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 observewild-

type and mutantMutations in the lactose operon

MUTANT SELECTION, ENRICHMENT, SCORING

Streptomycin sensitivity and resistanceHistidine auxotrophReplica plating

MUTATIONS

Substitutions, deletions, insertions, inversions, frameshiftsMutagens

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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3MUTATIONS AND MUTANTS

E. COLI 4.7 MEGA BASE PAIRSCIRCULAR CHROMOSOME~ 5,300 GENES~ 3 x 109 MOLECULAR WEIGHT

HAPLOIDEXAMPLE: TRYPTOPHAN OPERON trpA GENE

GENOTYPE:

GENE

mRNA

PROTEIN

WILD-TYPE trpA+ MUTANT trpA-

ACTIVEENZYME

INACTIVEENZYME

CATALYZES REACTION IN TRYPTOPHAN

SYNTHESIS

CANNOT CATALYZE ITS NORMAL REACTION

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PHENOTYPE WILD-TYPE MUTANT

GROWTH ON

GLUCOSE

GLUCOSEAND

TRYPTOPHAN

5LACTOSE OPERON

WILD - TYPE MUTANT

GENOTYPE: lacZ + lacZ -

GENE PRODUCT:

ACTIVEb-GALACTOSIDASE

[HYDROLIZES LACTOSE]

INACTIVEb-GALACTOSIDASE

[WILL NOT SPLIT LACTOSE]

PHENOTYPE:

GROWTH ONGLUCOSE

GROWTH ONLACTOSE

6USE 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

7MUTANT SELECTION

WILD-TYPE: STREPTOMYCIN-SENSITIVERIBOSOME PROTEIN

INHIBITOR- rpsL+

RICH MEDIUM

RICH + STREPTOMYCIN

~108 CELLS

CONFLUENT

PLATE:

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WILD-TYPE MUTANT

GENOTYPE: rpsL+ rpsL-

PHENOTYPE: STR-SENSITIVE STR-RESISTANT

rpsL MUTANTSSTREPTOMYCIN-RESISTANT

ALTERED RIBOSOME

PROTEIN

STILL FUNCTIONS IN

PROTEIN SYNTHESIS

NO LONGER RECOGNIZES

STREPTOMYCIN

SPONTANEOUSLY

OCCURRED

~10 rpsL-/ 1010 WILD TYPE

LET'S PLATE ~1010 CELLS

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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]

ISOLATE A HISTIDINE-REQUIRING MUTANT

[AUXOTROPH] his-

WILD-TYPE his+ GROWS WITHOUT HISTIDINE [i.e., ON GLUCOSE]

10SCORING-TESTING INDIVIDUAL COLONIES

FOR GENOTYPE/PHENOTYPEOR, HOW TO FIND 1 his- MUTANT CELL (OR COLONY)

AMONG 103 WILD-TYPE CELLS (OR COLONIES)

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:GLUCOSE AND HISTIDINE GLUCOSE

A

B

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:CDELETIONS

1 - 1,000sINSERTIONS

1 - 1000sINVERSIONSFRAME SHIFTS

+1, +2, -1, -2 BP

ANY NUMBER

OTHER THAN 3 OR

MULTIPLE OF 3MUTAGENS - • REACT WITH DNA• INCORPORATE INTO DNA

+

NITROUS ACID - REACTS WITH DNACONVERTS CYTOSINE URACIL

NITROUS ACID

G:C PAIR

G:U

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NITROUSACID

REPLICATION ANDBINARY FISSION

WILD-TYPE

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MUTANT

WILD-TYPE

trp GENE

= 1 NUCLEOTIDE

DELETION

SPONTANEOUS DELETION - IN trp OPERON

16HOW A DELETION OCCURS SPONTANEOUSLY

PARENTALSTRANDS

WILD-TYPE

WILD-TYPEMUTANT

AN PAIR DELETED

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