Download MICR 201 Microbiology for Health Related Sciences

Lecture 4:Microbial genetics, biotechnology, and recombinant DNA
Edith Porter, M.D.
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Microbial genetics
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Genotype and phenotype
DNA and chromosomes
Flow of genetic information
DNA replication, RNA and Protein synthesis
Bacterial gene regulation
Mutations
Gene transfer and recombination
Biotechnology and recombinant DNA
 Recombinant DNA technology
 Techniques in gene modification
 Applications or recombinant DNA
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Science of heredity
Study of genes, how genes
carry information, how
genes can be transferred,
how the expression of the
encoded information is
regulated, how genes render
specific characteristics to the
organism that harbors these
genes
 Genotype: collection of
genes
 Phenotype: collection of
proteins encoded by these
genes
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A gene is a specific sequence of nucleotides along
the DNA strand
Consists of a promotor, coding and terminator
region
Promoter
Binds RNA-polymerase
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Coding region
Terminator
Indicates end of gene
A gene can code for
 mRNA (used to make proteins from amino acids at
ribosomes)
 rRNA (synthesized in the nucleolus in eukaryotes)
 tRNA (brings specific single amino acids to the ribosomes)
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Sequence of nucleotides
 Base: Adenine, thymine,
cytosine, and guanine
 Deoxyribose
 Phosphate
Double helix associated with
proteins
 Strands held together by
hydrogen bonds between
AT and CG
 Strands antiparallel
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E. coli DNA ~ 1300 mm, the average cell ~
2-4 mm
Eukaryotic DNA ~ 1.8 m (= 1,800,000 mm),
the average cell ~ 15-30 mm
Supercoiling
Requires special enzymes to
 Supercoil
 Relax supercoiling (topoisomerases; e.g. gyrase
in prokaryotes)
 Unwind (helicases)
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Proteins to stabilize
 Histones in eukaryotes
 Histone-like proteins in prokaryotes
Ciprofloxacin:
Gyrase inhibitor
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Transfer of the genetic information to the next generation
 1 strand remains the parent strand, 1 strand is newly
synthesized
 Mistakes only in 1/ 1010 bases!
 Direction
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 In eukaryotes: uni-directional
 In prokaryotes: circular genome and bi-directional replication
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Origin may be
attached to the
cell membrane
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To copy DNA into RNA (synthesis of complimentary strand of
RNA from a DNA template)
RNA consists of base ribose and phosphate, single stranded
 Messenger RNA (mRNA)
▪ Information for proteins
▪ Thymine replaced with uracil
 Transfer RNA (tRNA): carries single specific amino acid residues
▪ Thymine in tRNA in eukaryotes and bacteria
▪ No thymine in archaea in tRNA
 Ribosomal RNA (rRNA): assists mRNA in binding to the ribosome
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Transcription begins when RNA polymerase binds to the
promotor sequence
Transcription proceeds in the 5'  3' direction
Transcription stops when it reaches the
terminator sequence
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Protein synthesis
Nucleotide language encoded
within mRNA is translated into
amino acid language
 mRNA is translated in codons
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The universal (degenerative) genetic code
 One codon consists of three
nucleotides
 One codon codes for one amino acid
Translation of mRNA begins at the
start codon: AUG
 Translation ends at a stop codon:
UAA, UAG, UGA
 tRNA has anticodons
complementary to the mRNA
codons
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In bacteria, first amino acid is always formyl methionine
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Elongation is
target for many
bacterial toxins
and antibiotics!
