Microbial Genetics

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Microbial Genetics
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Chromosomes

• Chromosome discrete cellular structure composed
  of a neatly packaged DNA molecule
• Eukaryotic chromosomes
• DNA wound around histones
• located in the nucleus
• diploid (in pairs) or haploid (single)
• linear appearance
• Prokaryotic chromosomes
• DNA condensed into a packet by means of
  histone-like proteins
• single, circular chromosome

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(No Transcript)
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Genes Related to Obesity in the Human Genome
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Map of E. colis 5000 Genes

• Notice it is single circular
• Does E. coli have 1 or 2 alleles of each gene?
  How do you know?
• Humans were first thought to function with
  100,000 genes and now the number has dropped to
  35,000 genes although this is still a hot topic
  in research

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Genome

• Genome sum total of genetic material of an
  organism
• most of the genome exists in the form of
  chromosomes
• some appears as plasmids or in certain organelles
  of eukaryotes
• genome of cells composed entirely of DNA
• genome of viruses can contain either DNA or RNA

E. coli cell disrupted to release its DNA
molecule.
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Gene

• A gene is a segment of DNA that contains the
  necessary code to make a protein or RNA molecule
• Three categories of genes
• structural genes code for proteins
• genes that code for RNA machinery used in
  protein production
• regulatory genes control gene expression

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Genetic Terms

• Genotype
• an organisms genetic makeup its entire
  complement of DNA
• Phenotype
• is the expression of the genes the proteins of
  the cell and the properties they confer on the
  organism.
• Size, shape, color, environment

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The DNA Code
Hydrogen bond
H
H
H
N
O
HN

• Nucleotide basic unit of DNA structure
• phosphate
• deoxyribose sugar
• nitrogenous base
• Nucleotides covalently bond to each other in a
  sugar-phosphate linkage

N
C
N
G
H
NH
N
N
H
O
N
Sugar
3'
H
OH
P
D
5'
4'
D
1'
5'
P
C
D
2'
G
P
D
3'
P
P
P
O
O
T
A
D
D
O
P
O
P
O
C
G
D
D
O
O
P
P
C
G
D
D
O
P
P
T
A
D
D
O
P
O
P
O
C
G
D
D
O
O
P
P
T
A
P
D
D
P
5'
D
D
3'
5'
H
OH
CH3
H
O
N
NH
N
A
T
N
H
N
H
N
N
O
H
Sugar
(a)
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Nitrogenous Bases and Base Pairing

• Pairing dictated by the formation of hydrogen
  bonds between bases
• Complementary Base Pairing if sequence of one
  strand known, sequence of other strand inferred
• Try it

TAC GTA ACG
ATG CAT TGC
Hydrogen bond
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Nature of the Double Helix

• Antiparallel arrangement one side of the helix
  runs in the opposite direction of the other
• One side runs from 5 to 3, and the other side
  runs 3 to 5
• This is a significant factor in DNA synthesis and
  protein production

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DNA Replication

• DNA ? DNA

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DNA Replication

• DNA replication involves unwinding a DNA double
  helix and using each strand as a template for a
  new, complementary strand
• DNA polymerase and over a dozen other enzymes and
  proteins are required to successfully replicate a
  single strand of DNA
• DNA replication is semi-conservative since each
  new chromosome will have one old and one new
  strand
• When does this occur??

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DNA Replication

• What is needed to replicate DNA
• Original DNA template
• Nucleotides
• a pool of nucleotides is free floating in the
  cytoplasm
• Enzymes
• DNA polymerase, ligase
• Energy
• ATP

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DNA Replication Prokaryotes

• Certain enzymes unwind the DNA.
• Then, DNA polymerase can read the parent strand
  and attach a complementary nucleotide to the new
  strand of DNA.
• Nucleotides are free in the cytoplasm.

