Microbial Genetics

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Microbial Genetics

• Dr. Gary Andersen, 913-279-2211
• Some slides used with permission from Curtis
  Smith, KCKCC
• Reference Chapter 7,8 from (Black, J., 2005)

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Basic Units of Genetics

• Genomes the total of the genetic material in a
  cell.
• Gene - The unit of heredity for a given genetic
  trait. The site on a DNA molecule that carries
  the code for a certain cell function.
• Viruses 4 or 5 genes, E. coli 4228 genes,
  Human 31,000 genes.

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A Big Question to Struggle With

• Which is more important nature or nurture?
  Genetics or the environment? In determining
  the characteristics and behavior of an organism?

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Nucleic Acids

• I. Nucleic acids are located in the nucleoid of
  bacteria, and the nucleus of eukaryotes. There
  are 2 kinds of nucleic acids RNA DNA.

Ruptured E. coli cell showing DNA
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DNA

• CHARACTERISTICS OF DNA
• DNA (deoxyribonucleic acid) is made of
  subunits called nucleotides. Nucleotides are
  made of 3 components. These 3 components are
  linked together with a covalent bond.
• E. Coli 4.6 million nucleotide pairs (1mm)
• Corn 2.5 billion nucleotide pairs
• Human 3 billion nucleotide pairs (2nm wide by 2
  meters long)

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Significance of DNA Structure

• Maintains the code with high degree of fidelity.
  (double strand assures accurate replication)
• Provides a method for introducing a high degree
  of variety. (unlimited variety of sequences
  possible)

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1. COMPONENT 1 - Phosphate

• Phosphate group - Phosphate functions as a
  structural part of nucleic acids.

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2. COMPONENT Ribose Sugar

• 2, DEOXYRIBONUCLEIC ACID
• Ribose - A five carbon sugar that functions
  as part of the DNA backbone (ie. structural).
  2, Deoxy means without oxygen on the number 2
  carbon atom.

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3. COMPONENT Nitrogen Bases

• NITROGEN CONTAINING BASES
• Function express genetic information.
• composition
• 2 PURINES ADENINE (A) GUANINE (G)
• double ring structures
• 2 PYRIMIDINES THYMINE(T) CYTOSINE(C)
• single ring structures

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Nucleotide Base
Composed of one Nitrogen base, one Deoxyribose,
and one Phosphate group
Adenine (Nitrogen base)
Deoxyribose
Phosphate
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4 Nucleotides
T
A

C
G
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DNA Structure

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DNA Structure

• 4. DNA is a double helix (there are 2 strands of
  DNA) which are intertwined with 5 base pairs per
  turn.
• 5. DNA has complimentarity
• that is A always bonds with T
• and G always with C
• 6. DNA is always antiparallel. The 2 strands of
  DNA are always oriented in opposite directions. (
  5 PO3 end 3 OH end)
• http//www.umass.edu/microbio/chime/dna/dna53.htm

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DNA Bonds

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3-D Image ofDNA
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B. RNA

• RIBONUCLEIC ACID
• Similar to DNA except
• 1. RNA is single stranded
• 2. RNA has a ribose sugar instead of
  deoxyribose. (Oxygen on 2 C).
• 3. RNA has URACIL (u) instead of thymine
• 4. RNA is always shorter than DNA, 1,000
  nucleotides in length

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C. FUNCTIONS OF RNA

• 1. rRNA (ribosomal) - comprises the ribosome
  (site of protein synthesis). (60 of a ribosome
  is made of RNA, the rest is protein).
• 2. tRNA (transfer) carries amino acids to the
  ribosome during protein synthesis. Also known
  as the ANTICODON
• 3. mRNA (messenger) - a complimentary strand of
  RNA equal in size to 1 gene (normally 1,000
  nucleotides). CODON - coded info from DNA
  (bound for the ribosome)

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THE CENTRAL DOGMA OF BIOLOGY Francis Crick
1956

