Ch 8: Microbial Genetics

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Ch 8 Microbial Genetics

• Genetics The study of what genes are, how they
  carry information, how information is expressed,
  and how genes are replicated

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Genetics Terms

• Gene A segment of DNA nucleotides that encodes a
  functional product, usually a protein
• Chromosome Structure containing DNA that
  physically carries hereditary information the
  chromosomes contain the genes
• Genome All the genetic information in a cell

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Genetics Terms

• Genomics The molecular study of genomes
• Genotype The genes of an organism
• Phenotype Expression of the genes

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DNA

• The DNA in a chromosome exists as one long double
  helix associated with various proteins that
  regulate genetic activity.
• Bacterial DNA is circular the chromosome of E.
  coli, for example, contains about 4 million base
  pairs and is approximately 1000 times longer than
  the cell.

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Determine Relatedness
Based on similarity of the viral envelope amino
acids
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Determine Relatedness

• Which strain is more closely related to the
  Uganda strain?

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Genetic Map of the Chromosome of E. coli
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The Flow of Genetic Information

• Information contained in the DNA is transcribed
  into RNA and translated into proteins.

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The Flow of Genetic Information
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DNA

• Polymer of nucleotides Adenine, thymine,
  cytosine, and guanine
• Double helix associated with proteins
• "Backbone" is deoxyribose-phosphate
• Strands are held together by hydrogen bonds
  between AT and CG
• Strands are antiparallel

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Semiconservative Replication

• Because each double-stranded DNA molecule
  contains one original and one new strand, the
  replication process is called semiconservative.

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DNA Replication

• During DNA replication, the two strands of the
  double helix separate at the replication fork,
• Each strand is used as a template by DNA
  polymerases to synthesize two new strands of DNA
  according to the rules of nitrogenous base
  pairing.
• The result of DNA replication is two new strands
  of DNA, each having a base sequence complementary
  to one of the original strands.

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DNA Synthesis
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DNA Synthesis

• DNA is copied by DNA polymerase
• In the 5' ? 3' direction
• Initiated by an RNA primer
• Leading strand is synthesized continuously
• Lagging strand is synthesized discontinuously
• Okazaki fragments
• RNA primers are removed and Okazaki fragments
  joined by a DNA polymerase and DNA ligase

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DNA Synthesis

• DNA polymerase proofreads new molecules of DNA
  and removes mismatched bases before continuing
  DNA synthesis.
• Each daughter bacterium receives a chromosome
  that is virtually identical to the parents.

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Synthesis in the Lab

• Mix DNA polymerase from a bacteria in a test tube
  with a human DNA template
• The DNA would be most similar to the original
  human DNA template with the bacterial DNA
  incorporated

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DNA Synthesis
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Replication of Bacterial DNA
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Replication of Bacterial DNA
ANIMATION DNA Replication Overview
ANIMATION DNA Replication Forming the
Replication Fork
ANIMATION DNA Replication Replication Proteins
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Transcription

• DNA is transcribed to make RNA (mRNA, tRNA, and
  rRNA)
• Transcription begins when RNA polymerase binds to
  the promoter sequence (AUG)
• RNA is synthesized from nucleotides containing
  the bases A, C, G, and U, which pair with the
  bases of the DNA strand being transcribed.

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Transcription

• Transcription proceeds in the 5' ? 3' direction
• Transcription stops when it reaches
  theterminator sequence

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Transcription
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The Process of Transcription
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The Process of Transcription
ANIMATION Transcription Overview
ANIMATION Transcription Process
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RNA Processing in Eukaryotes
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Translation

• Translation is the process in which the
  information in the nucleotide base sequence of
  mRNA is used to dictate the amino acid sequence
  of a protein.
• mRNA is translated in codons (three nucleotides)

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Translation

• Translation of mRNA begins at the start codon
  AUG
• Translation ends at nonsense codons UAA, UAG,
  UGA

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The Genetic Code

• The genetic code refers to the relationship among
  the nucleotide base sequence of DNA, the
  corresponding codons of mRNA, and the amino acids
  for which the codons code.
• 64 sense codons on mRNA encode the 20 amino acids

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The Genetic Code

• 3 are nonsense codons
• The genetic code is degenerate (most amino acids
  are coded for by more than one codon)
• tRNA carries the complementary anticodon (a base
  triplet)
• The base pairing of codon and anticodon at the
  ribosome results in specific amino acids being
  brought to the site of protein synthesis.