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Transcription

• DNA ? RNA

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DNA vs. RNA

• Contains ribose rather than deoxyribose
• RNA is single stranded
• There is no T in RNA. Instead it is a U
• AU in RNA
• Can assume secondary and tertiary levels of
  complexity, leading to specialized forms of RNA
  (tRNA and rRNA)

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Transcription RNA Synthesis

• What you need to synthesize RNA
• Original DNA template
• chromosome with a promoter site (DNA sequence
  indicating start site) and a terminator site
• 2. Nucleotides
• G, C, A, U Uracil is substituted for thymine
• 3. Enzymes
• RNA polymerase
• 4. Energy
• ATP

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Transcription

• RNA polymerase large, complex enzyme that
  directs the conversion of DNA into RNA
• Template strand only one strand of DNA that
  contains meaningful instructions for synthesis of
  a functioning polypeptide

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Transcription

• Many types of RNA can be transcribed
• Messenger RNA (mRNA)
• RNA molecule that serves as a message of the
  protein to be produced
• Transfer RNA(tRNA)
• Transfers amino acids to ribosome
• Ribosomal RNA (rRNA)
• Forms the ribosome
• Regulatory RNA
• micro RNAs, anti-sense RNAs, riboswitches, small
  interfering RNAs

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Transcription Initiation

• RNA polymerase recognizes promoter region
• RNA polymerase begins its transcription at a
  special sequence called the initiator
• As the DNA helix unwinds it moves down the DNA
  synthesizing RNA molecule

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Transcription Elongation
Direction of transcription
Early mRNA transcript
Nucleotide pool

• During elongation the mRNA is built, which
  proceeds in the 5 to 3direction (you do not
  need to know the direction of elongation for this
  class)
• The mRNA is assembled by the adding nucleotides
  that are complementary to the DNA template.
• As elongation continues, the part of DNA already
  transcribed is rewound into its original helical
  form.

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Transcription Termination
Elongation
Late mRNA transcript
At termination the polymerases recognize another
code that signals the separation and release of
the mRNA strand,or transcript.
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Practice Transcription

• DNA
• GCGGTACGCATTAAGCGCCC
• RNA

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Translation

• mRNA ? Protein

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Translation

• Decoding the language of nucleotides and
  converting/translating that information into the
  language of proteins.
• The nucleic acid language is in the form of
  codons, groups of three mRNA nucleotides.
• The protein language is in the form of amino
  acids

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Translation

• Translation occurs at the ribosome
• The green mRNA strand is threaded through the
  ribosome.
• The ribosome reads the mRNA strand codons with
  the help of the genetic code and tRNA

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tRNA

• Decoder molecule which serves as a link to
  translate the RNA language into protein language
• One site of the tRNA has an anticodon which
  complements the codon of mRNA
• The other site of the tRNA has an amino acid
  attachment site corresponding to a specific amino
  acid as noted in the genetic code

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Translation and the Genetic Code

• Triplet code that specifies a given amino acid
• We use the genetic code (at right) to translate
  mRNA nucleotide sequence (codons) into amino acid
  sequence which make up proteins.
• The genetic code is degenerate which allows
  for a certain amount of mutation. I.e. UUU and
  UUC both code for Phe

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Translation and the Genetic Code

• There is one start codon, AUG, that codes for the
  amino acid methionine.
• There are 3 stop codons, UAA, UAG and UGA that
  signal the ribosome to stop translation and let
  go of the polypeptide chain (protein).

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Practice Translation

• RNA
• CGCCAUGCGUAAUUCGCGGG
• 1st Step Find the start of the gene which is
  always indicated by AUG. Everything upstream
  from that can be ignored.

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Practice Translation

• RNA
• CGCCAUGCGUAAUUCGCGGG
• 1st Step Find the start of the gene which is
  always indicated by AUG. Everything upstream from
  that can be ignored.