• There are 3 parts to the flow of information in
  all cells.
• Transcription Translation
• DNA -------------?mRNA-----?protein
• Replication

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Central Dogma of Biology
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DNA REPLICATION

• 1. Where 2 parental strands of DNA are copied
  into 2 daughter strands. Rate 1,000 nucs per
  seconds without error. This leads to binary
  fission in bacteria.
• Cell Division) 2 daughter cells
• 2. Each cell receives 1 parental strand and 1
  daughter strand. (semiconservative replication)

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As the two replication forks meet, the two new
chromosomes separateeach containing one new and
one old strand
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Replication bonding

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Replication Fork

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1. EVENTS IN DNA REPLICATION

• DNA unwinds using the enzyme DNA Helicase
• SSBP holds the 2 strands apart (single strand
  binding proteins)
• Note 2 replication forks. DNA replication is
  considered bi-directional replication.

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DNA REPLICTIONCONTINUED

• Polymerization requires DNA Polymerase (POL III)
  which is an enzyme that synthesizes 2 nucleotide
  strands (daughter strands) from 2 parental
  (templates) strands.
• DNA exonuclease (POL I) removes any mistaken base
  pairs.
• DNA ligase seals any gaps and joins the 2 strands
  together.

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DNA Replication Enzymes at Work

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Steps in Replication

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Replication of DNA contd

• http//www.ncc.gmu.edu/dna/repanim.htm

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THE CENTRAL DOGMA OF BIOLOGY

• There are 3 parts to the flow of information in
  all cells.
• Transcription Translation
• DNA -------------?mRNA-----?protein
• Replication

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B. TRANSCRIPTION

• 2nd part of the central dogma of biology
• 1st step in gene expression (i.e.protein
  synthesis).
• The cells genetic plan contained in DNA is
  transcribed into a complimentary base sequence
  called messenger RNA (mRNA).
• The region of DNA that produces or serves as a
  template for mRNA is called a gene. A gene
  normally consists of around 1,000 base pairs. It
  is the smallest segment of DNA that codes for
  mRNA.

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TRANSCRIPTION CONTINUED

• 5. RNA polymerase is the enzyme responsible for
  making mRNA

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Transcription continued

• 7. Example
• DNA A T G C C G
• DNA T A C G G C
• mRNA A U G C C G
• 8. mRNA is a blueprint of DNA or a transcript
  or code.
• 9. One code word consists of three letters.

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Animation of Transcription

• http//www.ncc.gmu.edu/dna/mRNAanim.htm

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C. TRANSLATION

• Translation is the 3rd part of the central dogma
  of biology (2nd step in gene expression or
  protein synthesis).
• After transcription, the coded information in
  mRNA is translated into an enzyme (protein).
• This process takes place on the ribosome. Note
  that the ribosome is made of rRNA and protein.

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Translation Graphic
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TRANSLATION CONTINUED

• tRNA STRUCTURE
• tRNA utilizes the information in mRNA to
  determine the sequence of amino acids in a
  protein. tRNA has a cloverleaf shape. The amino
  acid end binds one specific amino acid in the
  cytoplasm. The anticodon end pairs with the
  codon on mRNA.

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Transfer RNA Structure
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TRANSLATION CONTINUED

• The mechanics of translation
• Initiation mRNA bumps into the small subunit and
  triggers the two ribosomal subunits to bind
  together. The first tRNA anticodon (UAC)
  carrying the amino acid methionine hydrogen bonds
  with the codon AUG on mRNA.

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TRANSLATION CONTINUED

• b. Elongation The second tRNA binds to the
  second code word on mRNA. A peptide bond forms
  between the two amino acids. The first tRNA
  leaves, and the enzyme translocase moves the
  ribosome down one code word of mRNA at a time.
  This repeats 300X.

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TRANSLATION CONTINUED

• C. In termination, one of three possible stop
  codons is reached. The last tRNA falls away and
  the two ribosomal subunits fall apart.

