY) AMONG 103 WILD-TYPE CELLS (OR COLONIES) A PLATE MIXTURE ON GLUCOSE AND HISTIDINE his- MUTANTS GROW INTO COLONIES his+ WILD-TYPE ALSO GROWS INTO COLONIES [PLATE ENOUGH PLATES TO GET 2000 - 3000 COLONIES] TRANSFER ~3,000 INDIVIDUAL COLONIES TO TWO PLATES: B GLUCOSE AND HISTIDINE GLUCOSE his- GROWS and his+ GROWS [THEY LOOK THE SAME] his- MUTANTS FAIL TO GROW

MUTATIONS - CHANGES IN DNA NUCLEOTIDE SEQUENCE 11 MUTATIONS - CHANGES IN DNA NUCLEOTIDE SEQUENCE BASE PAIR SUBSTITUTIONS A:T G:C DELETIONS 1 - 1,000s INSERTIONS 1 - 1000s INVERSIONS FRAME SHIFTS +1, +2, -1, -2 BP ANY NUMBER OTHER THAN 3 OR MULTIPLE OF 3 MUTAGENS - REACT WITH DNA INCORPORATE INTO DNA +

NITROUS ACID - REACTS WITH DNA CONVERTS CYTOSINE URACIL 12 NITROUS ACID - REACTS WITH DNA CONVERTS CYTOSINE URACIL G:C PAIR NITROUS ACID G:U

13 NITROUS ACID REPLICATION AND BINARY FISSION WILD-TYPE

SPONTANEOUS DELETION - IN trp OPERON 15 SPONTANEOUS DELETION - IN trp OPERON = 1 NUCLEOTIDE DELETION trp GENE WILD-TYPE MUTANT

HOW A DELETION OCCURS SPONTANEOUSLY 16 HOW A DELETION OCCURS SPONTANEOUSLY PARENTAL STRANDS WILD-TYPE MUTANT WILD-TYPE AN PAIR DELETED

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