Genetics PCB 3063

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Genetics - PCB 3063

• Todays focus
• Bacterial Genetics and Bacteriophage Genetics
• We will focus on three major questions today
• How does bacterial genetics differ from
  eukaryotic genetics?
• How do bacterial mutants arise?
• What are the processes of conjugation,
  transformation, and transduction?

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Problem Set

• Chapter 7
• Do problems 8, 17, and 20
• Chapter 8
• Do problems 14, 21, 24, 26

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Bacteria and Eukaryotes

• Bacteria differ from eukaryotes because they lack
  a nucleus and other membrane-bound organelles.
• The presence of a nucleus is the defining feature
  of eukaryotes, and those organisms that lack a
  nucleus are called PROKARYOTES.
• Many bacterial genomes are circular, unlike the
  linear genomes of eukaryotes.
• Bacteria typically have many fewer genes than
  eukaryotes as well, ranging from 1800 for
  bacteria with small genomes (e.g. H. influenzae)
  to 4200 for bacteria with larger genomes (e.g.,
  E. coli, B. subtilis).
• For comparison, a eukaryotes will small genomes
  (like the yeasts S. cerevisiae and S. pombe) have
  4500 to 6000 genes and complex eukaryotes
  (e.g., vertebrates) have 40000 genes the exact
  number is still unclear.

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Bacteria and Eukaryotes

• Bacteria divide by binary fission and exchange
  genetic material in a manner distinct from
  eukaryotic sex.
• Thus, the processes of mitosis and meiosis do not
  occur in prokaryotes.
• Instead, the bacterial chromosome is partitioned
  into daughter cells by attachment to the membrane
  (no mitotic spindle).
• Instead of sexual reproduction, there are 3
  processes that result in the exchange of genetic
  material for bacteria
• CONJUGATION - transfer of genetic material
  through physical contact between bacteria.
• TRANSFORMATION - uptake of genetic material
  directly from the environment.
• TRANSDUCTION - transfer of genetic material
  through phages (bacterial viruses).

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The Advantages ofBacteria for Genetics

• Bacteria were adopted as model organisms for
  genetics because they have desirable features.
• Bacteria are small and rapidly growing, allowing
  large numbers of bacteria to be cultured.
• Bacteria grow as clones, so large numbers of
  identical individuals can be obtained.
• Many different kinds of bacterial mutants have
  been obtained.
• Mutants resistant to antibiotics or infection
  with specific phages. These would include mutants
  resistant to phages like T4 or antibiotics like
  ampicillin (Ampr).
• Mutants that cannot grow at a restrictive
  temperature (either high temperature sensitive or
  cold sensitive).

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The Advantages ofBacteria for Genetics

• Bacteria were adopted as model organisms for
  genetics because they have desirable features.
• Bacteria are small and rapidly growing, allowing
  large numbers of bacteria to be cultured.
• Bacteria grow as clones, so large numbers of
  identical individuals can be obtained.
• Many different kinds of bacterial mutants have
  been obtained.
• Mutants unable to utilize specific nutrients.
  These would include mutants unable to use sugars
  such as galactose (gal-) or lactose (lac-).
• Mutants unable to synthesize specific nutrients.
  These mutants are called auxotrophic mutants.
  These would include mutants unable to make amino
  acids like tryptophan (trp-).

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How do Bacterial Mutants Arise?

• Initially, it was not clear whether bacterial
  mutants arise spontaneously or in response to
  selection.
• The idea that mutants can occur in response to
  selection is essentially the application of the
  idea that acquired characters can be inherited to
  bacteria, a discredited idea originally put forth
  by Jean Baptiste de Lamarck.
• It is important to remember that mutants arise in
  a manner independent of selection - the theory of
  evolution by natural selection postulates that
  variation already exists, and only those variants
  that reproduce successfully make it into the next
  generation.
• Thus, variants that are favored in a certain
  environment are not any more likely to arise in
  that environment - however, the variants that are
  favored will be successful.
• S. Luria and M. Delbrück established that E. coli
  mutants resistant to the bacteriophage T1 (T1-r
  mutants) arose independently of selection using
  the FLUCTUATION TEST.
• Delbrück was a physicist who moved into biology.