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d. The Genetic Code

• 61 sense codons for 20 amino acids
• 3 nonsense (or stop codons)
• total codons
• Pg 180 (Black, J., 2005)

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The Genetic Code

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Steps in Protein Synthesis
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Steps in Protein Synthesis
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Steps in Protein Synthesis
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Steps in Protein Synthesis
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Steps in Protein Synthesis
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Protein Synthesis
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Translation - Animation

• http//www.ncc.gmu.edu/dna/ANIMPROT.htm

Translation Animation - http//www.wehi.edu.au/weh
i-tv/dna/movies/Translation.mov.gz
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Translation Blockers

• Streptomycin (SM) blocks assembly of the
  ribosome during initiation.
• Chloroamphenicol (CA) blocks peptide bond
  formation during elongation.
• Tetracycline TC blocks the 2nd site on the
  ribosome during elongation.
• Erythromycin EM blocks translocase during
  elongation.

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Gene Regulation

• How can genes be turned off and on?
• Examples from E. coli
• Inducer example is lactose (lac operon), pg 187
  of (Black, J., 2005)
• Repressor argenine (arg operon), pg 187-188 of
  (Black, J., 2005)

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Induction - Lac operon
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Repression - Trp operon
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III. 5 Ways of Creating Genetic Diversity in
Bacteria

• A. Mutations
• B. Transformation
• C. Conjugation
• D. Transposition
• E. Transduction

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A. Mutations

• 1. Changes in the nucleotide sequence
  usually due to an error in DNA replication.
  These occur naturally at low levels (also known
  as spontaneous mutations) or by the effects of
  chemical agents called mutagens or by physical
  agents like radiation.

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Results of Mutations

• 2. Most mutations are neutral - they have no
  effect on the polypeptide. Some mutations result
  in a less active product Less often an inactive
  product Very few mutations are beneficial.
  However, these would be passed on!

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Types of Mutations

• 3. Point mutations - a one base change in DNA.
  There are 3 types
• a. silent mutations - single base substitution
  in the 3rd base nucleotide position of a codon.
  This results in NO change in amino acid. Note
  that the first 2 letters of the genetic code are
  the most critical.

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• b. missense mutations - single base substitution
  in 1st or 2nd base nucleotide position. This
  results in a changed amino acid. A change in one
  amino acid usually will have little effect
  depending on where in the polypeptide it occurs.
• nonsense mutations - single base substitutions
  that yield a stop codon. Note there are 3
  nonsense codons in the genetic code NO PROTEIN
• 4. Frame Shift Mutations - the addition or
  deletion of 1 or more bases. These are due to
  powerful mutagens chemical or physical.

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• a. Chemical mutagens - (used in research to
  study mutagenesis). There are 3 kinds of
  chemical mutagens.
• 1. alkylating agents. Adds alkyl group,
  CnH(2n1) Ex. formalin, nitrogen, mustard, and
  ethylene oxide (reacts with G changing it to bind
  with T).
• 2. base analogs. Mimics a nitrogen base. Ex.
  AZT is a modified sugar that substitutes for T.
  Ex. 5 - bromouracil binds with A or G.
• 3. intercalating agents. Inserts into DNA
  and pushes bases apart. Ex. AFLATOXIN - a
  chemical produced by peanut and grain molds. The
  mold is Aspergillus flavus (fungus).

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• b. Physical mutagens
• 1. nonionizing radiation - Causes the formation
  of T T dimers. UV light _at_ 260 nm.
• 2. Ionizing radiation - damages DNA by causing
  the formation of free radicals leading to
  mutations. 3 Ex. X-rays. Gamma rays from
  radioactive fallout penetrates the body. Alpha
  rays from inhaled dust containing radioactive
  fallout.

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B. TRANSFORMATION

• The passage of homologous DNA from a dead donor
  cell to a living recipient cell. Occurs in
  Streptococcus pneumoniae. When S. pneumo dies
  the DNA can be absorbed by a living S. pneumo and
  recombined into the chromosome. The gene for
  capsule formation is obtained in this way, as is
  a gene for penicillin resistance. Discovered in
  1929 by Fredrick Griffith.