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Practice Translation

• RNA
• AUG/CGU/AAU/UCG/CGG/G
• 2nd Step To make it easier to track the codons I
  separate each with a slash

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Practice Translation

• RNA
• AUG/CGU/AAU/UCG/CGG/G
• 3rd Step Use genetic code to translate mRNA
  message into amino acid language

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Translation at the Molecular Level Initiation

• Ribosomes bind mRNA near the start codon (ex.
  AUG)
• tRNA anticodon with attached amino acid binds to
  the start codon

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Translation at the Molecular Level Elongation

• Ribosomes move to the next codon, allowing a new
  tRNA to bind and add another amino acid

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Translation at the Molecular Level Elongation

• Two amino acids form peptide bonds

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Translation at the Molecular Level Termination

• Stop codon terminates translation

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Videos

• https//www.youtube.com/watch?v41_Ne5mS2ls
• https//www.youtube.com/watch?v5bLEDd-PSTQlistP
  L1AD35ADA1E93EB6Findex2

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Polyribosomal Complex

• A single mRNA is long enough to be fed through
  more than one ribosome
• Permits the synthesis of hundreds of protein
  molecules from the same mRNA transcript
• Would you see this in Eukaryotes?

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Transcription and Translation in Eukaryotes and
Prokaryotes

• Similar to prokaryotes except
• AUG encodes for a different form of methionine
• Transcription and translation are not
  simultaneous in eukaryotes
• Eukaryotes must splice out introns to achieve a
  mature mRNA strand ready to go to the ribosome.

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Operons and Gene Regulation

• Only found in bacteria
• Coordinated set of genes to make proteins that
  are needed at the same time
• all regulated as a single unit
• either inducible or repressible

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lac Operon

• Most studied operon
• When lactose is absent the repressor blocks RNA
  Polymerase from binding to the operator and
  transcribing downstream genes.
• When lactose is present it binds to the repressor
  and it falls off the operator allowing RNA
  Polymerase to bind.
• The downstream genes are responsible for
  digesting lactose and are only on when lactose is
  present.

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Phase Variation

• Bacteria turn on or off a complement of genes
  that leads to obvious phenotypic changes
• New environment new phenotype!
• Most often traits affecting the bacterial cell
  surface
• Examples
• Neisseria gonorrhoeae production of attachment
  fimbriae
• Streptococcus pneumoniae production of a capsule

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Mutations

• A change in the sequence of DNA
• Possible effects of mutations
• No effect--gtno change in a.a. sequence
• Good--gtnew aa. Seq
• Increases variability in the gene pool, this is
  evolution!
• Bad--gtnew aa. Seq
• Cancer can be the product of a combination of bad
  mutations.

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Types of Mutations

• Point Mutation
• put the cat out---gtpuc the cat out
• put the cat out---gtput
• Frameshift (reading frame of mRNA shifts)
• put the cat out---gtput hec ato ut
• Deletion
• Addition
• Duplication

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The Effects of a Point Mutation

• When a base is substituted in DNA the mutation
  may have 2 effects
• Changes the amino acid
• Does not change the amino acid
• Why doesnt a mutation always change the amino
  acid sequence?

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The Effects of Frameshift Mutations

• The addition, deletion or insertion of one or
  more nucleotides drastically changes the amino
  acid sequence.

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Mutation Rates

• Normal Mutation Rate- 1/1 million per gene
• Mutations are constantly occurring since our
  enzymes are not 100 perfect.
• Mutagen- chemical or radiation that bring about
  mutations.
• Mutagen Mutation Rate 1/1000-1/100,000 per gene
  (10-1000X the normal rate)

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Mutagen Examples

• 5-Bromouracil and acridine are 2 mutagen examples
  that can insert themselves in DNA and cause
  errors in DNA replication, transcription and
  translation.
• Notice how similar in structure mutagens can be.
  There is just one change to thymine that can have
  dire consequences

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Thymine Dimers Caused by Radiation

• Radiation, such as X-rays and UV rays, can cause
  dimers to form in DNA.
• Thymine dimers can interfere with DNA
  replication, transcription and translation.

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What is the connection to cancer?

• Cancer is a genetic disease. It is the
  consequence of a change in DNA sequence.
• Carcinogensubstance that causes cancer
• Are mutagens also carcinogens? Yes
• Are all carcinogens also mutagens? No, alcohol
  and estrogen are carcinogens that speed up
  mitosis but do not directly cause mutations.

