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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

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MUTATIONS/MUTANTS

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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)

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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

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MUTANT

PHENOTYPE

WILD-TYPE

GROWTH ON

GLUCOSE

GLUCOSE

AND

TRYPTOPHAN

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LACTOSE OPERON

PHENOTYPE:

GROWTH ON

GLUCOSE

GROWTH ON

LACTOSE

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USE OF INDICATOR MEDIUM TO SCORE PHENOTYPE

lacZ +

lacZ -

MAC CONKEY'S INDICATOR MEDIUM AND LACTOSE

WHITE COLONIES

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  • 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
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MUTANT SELECTION

WILD-TYPE: STREPTOMYCIN-SENSITIVE

RIBOSOME PROTEIN INHIBITOR- rpsL+

PLATE:

RICH MEDIUM

RICH + STREPTOMYCIN

~108CELLS

CONFLUENT

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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

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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]

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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

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+

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
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NITROUS ACID - REACTS WITH DNA

CONVERTS CYTOSINE URACIL

G:C PAIR

NITROUS ACID

G:U

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NITROUS

ACID

REPLICATION AND

BINARY FISSION

WILD-TYPE

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= 1 NUCLEOTIDE DELETION

SPONTANEOUS DELETION - IN trp OPERON

trp GENE

WILD-TYPE

MUTANT

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HOW A DELETION OCCURS SPONTANEOUSLY

PARENTAL

STRANDS

WILD-TYPE

MUTANT

WILD-TYPE

AN PAIR DELETED