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Transformation Graphic

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Griffiths Transformation Experiment

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C. CONJUGATION

• 1. A mating process between a donor F
  (bacteria with fertility factor plasmid) and an
  F- recipient cell.
• 2. Occurs in Gram - enteric bacteria like E.coli
• 3. Discovered in 1946 by Joshua Lederberg and
  Edward Tatum.
• 4. Plasmids carry genes that are nonessential
  for the life of bacteria. Ex. gene for pili (sex
  pilus). Ex. plasmid replication enzymes. Ex.
  Medical Problem R-Factor antibiotic
  resistance!

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Conjugation continued

• Normal - Sex plasmid transfer (usually
  20 of 100 genes).
• a. Requires a sex pilus
• b. F bacteria transmits a copy of the
  plasmid to F- bacteria. This converts the F-
  cell into an F cell. Medical Problem The
  R factor (antibiotic resistance) on the F factor
  is transmitted! http//www.cat.cc.md.us/courses/bi
  o141/lecguide/unit4/genetics/recombination/conjuga
  tion/f.html

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• 6. Hfr (High Frequency Recombination)
• a. Hfr- bacterial plasmid integrates into the
  chromosome.
• b. Medical Problem Hfr antibiotic
  resistance genes are passed during binary
  fission (every time the cell divides).
  Therefore, antibiotic resistance spreads very
  rapidly!
• c. When Hfr mate with F bacteria, only
  the bacterial genes cross NOT plasmid genes.
  Genetic diversity results in this case due to
  recombination. http//www.cat.cc.md.us/courses/bi
  o141/lecguide/unit4/genetics/recombination/conjuga
  tion/hfr.html

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D. TRANSPOSITION p 285

• Transposons (jumping genes) are big
  chunks of DNA that randomly excise and relocate
  on the chromosome.
• Transposons were discovered in 1950 by Barbara
  McLintock in corn.
• Causes antibiotic resistance in Staph. aureus,
  the famous methicillin resistant Staphlococcus
  aureus (MRSA) strain!

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E. TRANSDUCTION

• the transfer of genetic material from donor
  bacteria to recipient bacteria via a transducing
  agent (virus!). Bacterial viruses are called
  bacteriophage.
• 1. Discovered in 1952 by Zinder
  Lederberg.
• 2. Two kinds of transduction generalized
  and specialized.

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• 2. Generalized transduction Starts with the
  LYTIC CYCLE where a T- even phage (Fig. 8.5 pg
  210) infects E.coli killing the host cell, and
  synthesizing 2,000 copies of itself. The T-even
  phage randomly packages bacterial DNA in a few
  defective phages. Once a T even phage infects
  another E. coli, this genetic information can be
  recombined into the host cell without causing the
  lytic cycle. New genetic information is thereby
  transduced from one bacteria to another.

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Generalized Transduction

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Generalized Transduction

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Specialized Transduction

• 3. Specialized transduction
• Lambda phage infects E.coli. The phage does not
  lyse the cell immediately. Instead it integrates
  into chromosome of the bacteria as a prophage and
  remains dormant. This is called the LYSOGENIC
  CYCLE. Phage genes are replicated and
  passed to all daughter cells until the bacteria
  is under environmental stress, from lack of
  nutrients, etc. Then phage gene will excise from
  the nucleoid and enter the LYTIC CYLE taking one
  adjacent gene for galactose metabolism.

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Specialized Transduction Cont.

• The gal transducing phage (lambda) makes
  2,000 copies of itself with the gal gene, and
  infects other E.coli. When gal integrates into
  the nucleoid of other E. coli, it may provide
  these bacteria with a new capacity to metabolize
  galactose.

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Specialized Transduction Graphic
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Comparison of Bacteriophage

• 3. Comparison of bacteriophage transduction in
  E.coli.
• Generalized Specialized
• T even phage lambda phage
• lytic cycle lysogenic
• random packaging specific gal gene

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End of Slides