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How do Bacterial Mutants Arise?

• Initially, it was not clear whether bacterial
  mutants arise spontaneously or in response to
  selection.
• S. Luria and M. Delbrück established that E. coli
  mutants resistant to the bacteriophage T1 (T1-r
  mutants) arose independently of selection using
  the FLUCTUATION TEST.
• To conduct this experiment, T1 sensitive E. coli
  were diluted to a concentration of 103 cells/ml.
• These cells were split into 20 cultures of 0.2 ml
  each and one 10 ml culture.
• These are called the small and large cultures.
• The cells were grown for 21 generations (to a
  final concentration of 2.8x109 cells/ml) and a
  defined number plated (5.6x108 cells)
• The number of T1-r mutants were counted.
• Any T1 sensitive cells were killed, because a
  large number (1010) of T1 phages were added to
  the plates.
• So, the only colonies that could grow were T1-r
  mutants.

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The Fluctuation TestWhat do we Expect?

• Lets state first model - mutants arise randomly
  in the absence of selection for the mutation.
• Selection - natural or artificial - may favor
  certain variants, but those variants arise in a
  random fashion.
• If this model is correct, then we expect a high
  variance in the number of mutants for small
  cultures and a low variance for the large
  culture.
• Large and small refer to the number of cells
  starting the culture.
• The high variance for small cultures reflects the
  fact that the T1-r mutants can arise at any time
  - and that they can take over a small culture
  some of the time.
• Note The cultures shown as being started with a
  single cell and mutations to T1-r are indicated
  with the arrow.

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The Fluctuation TestWhat do we Expect?

• If this model is correct, then we expect a high
  variance in the number of mutants for small
  cultures and a low variance for the large
  culture.
• In contrast, the T1-r mutants will often be found
  in large cultures but they seldom occur early
  enough to time take over the culture.
• Note The cultures shown as being started with
  four cells and mutations are indicated as above.

• Thus, higher variance in the number of T1-r cells
  for small cultures is expected under this model.
• So, the approach that Luria and Delbrück used was
  to conduct the fluctuation test by starting these
  large and small cultures, then counting the
  numbers of mutants.

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The Fluctuation TestWhat do we Expect?

• The other model is mutants arise in response to
  selection.
• Similar variances for the number of mutants
  obtained from small cultures and large culture.
• This reflects the fact that the T1-r mutants
  would have arisen only after the selection.
• Note The cultures shown as being started with a
  single cell and mutations to T1-r are indicated
  with the arrow.
• Imagine the selection coming at the time of the
  first arrow...

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The Fluctuation TestWhat do we Expect?

• So we have two models
• Model 1 - Mutants arise randomly in the absence
  of selection for the mutation.
• Model 2 - Mutants arise in response to
  selection.
• Model 2 would be a Lamarkian model for
  bacteria.
• The models make distinct predictions.
• Model 1 - Higher variance in the numbers of T1-r
  mutants in small cultures (those with a small
  founder population) than in large cultures (those
  with a large founder population).
• Model 2 - Similar numbers of T1-r mutants in both
  small and large cultures, and similar variances
  in both cultures.
• So what were the results of the experiment?

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Small
Large
Bacterial Mutation is Independent of Selection

• Luria and Delbrück found that the variance of the
  number of mutants from small cultures was larger
  than the variance from large cultures.
• This is consistent with the first model, in which
  mutants arise in the absence of selection.
• Selection is still very important, but only after
  the mutation has arisen, nor in favor the mutant
  that will arise.

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The Origin of Mutants

• Luria and Delbrück found that E. coli cells
  accumulate mutations at random.
• After mutants arise the frequency of these
  mutations can change due to selection.
• These results are consistent with modern views of
  evolution - variation is generated at random
  processes and variants that are advantageous
  under specific conditions will increase in
  frequency in the population.
• It is possible to calculate the mutation rate
  using the results of the fluctuation test.
• We will not cover the issues with calculating the
  mutation rate.
• However, it is important to note that phage
  resistance (or antibiotic resistance) is a lethal
  selection.
• So the cells dont have much chance to respond to
  selection.

