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					MICROBIOLOGY CHAPTER 7
        INHERITANCE
Gregor Mendel (1866)
• Particulate theory of inheritance
   – Traits are caused by “heritable units”
      • Now called “genes”
   – Genes are transmitted from one
     generation to the next
   – Of what are genes made?
      • Mendel did not know
       INHERITANCE
Walter Sutton (1902)
• Columbia University
• Chromosome theory of inheritance
  – Noted that chromosomes segregate in
    a manner similar to Mendel’s genes
  – Proposed that genes reside upon
    chromosomes
     • Genes segregate as a result of
       chromosomal segregation
INHERITANCE
Thomas Hunt Morgan (1909)
• “Lord of the Flies”
• Established that genes reside
  upon chromosomes
   – Received Nobel Prize in 1933
• Used fruit flies
   –   Drosophila melanogaster
   –   Red-eyed vs. white-eyed mutant
   –   Mapped gene to X-chromosome
   –   Later mapped many more genes
   –   Genetic distance measured in
       “Morgans”
 GENETIC MATERIAL
Frederick Griffith (1928)
• English bacteriologist
• Experiments indicated that DNA is the genetic
  material
  – Most thought that protein was a more likely
    candidate
     • Why do you think this might be?
  – Used Streptococcus pneumoniae
     • Pneumonia-causing bacterium
 GENETIC MATERIAL
Frederick Griffith (1928)
• Streptococcus pneumoniae
  – Two strains
     • “S” strain
         – Smooth colonies
           (macroscopic)
         – Pathogenic
     • “R” strain
         – Rough colonies
           (macroscopic)
         – Non-pathogenic
  – What do you think is the basis for this difference?
 GENETIC MATERIAL
Frederick Griffith (1928)
• R-strain bacteria + mouse  alive
• S-strain bacteria + mouse  dead
• Heat-killed S-strain bacteria + mouse  alive
• Conclusions?
GENETIC MATERIAL
Frederick Griffith (1928)
• Heat-killed S + live R + mouse  dead mouse!!!
  – Live S cells present in blood
     • R transformed into S
     • “Bacterial transformation”
 GENETIC MATERIAL
Frederick Griffith (1928)
• Bacterial transformation
  – A chemical substance was transferred from the
    dead S bacteria to the live R bacteria
  – What is this substance?
     • Why is DNA a more likely candidate than protein?
     • Most of the world still thought that protein was the
       genetic material
 GENETIC MATERIAL
Oswald Avery (1944)
• American bacteriologist
• Furthered Griffiths’ experiments
• Determined that the transforming substance
  was DNA
  – Thus, DNA is the genetic material
  – People still not convinced
GENETIC MATERIAL
Oswald Avery (1944)
• Purified various
  macromolecules
  from S bacteria
  –   DNA
  –   RNA
  –   Protein
  –   Sugars
• Attempted to transform R bacteria with each
 GENETIC MATERIAL
Oswald Avery (1944)
• S sugars + live R  no live S
• S protein + live R  no live S
• S RNA + live R  no live S
• S DNA + live R live S!!! (dead mouse)

• DNA is the genetic material!
  – People still not convinced
GENETIC MATERIAL
Oswald Avery (1944)
• Critics of Avery
  – DNA was contaminated with small amounts of
    protein
  – Enzymes are active in small amounts
  – Protein contaminants in the DNA preparation are
    the transforming material
  – (Wrong, but rational)
GENETIC MATERIAL
Oswald Avery (1944)
• Response to critics
  – S DNA + live R live S
  – Include protease live S!!!
     • Protein enzymatically degraded
  – Include DNAse no live S!!!
