MUTATIONS/MUTANTS

1. 1 MUTATIONS/MUTANTS

2. 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)

3. 3 MUTATIONS AND MUTANTS 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

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

5. 5 LACTOSE OPERON PHENOTYPE: GROWTH ON GLUCOSE GROWTH ON LACTOSE

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

7. 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

8. 7 MUTANT SELECTION WILD-TYPE: STREPTOMYCIN-SENSITIVE RIBOSOME PROTEIN INHIBITOR- rpsL+ PLATE: RICH MEDIUM RICH + STREPTOMYCIN ~108CELLS CONFLUENT

9. 8 LET'S PLATE ~1010 CELLS rpsL MUTANTS STREPTOMYCIN- RESISTANT ALTERED RIBOSOME PROTEIN STILL FUNCTIONS IN PROTEIN SYNTHESIS NO LONGER RECOGNIZES STREPTOMYCIN SPONTANEOUSLY OCCURRED ~10 rpsL-/ 1010 WILD TYPE

10. 9 ISOLATE A HISTIDINE-REQUIRING MUTANT [AUXOTROPH] his- WILD-TYPE his+ GROWS WITHOUT HISTIDINE [i.e., ON GLUCOSE] SCORE TO FIND 1/103: PLATE SO EVERYBODY GROWS [GLUCOSE AND HISTIDINE] TRANSFER COLONIES TO GLUCOSE ONLY [NO HISTIDINE]

11. 10 SCORING-TESTING INDIVIDUAL COLONIES FOR GENOTYPE/PHENOTYPE OR, HOW TO FIND 1 his- MUTANT CELL (OR COLONY) AMONG 103WILD-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

12. 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

13. 12 NITROUS ACID - REACTS WITH DNA CONVERTS CYTOSINE URACIL G:C PAIR NITROUS ACID G:U

14. 13 NITROUS ACID REPLICATION AND BINARY FISSION WILD-TYPE

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

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

17. 17

18. 18

19. 19

20. 20