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The Origin of Mutants Revisited

• In 1988, J. Cairns and his colleagues examined a
  non-lethal selection - reversion of lac- cells to
  lac when lactose is the sole carbon source.
• lac- mutants cannot grow but they are not killed.
• Cairns et al. suggested that mutants might arise
  under these conditions in response to selection.
• This conclusion was highly controversial - there
  were 6 direct replies to the paper when it was
  published.
• There are a large number of technical issues - it
  is difficult to make medium that is unable to
  support any growth (due to small amounts of
  contaminating carbon sources, etc.) so counting
  the number of cells in the experiment is
  difficult.
• It now appears that Cairns et al. may have found
  that cells under nutritional stress accumulate
  mutations at a different rate - but the location
  of the mutations in the genome still appears
  random!

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Bacterial Conjugation

• In 1946 J. Lederberg and E. Tatum found that E.
  coli cells could exchange genes - they found
  evidence for the transfer of auxotrophic markers.
• A thr leu thi strain mixed with a met bio strain
  resulted in the generation of prototrophic cells
  at a relatively high frequency (1 in 107 cells).
• This reversion required physical contact between
  cells.
• Simply sharing the same medium in a container
  separated by a filter that allows small molecules
  and phages to pass through is not sufficient.
• The apparent genetic transfer reflected the
  presence of a population of cells now called Hfr
  cells (for HIGH FREQUENCY OF RECOMBINATION).
• The generation of Hfr cells is dependent upon the
  presence of a CONJUGATIVE PLASMID.
• Hfr cells arise from cells containing the
  F-plasmid (F cells).

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Plasmids

• Plasmids are small, almost always circular
  segments of DNA that contain a few genes.
• A replication origin is necessary for plasmids.
• The types of plasmids include
• F-plasmids relatively large (95 kbp) plasmids
  that are transferred between bacteria by
  conjugation.
• Col-plasmids smaller plasmids that encode toxins
  called colicins and also provide resistance to
  the colicins.
• R-plasmids plasmids of variable size that
  provide resistance to antibiotics.
• Specialized plasmids plasmids of variable size
  that provide specific functions, such as host
  interaction for symbionts or pathogens.
• Plasmids can have distinct host ranges, sometimes
  broad and other times quite narrow.

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The F-Plasmid

• F-plasmids encode proteins necessary to generate
  F-pili
• Singular - F-pilus, also called the sex pilus.
• The F-plasmid has a narrow host range.
• The F-plasmid encodes genes for the F-pilus.
• This structure allows the F cells (males) to
  attach to F- cells.
• One DNA strand is transferred to the F- cell.
• The DNA is replicated to generate F-plasmids in
  each cell.

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The F-Plasmid

• After transfer of a single strand of the F
  plasmid, the plasmid DNA is replicated to
  generate F-plasmids in each cell .
• At this point, two F cells have been generated.
• Some conjugative plasmids repress their transfer
  - but the F-plasmid does not.
• Conjugation represents a major mechanism for the
  transfer of antibiotic resistance in bacterial
  populations.
• This transfer will occur if the antibiotic
  resistance gene is present on the conjugative
  plasmid.

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Hfr Cells

• Hfr cells are generated by recombination of the
  F-plasmid with the bacterial genome.
• This occurs by homologous recombination within
  insertion sequences (IS).

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Hfr Cells

• Since insertion sites for the F-plasmid are
  spread throughout the E. coli genome, many
  different Hfr strains can form.

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Hfr Cells can be usedto Map Genes in E. coli

• Since Hfr cells represent E. coli that have an
  F-plasmid integrated into the genome, the
  complete genome can be transferred.
• This can be used to map genes by cutting off the
  genome transfer at specific times.
• This procedure is called INTERRUPTED MATING.
• Hfr cells are mixed with other cells, then the
  mating is stopped by mechanical means.
• Then Hfr strains with specific markers that
  introduce those marker into recombinant
  exconjugant cells allow mapping.
• Positions in the genome are given in minutes.
• In 1958, F. Jabob and E. Wollman used this method
  to demonstrate that the E. coli genome was
  circular.