ANIMATION Translation Overview
ANIMATION Translation Genetic Code
ANIMATION Translation Process
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The Genetic Code
The start codon, AUG, codes for methionine.
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Simultaneous Transcription Translation
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The Process of Translation
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The Process of Translation
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The Process of Translation
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The Process of Translation
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The Process of Translation
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The Process of Translation
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The Process of Translation
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The Process of Translation
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Regulation

• Regulating protein synthesis at the gene level is
  energy-efficient because proteins are synthesized
  only as they are needed.
• Constitutive genes are expressed at a fixed rate
  (Examples are genes for the enzymes in
  glycolysis.)
• Other genes are expressed only as needed
• Repressible genes
• Inducible genes
• Catabolite repression

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Repression

• Repression controls the synthesis of one or
  several (repressible) enzymes.
• When cells are exposed to a particular
  end-product, the synthesis of enzymes related to
  that product decreases
• Synthesis of the repressor enzyme is stopped by
  the corepressor-repressor binding to the operator

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Induction

• In the presence of certain chemicals (inducers),
  cells synthesize more enzymes. This process is
  called induction.
• An example of induction is the production of
  b-galactosidase by E. coli in the presence of
  lactose lactose can then be metabolized.
• lactose is the substrate that induces the
  production

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The Operon Model of Gene Expression

• In bacteria, a group of coordinately regulated
  structural genes with related metabolic
  functions, plus the promoter and operator sites
  that control their transcription, are called an
  operon.
• When the inducer is absent, the repressor binds
  to the operator, and no mRNA is synthesized.

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The Operon Model of Gene Expression

• When the inducer is present, it binds to the
  repressor so that it cannot bind to the operator
  thus, mRNA is made, and enzyme synthesis is
  induced.
• In repressible systems, the repressor requires a
  corepressor in order to bind to the operator
  site thus, the corepressor controls enzyme
  synthesis.

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Operon
ANIMATION Operons Overview
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Induction
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Induction
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Repression
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Repression
ANIMATION Operons Induction
ANIMATION Operons Repression
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Catabolite repression

• Allows bacteria to adapt quickly to a preferred
  (rapidly metabolisable) carbon and energy source
  first
• Usually achieved through inhibition of synthesis
  of enzymes involved in catabolism of carbon
  sources other than the preferred one

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Escherichia coli

• Grows faster on glucose than on any other carbon
  source
• If E. coli is placed on an agar plate containing
  only glucose and lactose, the bacteria will use
  glucose first and lactose second
• The absence of glucose will "turn off" catabolite
  repression

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Catabolite Repression
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• Lactose present, no glucose

• Lactose glucose present

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Positive Regulation

• Transcription of structural genes for catabolic
  enzymes (such as b-galactosidase) is induced by
  the absence of glucose. Cyclic AMP and CRP must
  bind to a promoter in the presence of an
  alternative carbohydrate.
• The presence of glucose inhibits the metabolism
  of alternative carbon sources by catabolite
  repression.

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Mutation

• A change in the genetic material
• Mutations may be neutral, beneficial, or harmful
• Mutagen Agent that causes mutations (permanent
  DNA changes)
• Spontaneous mutations Occur in the absence of a
  mutagen
• Many mutations are neutral, some are
  disadvantageous, and others are beneficial.

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Mutation

• Base substitution change in one base by another
  base (point mutation)
• Result in change in amino acid

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Mutation

• Cause amino acid substitutions) or nonsense
  mutations
• (which create stop codons).

• Nonsense mutation

• Results in a nonsense codon

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Mutation

• Frameshift mutation one or a few base pairs are
  deleted or added to DNA.

• Insertion or deletion of one or more nucleotide
  pairs

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The Frequency of Mutation

• Spontaneous mutation rate 1 in 109 replicated
  base pairs or 1 in 106 replicated genes
• Mutagens increase to 105 or 103 per replicated
  gene
• Occur randomly along a chromosome.
• A low rate of spontaneous mutations is beneficial
  in providing the genetic diversity needed for
  evolution.

ANIMATION Mutations Types
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Cancer

• Cancerous cells are growing faster than normal
  cells
• Mutations have a greater effect when a cell is
  growing because it is synthesizing DNA and
  enzymes.
• The probability of a lethal mutation also is
  increased in rapidly growing cells.

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Chemical Mutagens

• Include base-pair mutagens, nucleoside analogs,
  and frameshift mutagens.

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Chemical Mutagens
ANIMATION Mutagens
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Radiation

• Ionizing radiation (X rays and gamma rays) causes
  the formation of ions that can react with
  nucleotides and the deoxyribose-phosphate
  backbone
• Initial effect causes formation of highly
  reactive ions in a cell
• Base substitutions or breakage of the
  sugar-phosphate backbone results.