     • DNA enzymatically degraded
  – DNA is the genetic material
     • People still not convinced (Thick skulls, I guess)
 GENETIC MATERIAL
Hershey & Chase (1952)
• Established that DNA is the
  genetic material
• Used T2 bacteriophage
   – Virus infecting bacteria
   – Composed of DNA & protein
   – Reprograms infected cell
      • Reprogramming requires
        genetic material
  GENETIC MATERIAL
Hershey & Chase (1952)
• Radiolabeled phage
  with 35S
  – Labels protein, not DNA
• Radiolabeled phage
  with 32P
  – Labels DNA, not protein
 GENETIC MATERIAL
Hershey & Chase (1952)
• Infect E coli with 35S-labeled phage
• Disrupt, centrifuge
   – Supernatant hot
   – Pellet cold
• Protein does not enter cell
GENETIC MATERIAL
Hershey & Chase (1952)
• Infect E coli with 32P-labeled phage
• Disrupt, centrifuge
   – Supernatant cold
   – Pellet hot
• DNA enters cell
   – DNA is the genetic material
   – People are finally convinced!!!
    DNA STRUCTURE
Crick & Watson (1953)
• Determined the 3-D structure of DNA
  – Structure of single strand already known
    DNA STRUCTURE
Crick & Watson (1953)
• Relied heavily on work by
  – Erwin Chargaff
  – Rosalind Franklin
  – Maurice Wilkins

    How many experiments do you imagine
         Crick & Watson performed?
     Who shared the Nobel in 1962 with
              Crick & Watson?
    DNA STRUCTURE
Crick & Watson (1953)
• Chargaff’s Rules
  – For any organism’s DNA
     • [A] = [T]
     • [G] = [C]
• Crick & Watson’s realization
  – This indicates a specific
    relationship between A & T,
    and between G & C
   DNA STRUCTURE
Crick & Watson (1953)
• Maurice Wilkins & Rosalind Franklin
  – X-ray crystallography
  – DNA is helical
  – Full turn every
    3.4 nanometers
  – Diameter of
    helix is 2
    nanometers
DNA DOUBLE HELIX
 DNA REPLICATION
Crick & Watson (1954)
• The second DNA strand is redundant
• Why are there two strands?
  – Important for replication
     • “Semiconservative replication”
 DNA REPLICATION
• Begins at specific sites
   – “Origins of replication”
      • One (prokaryotes) or several (eukaryotes)
• Bidirectional
 DNA REPLICATION




• 5’  3’ growth
  – One strand synthesized continuously
  – One strand synthesized discontinuously
  DNA REPLICATION
• Various enzymes involved
  – DNA gyrase, helicase
     • Unwind helix
  – DNA polymerase
     • Polymerizes nucleotides
  – Primase
     • Synthesizes RNA primer, from which DNA synthesis
       begins
  – DNA ligase
     • Covalently connects DNA fragments
DNA REPLICATION
 DNA REPLICATION
Energy requirements
• Deoxynucleotides supplied as triphosphates
   – Two phosphates
     removed
   – Breaking of
     phosphate bond
     releases energy
 GENE EXPRESSION
Archobold Garrod (1909)
• Suggested that genes dictate phenotypes
  through enzymes
  – These enzymes catalyze specific chemical
    processes
  – Inherited diseases involve the inability to make a
    particular enzyme
     • “Inborn errors of metabolism”
• Later experiments by others showed Garrod to
  be correct
GENE EXPRESSION
Beadle & Tatum (1930s)
• Isolated nutritional mutants of the bread
  mold Neurospora crassa
  – Wild-type grows on
    minimal media
  – Mutants require arginine
     • “Auxotrophs”
     • Deficient in arginine
       synthesis
     • “Arg- mutants”
 GENE EXPRESSION
Beadle & Tatum (1930s)
• Arg- mutants
  – Will not grow without arginine
  – Will grow when arginine is supplied
• Three Arg- classes identified
  – Grow when supplied with
     • I: arginine, citrulline, or ornithine
     • II: arginine or citrulline
     • III: arginine only
 GENE EXPRESSION
Beadle & Tatum (1930s)
• Arginine biosynthesis
  – Precursor  ornithine  citrulline  arginine
  – Each step catalyzed by a specific enzyme
  – Each mutant class lacks a different enzyme
     • I: precursor  ornithine
     •              II: ornithine  citrulline
     •                            III: citrulline  arginine
BEADLE & TATUM
 GENE EXPRESSION
Beadle & Tatum (1930s)
• Arg- phenotype is heritable
  – Genetic defect
• Enzyme lacking
  – Result of genetic defect
     • Garrod’s “Inborn error of metabolism”
• One gene : One enzyme
  – (One gene : One protein)
  – (One gene : One polypeptide)
 GENE EXPRESSION
What is a gene?