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Ames Test
The Ames Test uses bacteria to identify possible
carcinogens by looking for mutations to occur.
Once a mutagen is identified, it is tested in
animals to test if it is a carcinogen.
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Genetic Recombination

• During meiosis of human gametes
• In bacteria, occurs when DNA is transferred
  between bacteria.
• Increases diversity in gene pool
• End result is a new strain different from both
  the donor and the original recipients
• Vertical gene transfer-
• Genes/DNA passed from an organism to its
  offspring
• Horizontal gene transfer-
• Genes/DNA transferred between organisms

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Genetic Recombination

• Depends on the fact that bacteria have plasmids
  and are adept at interchanging genes
• Provide genes for resistance to drugs and
  metabolic poisons, new nutritional and metabolic
  capabilities, and increased virulence and
  adaptation to the environment

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Plasmids

• Self-replicating circular pieces of DNA
• 1-5 the size of bacterial chromosome
• mini-chromosome
• Bacteria can store up many different plasmids for
  their use can transfer these to other bacteria.
  
• They can contain any gene that the bacteria dont
  require but are useful to the survival of the
  bacteria. For example antibiotic resistance
  genes, toxin production, etc.

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Antibiotic Resistance (R) Plasmids

• Some plasmids can carry many antibiotic
  resistance genes.
• When bacteria collect many plasmids and these
  plasmids have many antibiotic resistance genes, a
  superbug may originate.

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Three Types of Genetic Transfer (Recombination)
in Bacteria

• Conjugation
• Transformation
• Transduction

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Conjugation

• A donor cell contains a F (fertility) plasmid
  making it F.
• A conjugation pilus (genes for which are on the
  F plasmid) forms and the donor cell transfers a
  copy of the F plasmid to the recipient.
• Now, both cells have a F plasmid
• F plasmids can have other genes on them too, for
  example antibody resistance containing genes

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Hfr Conjugation

• High frequency recombination (Hfr) donors contain
  the F factor in the chromosome
• Donor gives part of its chromosome to the
  recipient
• This transfers more genes to the recipient
  bacteria
• Very fast evolution for the recipient!

Donor Hfr cell
Partial copy of donor chromosome
Bridge broken
Integration of F factor into chromosome
Pilus
Donated genes
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Transformation

• Occurs when naked DNA fragments of one bacteria
  are close to another living cell.
• Some bacteria have the ability to pick up naked
  DNA fragments and recombine the DNA into their
  own DNA
• The new recombinant cell now has some new DNA
  from the disintegrating cell.
• The now transformed bacteria could have just
  picked up a new virulence factor or antibody
  resistance

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Griffiths Classic Experiment to Test
Transformation Principle
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Mechanism of Transduction

• Virus mediated gene transfer
• The virus injects its genetic material into the
  bacteria
• The bacterial DNA is fragmented

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Mechanism of Transduction

• Viral particles are produced by the bacteria
• When the cell lyses, the viral particles which
  have picked up DNA from the original bacterial
  cell now insert that DNA into a new cell.
• The new cell may or may not insert the new DNA
  sequence into its chromosome.
• Transduction can be a problem when the inserted
  DNA codes for an antibiotic resistance gene.

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Transformation and Transduction in Research
A way to get the genes you want to work with into
bacteria. Used in all types of molecular
genetics research
Electroporation
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Transposons

• Transposons-
• Small segments of DNA that can move (be
  transposed) from one region of a DNA molecule to
  another.
• jumping genes
• Involved in
• Changes in traits such as colony morphology,
  pigmentation, and antigenic characteristics
• Replacement of damaged DNA
• Intermicrobial transfer of drug resistance (in
  bacteria)

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Genes Evolution

• Genes are continually altered due to mutation,
  recombination, and transposition
• These changes increase genetic diversity of the
  gene pool and then through natural selection
  adventitious genes may be selected for to ensure
  survival in many different habitats.
• For pathogens that means they are more virulent!