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Radiation

• UV radiation causes thymine dimers
• Nonionizing it causes bonding between adjacent
  thymines.
• Can be repaired by enzymes that cut out and
  replace the damaged portion of DNA.

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Repair

• Photolyases (light repair enzymes) separate
  thymine dimers
• Nucleotide excision repair

ANIMATION Mutations Repair
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Selection

• Positive (direct) selection detects mutant cells
  because they grow or appear different
• Negative (indirect) selection detects mutant
  cells because they do not grow
• Replica plating used for negative selectionto
  detect, for example,
• auxotrophs that have nutritional requirements not
  possessed by the parent (nonmutated) cell which
  is called a prototroph

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Replica Plating
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Interpret Replica Plating

• The colonies are Ampicillin sensitive because the
  did not grow on the replica plate containing
  Ampicillin. If they did grow, we would classify
  them as Ampicillin resistant.

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Ames Test for Chemical Carcinogens

• relatively inexpensive and rapid test for
  identifying possible chemical carcinogens.

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Ames Test for Chemical Carcinogens

• assumes that a mutant cell can revert to a normal
  cell in the presence of a mutagen and that many
  mutagens are carcinogens.

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Genetic Recombination

• Exchange of genes between two DNA molecules
• Usually involves DNA from different organisms
• Contributes to genetic diversity.
• When some of the donors DNA has been integrated
  into the recipients DNA, the resultant cell is
  called a recombinant.

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Genetic Recombination

• Crossing over occurs when two chromosomes break
  and rejoin
• Genes from two chromosomes are recombined into
  one chromosome containing some genes from each
  original chromosome.

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Genetic Recombination

• Vertical gene transfer Occurs during
  reproduction between generations of cells.
• Horizontal gene transfer The transfer of genes
  between cells of the same generation.

ANIMATION Horizontal Gene Transfer Overview
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Genetic Recombination
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Genetic Transformation

• genes are transferred from one bacterium to
  another as naked DNA in solution.

ANIMATION Transformation
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Bacterial Conjugation

• Requires contact between living cells
• One type of genetic donor cell is an F
• Recipient cells are F
• F cells contain plasmids called F (fertility)
  factors that are transferred to the F- cells
  during conjugation

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Conjugation in E. coli

• When the plasmid becomes incorporated into the
  chromosome, the cell is called an Hfr (high
  frequency of recombination) cell
• Conjugation differs from reproduction because it
  transfers DNA horizontally to cells in the same
  generation

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Conjugation in E. coli

• During conjugation, an Hfr cell can transfer
  chromosomal DNA to an F cell. Usually, the Hfr
  chromosome breaks before it is fully transferred.

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Conjugation in E. coli
ANIMATION Chromosome Mapping
ANIMATION F Factor
ANIMATION Conjugation Overview
ANIMATION Hfr Conjugation
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Gene Mapping

• Conjugation can be used to map the location of
  genes of bacterial chromosomes
• The time allowed for conjugation to occur can
  help determine the order of the genes
• Ex If the F- acquires the ability to synthesize
  threonine, the thr gene must be located early in
  the chromosome

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Transduction by a Bacteriophage

• DNA is passed from one bacterium to another in a
  bacteriophage (virus that infects a bacteria) and
  is then incorporated into the recipients DNA
• In generalized transduction, any bacterial genes
  can be transferred.

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Transduction by a Bacteriophage
ANIMATION Generalized Transduction
ANIMATION Specialized Transduction
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Plasmids

• Plasmids are self-replicating circular molecules
  of DNA carrying genes that are not usually
  essential for the cells survival.
• Conjugative plasmid Carries genes for sex pili
  and transfer of the plasmid
• Dissimilation plasmids Encode enzymes for
  catabolism of unusual compounds
• R factors Encode antibiotic resistance

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R (resistance)Factor, a Type of Plasmid
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Transposons

• Segments of DNA that can move from one region of
  DNA to another (same or different chromosome)
• Contain insertion sequences for cutting and
  resealing DNA (transposase)
• Complex transposons carry other genes

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Transposons

• Complex transposons can carry any type of gene,
  including antibiotic-resistance genes, and are
  thus a natural mechanism for moving genes from
  one chromosome to another.

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Transposons
ANIMATION Transposons Overview
ANIMATION Transposons Insertion Sequences
ANIMATION Transposons Complex Transposons
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Genes and Evolution

• Diversity is the precondition for evolution
• Mutations and recombination provide diversity
• Fittest organisms for an environment are selected
  by natural selection

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Difference Between Prokaryotic Eukaryotic DNA