• Mendel
  – Unit of inheritance conferring a phenotype
• Beadle & Tatum
  – Unit of DNA directing the synthesis of a
    polypeptide
• Modern definition
  – Unit of DNA directing the synthesis of a
    polypeptide or functional RNA molecule
GENE EXPRESSION
How is a gene expressed?
  DNA  RNA  protein


• DNA  RNA
  – Transcription
• RNA  protein
  – Translation
GENE EXPRESSION
Transcription
• Synthesis of RNA from a DNA template
• Similar to DNA
  synthesis
• Some differences
 GENE EXPRESSION
Transcription
• How is it different from DNA synthesis?
  – Start at promoter
       • Unidirectional
  –   Single strand transcribed
  –   Continuous (not discontinuous)
  –   Catalyzed by RNA polymerase
  –   Termination sequence
  –   No mismatch repair or ligation
TRANSCRIPTION
TRANSCRIPTION
TRANSCRIPTION
TRANSCRIPTION
  GENE EXPRESSION
Translation
• Synthesis of a protein
  from an mRNA template
• Requires ribosomes,
  tRNAs, and various
  other factors
• Utilizes genetic code
 GENE EXPRESSION
Translation
• Messenger RNA
  – mRNA
  – Temporary copy of gene
  – Information deciphered
    using the genetic code
  – Functional unit is codon
     • Three consecutive
       nucleotides
     • “Triplet”
 GENE EXPRESSION
Translation
• mRNA codons
  – 3 consecutive nucleotides
  – 64 different codons (43)
     • 61 encode specific amino
       acids
         – 61 > 20; some duplication
         – One of these (AUG) is a
           start codon
     • 3 are stop codons (UAG,
       UGA, UAA)
         – Terminate translation
  – Recognized by tRNAs
GENE EXPRESSION
    Translation
    • Transfer RNA (tRNA)
      – Short segments of RNA
         • ~80 nucleotides long
      – Two important regions
         • Anticodon
             – Nucleotide triplet
             – Complementary to codon of mRNA
         • Amino acid attachment site
             – Amino acid corresponding to codon
               is covalently attached to the tRNA
GENE EXPRESSION
Translation
• Ribosomes
  – Cellular organelles
  – Workhorse
  – Site of translation
  – Facilitate interaction between
    mRNA and tRNA
  – Facilitate polymerization of
    amino acids
TRANSLATION
TRANSLATION
TRANSLATION
GENE EXPRESSION
Differences between prokaryotes & eukaryotes
• Transcription
  – Eukaryotes process RNA, prokaryotes do not
     • Splicing
         – Removal of introns
           (junk)
     • Capping
         – Backwards g-residue
           at 5’ end
     • Polyadenylation
         – Addition of a-residues at 3’ end
 GENE EXPRESSION
Differences between prokaryotes &
  eukaryotes
• mRNA organization
  – One protein per mRNA in eukaryotes
  – Multiple proteins per mRNA in prokaryotes
     • “Polycistronic”
GENE EXPRESSION
Differences between prokaryotes & eukaryotes
• Transport from nucleus
  – Eukaryotes have a nucleus
  – Prokaryotes lack a nucleus
GENE EXPRESSION
Differences between prokaryotes & eukaryotes
• Translation
  – Begins during transcription in
    prokaryotes
  – Follows transcription, processing,
    transport in eukaryotes
  GENE EXPRESSION
Differences between prokaryotes & eukaryotes
• Translation initiation
   – Eukaryotic ribosome binds to cap and scans for start codon
   – Prokaryotic ribosome lands on internal landing pads
      • Often multiple landing pads
• Translation termination
   – Different mechanisms
   – Same result
              MUTATION
Mutation
• Heritable
  – Change in DNA sequence
• Relatively rare
• Typically occurs during
  DNA replication or repair
• May affect gene
  expression
           MUTATION
Mutation
• Different effects
  – Harmful
  – Neutral
  – Beneficial
• Most are neutral or harmful
• A minority are beneficial
• Ultimate source of genetic
  variation
  – Natural selection acts upon variation
           MUTATION
Single gene mutations
• Affect only a single gene
• “Point mutation”
  – Affects a single base pair
• Various types
  – Base substitution
  – Base insertion
  – Base deletion
          MUTATION
Single gene mutations
• How do mutations
  affect gene expression?
