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. 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

4. PHENOTYPE WILD-TYPE MUTANT4
PHENOTYPE
WILD-TYPE
MUTANT
GROWTH ON
GLUCOSE
GLUCOSE
AND
TRYPTOPHAN

5. [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

6. USE OF INDICATOR MEDIUM TO SCORE PHENOTYPE6
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. MUTANT SELECTION 7 WILD-TYPE: STREPTOMYCIN-SENSITIVE
RIBOSOME PROTEIN INHIBITOR- rpsL+
PLATE:
RICH MEDIUM
RICH + STREPTOMYCIN
~108 CELLS
CONFLUENT

9. 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

10. 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]

11. SCORING-TESTING INDIVIDUAL COLONIES FOR GENOTYPE/PHENOTYPE10
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 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. MUTATIONS - CHANGES IN DNA NUCLEOTIDE SEQUENCE11
MUTATIONS - CHANGES IN DNA NUCLEOTIDE SEQUENCE
BASE PAIR SUBSTITUTIONS A:T G:C
DELETIONS ,000s
INSERTIONS s
INVERSIONS
FRAME SHIFTS , +2, -1, -2 BP
ANY NUMBER OTHER THAN 3 OR MULTIPLE OF 3
MUTAGENS -
REACT WITH DNA
INCORPORATE INTO DNA +

13. NITROUS ACID - REACTS WITH DNA CONVERTS CYTOSINE URACIL12
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. SPONTANEOUS DELETION - IN trp OPERON15
SPONTANEOUS DELETION - IN trp OPERON
= 1 NUCLEOTIDE DELETION
trp GENE
WILD-TYPE
MUTANT

16. HOW A DELETION OCCURS SPONTANEOUSLY16
HOW A DELETION OCCURS SPONTANEOUSLY
PARENTAL
STRANDS
WILD-TYPE
MUTANT
WILD-TYPE
AN PAIR DELETED

17. 17

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