1 / 20

MUTATIONS/MUTANTS

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

jase
Download Presentation

MUTATIONS/MUTANTS

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related