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Hfr Cells can be usedto Map Genes in E. coli

• The F-plasmid can excise from the genome in Hfr
  cells.
• Imprecise excision will generate F PLASMIDS
  (F-prime plasmids).
• F plasmids are versions of F plasmids that have
  one or more bacterial genes.
• Bacteria with F plasmids can be PARTIAL DIPLOIDS
  (also called MEROZYGOTES), since these cells have
  two copies of the genes that are present on the
  F plasmid.
• These cells are partial diploids because they
  have plasmid and genomic copy of the genes on the
  F plasmid.
• They are partial diploids because they are
  haploid for most genes in the genome.
• F plasmids can be very large - plasmids covering
  much of the genome have been identified.
• These plasmids have also been used to hold large
  inserts for biotechnology (BAC Bacterial
  Artificial Chromosome).

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Transformation

• Bacterial TRANSFORMATION involves genetic
  transfer by the direct uptake of genetic material
  (DNA) from the environment.
• Bacteria that can take up DNA from the
  environment are called COMPETENT.
• Some bacterial species are naturally competent
• Examples of naturally competent bacterial species
  include B. subtilis, N. gonorrhoeae, H.
  influenzae, and S. pneumoniae.
• In some cases the competent phenotype will
  develop only under certain conditions (e.g.,
  nutritional status).
• Other species can be treated to make the cells
  competent
• E. coli can be competent after treatment with
  certain ions (Ca2 or Rb ).
• Transformation can be used to map bacterial genes
  by looking for co-transformation.

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Transformation

• Transformation can be used to map bacterial genes
  by looking for co-transformation.
• If DNA is prepared by lysing cells, it is
  typically sheared in a random fashion.
• So, isolation of DNA from cells does not result
  in the isolation of intact chromosomes - instead,
  random fragments of DNA of some mean size will be
  isolated.
• In some cases, shearing the DNA by passing it
  through a hypodermic needle or other methods may
  be desirable.
• Transformants are relatively rare, so the
  probability that two markers on different
  fragments will be CO-TRANSFORMED is extremely
  low.
• Thus, finding that two genes are often
  co-transformed indicates that they are very close
  to each other in the genome.
• As the distance between markers increases, the
  frequency of co-transformation decreases.

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Bacteriophage Genetics

• BACTERIOPHAGES (often simply called PHAGES) are
  viruses that infect bacteria.
• Phages cannot replicate on their own, just like
  the viruses that infect eukaryotes.
• Phages contain genetic material (either DNA or
  RNA) and encode proteins that necessary for
  packaging the genetic material.
• We will be discussing DNA phages.
• Excellent general information on phages can be
  found at
• http//www.asmusa.org/division/m/M.html
• Phages can be either VIRULENT or TEMPERATE.
• VIRULENT PHAGES enter the LYTIC CYCLE. This
  involves the replication of phage and generation
  of more phage particles, ultimately killing the
  bacterial host.

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Bacteriophage Genetics

• BACTERIOPHAGES (often simply called PHAGES) are
  viruses that infect bacteria.
• Phages can be either VIRULENT or TEMPERATE.
• VIRULENT PHAGES enter the LYTIC CYCLE. This
  involves the replication of phage and generation
  of more phage particles, ultimately killing the
  bacterial host.
• TEMPERATE PHAGES infect the cell and integrate
  into the bacterial genome as a PROPHAGE. This is
  called LYSOGENIC INFECTION.
• Lysogenic phages can be triggered to enter the
  lytic cycle under some conditions.
• For example, l phage is a lysogenic phage that
  integrates into the bacterial genome as a
  prophage.
• Exposure of prophage containing cells to UV
  radiation will trigger entry into the lytic cycle.

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The Lytic Cycle and Lysogeny

• I will use bacteriophage l to illustrate the
  difference between LYSIS and LYSOGENY.
• l is a temperate phage that can enter either
  cycle.

• When phages are grown on plates, they are usually
  grown on lawns of bacteria and form PLAQUES -
  clear areas on the lawn.