4. Bacterial Genetics I

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4. Bacterial Genetics I
2011-10-03 16:25:50
PMB 112 midterm1

general microbiology midterm 1
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  1. gene
    segment of DNA that encodes a functional product, along with the regions that regulate its expression
  2. selectable mutations
    confer a distinct advantage on the mutant strain under some environmental/growth conditions
  3. nonselectable mutations
    may result in very clear, obvious changes, but they do not confer any growth advantage
  4. selection
    • establish conditions where only the mutants you want will grow
    • parent strain and unrelated mutants die
    • very efficient, can select desired mutants from huge populations of unrelated ones
    • selection isn't always possible
  5. screen
    • after mutagenesis, grow all survivors under permissive conditions, then test all surviving mutants for growth/behavior in nonpermissive conditions - compare two plates
    • must first be grown as independent colonies - takes a lot more plates and time
    • less powerful but may be only option
  6. essential genes
    • genes whose function is required under all known growth conditions
    • can only isolate mutations in essential genes by screening for conditional loss-of-function mutants (most common is temperature sensitivity)
  7. mutation rate
    probability that a given gene will acquire a mutation in one generation
  8. spontaneous mutations
    arise from errors in DNA replication
  9. mutagens
    • can increase mutation rate
    • chemicals
    • radiation
    • transposons
  10. base pair substitutions (microlesions)
    • missense mutation - codes for different amino acid
    • nonsense mutation - codes for stop codon
    • silent mutation - doesn't change codon
  11. frame-shift mutations (microlesions)
    shifts in the reading frame of messenger RNA caused by insertion or deletions mutations in DNA
  12. mutagenesis
    • increase the rate of mutation using mutagens
    • do to increase our chances of finding desired mutants
  13. chemical mutagens
    nucleotide base analogs - create mutations when they are incorporated into DNA during replication, look similar but do not always base-pair correctly, create single-base substitutions

    alkylating agents - chemically alter the amino, carboxyl and hydroxyl groups of nucleotide bases, cause base-pair substitutions

    intercalating agents - planar ring-containing molecules that insert between two adjacent DNA base pairs, causes small insertions or deletions during replication
  14. UV radiation
    • UV absorption causes covalent crosslinking of adjacent pyrimidine bases (dimers)
    • can't replication past lesion

    SOS response - UV damage is detected by RecA, induces response

    • translesion synthesis - polymerase can synthesize DNA from a template with pyrimidine dimers
    • adds random bases across from lesion, making mutations
  15. transposons
    • naturally occurring mobile DNA elements
    • can move from place to place in the genome
    • consist of two identical insertion elements bracketing other genes, often for antibiotic resistance
    • if "hops" into a gene, usually disrupts its expression and causes a loss-of-function mutation
  16. insertion sequences
    inverted repeat sequences flanking a gene for the transposase enzyme
  17. transposase
    • recognizes IR sequences
    • cuts donor and target DNAs
    • ligates the transposon into a new site
    • each transposase is specific for its own IR sequences
  18. mini-transposons
    • transposase is encoded outside of the transposon
    • plasmid cannot replicate in the host strain (suicide vector)
    • inserted mini-Tn cannot move again, because transposase was only encoded on the plasmid
    • can create random loss-of-function mutations
    • have to select or screen for mutants with desired phenotype
    • cannot expect to create mutations in essential genes using transposon
    • mutated gene is easy to identify - know mini-Tn sequence, design DNA sequencing primers to read out surrounding DNA