• A change in DNA can
  give rise to a change in
  RNA codons, which can
  encode different amino
  acids
           MUTATION
Base substitution mutations
• One base pair altered
• Various effects
  – Silent mutation
  – Missense mutation
     • Neutral mutation
  – Nonsense mutation
  – Frameshift mutation
           MUTATION
Silent mutations
• No change in amino acids
  – e.g., CCC  CCG (pro  pro)
  – Genetic code is degenerate
• No effect on phenotype
     MUTATION
Missense mutations
• Change in amino acids
• e.g., GAA  GTA (glu  val)
• Phenotype may be affected
          MUTATION
Neutral mutations
• Type of missense mutation
• Change in amino acids
  – e.g., CTT  ATT (leu  ile)
• Protein function unaffected
          MUTATION
Nonsense mutations
• Normal codon changed to
  termination codon
  – e.g., AAA  AAG (lys  stop)
• Premature termination
  – Truncated protein
• Protein function affected
           MUTATION
Frameshift mutations
• Insertion or deletion of base pair(s)
   – e.g., GGA  GAGA (gly  glu)
• Alter reading frame
   – All downstream amino acids altered
• Protein function affected
• Effect similar to nonsense mutation
MUTATIONS
 GENE REGULATION
• All genes are not expressed in all cells at all
  times
  – Not all proteins are needed at all times
  – Expressing proteins when they are not needed is
    wasteful of energy
  – Cells wasting energy are at a selective
    disadvantage
  – Gene expression is regulated
 GENE REGULATION
What types of proteins are not always
  needed?
• Enzymes involved in the metabolism of a
  particular sugar are needed only when the
  sugar is available
• Enzymes involved in the synthesis of a
  particular amino acid are needed only when
  the cell is deficient in this amino acid
• Etc.
 GENE REGULATION
How are genes regulated?
• The operon is a key unit of gene regulation in
  bacteria
• An operon is a group of genes with related
  functions regulated as a group
 GENE REGULATION
trp operon
• Involved in the synthesis of the amino acid
  tryptophan
• Expression normally on, but can be turned off
  in the presence of sufficient levels of
  tryptophan
  – “Repressed”
GENE REGULATION
lac operon
• Involved in the metabolism of the sugar lactose
• Expression normally off, bur can be turned on in
  the presence of lactose
   – “Induced”
GENE REGULATION
lac operon
• Repressor binds to
   operator
   – No expression
• Lactose removes repressor
   – expression
             GENOMICS
• DNA sequencing now automated, very rapid
• Entire genomes can now be sequenced
  – >75 completed so far
  – Many viruses also
• Analysis more cumbersome
              GENOMICS
• Analysis of sequenced DNA
  – Search for open reading frames (ORFs)
     • Start codon  …  stop codon
  – Compare DNA and inferred protein sequence to
    database
     • Homology
     • Putatively determine function

				
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