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Usually a number of
ribosomes are attached
to one mRNA molecule
 Multiple protein copies
from one mRNA
molecule
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Different enzymes
In eukaryotes exons, introns, repetitive sequences
 Introns are transcribed but not translated nucleotide sequences
 Cut out by ribozymes (RNA with enzymatic activity)
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In prokaryotes exons only
 Exceptions: archaea and cyanobacteria
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In eukaryotes mRNA must exit nucleus and therefore must
be completed before translation can begin
In prokaryotes simultaneous transcription and translation
Gene overlap
 Never in eukaryotes, sometimes in prokaryotes, often in viruses
Gene 1
Gene 2
Gene 3
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Of all genes 60 – 80% are constitutive (always
expressed)
20 – 40% are regulated (expressed only when
needed)
One form of gene regulation is negative regulation
by means of operators and repressors inserted
between the promoter and coding gene region
Promoter
Binds RNA-polymerase
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Coding region
Terminator
Indicates end of gene
RNA-polymerase cannot bind to promoter or
cannot proceed when operator is occupied by
repressor
The unit consisting of a promoter, operator and the
structural gene is called operon
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An operon consists of promoter, operator and
the associated structural genes that need to
be regulated
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During base line metabolism
 Operator is occupied by an active repressor
 Gene is turned off
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When needed
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Inducer binds to active repressor
Repressor is inactivated
Repressor cannot bind anymore to operator
RNA –polymerase can bind to promoter and proceed with
transcription
 Gene is turned on
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During base line metabolism constant need of
gene product
 Operator is not occupied by a repressor
 Inactive repressor cannot bind to operator
 RNA–polymerase binds to promoter and proceed with
transcription
 Gene is turned on
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When gene product is not needed anymore
 Co-repressor (typically the gene product) binds to the
inactive repressor
 Repressor is activated
 Now repressor can bind to operator
 Gene is turned off
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Mutations
Gene transfer and recombination
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Not-corrected errors during DNA replication
Occur spontaneously rarely at 1/109 replicated base pairs
Lead to permanent changes in genotype
 If coupled to changes in proteins with altered function: changes in
phenotype
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Base substitutions (point mutations) can lead to
 Missense: one amino acid change with major consequences
▪ A T leads to glutamic acid  valine in hemoglobin: sickle cell disease
 Nonsense: can lead to stop of transcription
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Deletion or insertion of a few base pairs
 Frame shift mutation: shift translational reading frame, major
alterations in amino acid sequence, almost always dysfunction protein
results
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Increased antibiotic resistance or loss of
antibiotic resistance
Increased pathogenicity or loss of
pathogenicity
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Natural mutation rate is ~ 1 in
109 replicated base pairs (or in 106
replicated genes)
Mutagens increase the rate of
mutations by factor 10 – 1000
Chemical
 Point mutations
▪ Nitrous acid
▪ Nucleosid analogs
 Frame shift mutations
▪ Benzpyrene (smoke)
▪ Aflatoxin (Aspergillus flavus toxin)
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Physical
 UV- radiation
▪ Thymine dimerization
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Auxotrophic mutants
Cannot grow without the presence of a
particular nutrient, e.g. histidine
When exposed to mutagens development of
revertants
 Can grow in the absence of this nutrient
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Assay performed with addition of liver extract
 Some mutagens are only formed after
metabolisation by liver
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Vertical transfer
 Passing genes to off springs
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Horizontal transfer
Passing genes laterally to representatives of
the same generation
Donor cell passes genes which will be
integrated into recipient’s DNA
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Transformation
 Uptake of naked DNA
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Conjugation
 Plasmid uptake through Sex-Pili
 Requires cell to cell contact and two mating
types
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Transduction
 Uptake of foreign DNA through a
bacteriophage
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DNA replication DNA  DNA
 In bacteria, bi-directional
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Transcription: DNA  RNA
Translation: RNA  Protein
 In bacteria, transcription and translation occur simultaneously
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Bacterial gene regulation utilizes operons
 Inducible genes
 Repressible genes
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Mutations are permanent, inheritable changes of the genetic
informati0n
 Missense (protein with altered amino acid sequence may result)
 Nonsense (protein synthesis is aborted)
 Frameshift (entirely different protein results)
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Mutagens increase the frquency of mutations
Genetic transfer and recombination can be achieved by
 Transformation (uptake of naked DNA)
 Conjugation (uptake via cell to cell contact and sex pili)
 Transduction (genetic exchange via a bacteriophage)
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Biotechnology: the use of microorganisms,
cells, or cell components to make a
product that is not naturally produced
 Foods, antibiotics, vitamins, enzymes
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Recombinant DNA technology: insertion
or modification of genes to produce
desired proteins
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Genetic engineering
Technique for artificial DNA recombination
Examples:
 Higher vertebrate genes (animal including human)
inserted into a bacterial genome
▪ Human growth hormone gene inserted into E. coli
 Viral gene into yeast cells
▪ Hepatitis B gene inserted into yeast cells for vaccine production
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DNA with the gene of interest
 Selection
 Mutation
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Vector DNA
Restriction enzymes
 Discovered when studying viruses
▪ Some bacteria can degrade viruses with these enzyme and are
protected against these viruses
 Cut at certain nucleotide sequences
▪ Recognize 4, 6, or 8 base pairs
▪ Produce “sticky ends”
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Ligases to join the DNA fragments
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Self replicating DNA
Must not be destroyed by recipient cell
 Circular DNA like plasmids
 Virus which is rapidly integrated into host genome
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Vectors contain marker genes
 Tag to identify vector
 Often antibiotic resistance genes or enzyme carrying out easily
identifiable reactions
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Can be used for cloning
Shuttle vectors
 Can exist in several different species
▪ Bacteria, yeasts, mammals
▪ Bacteria, fungi, plants
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To make numerous (unlimited) identical
copies of one original
Cell cloning: 1 single cell multiplied
Gene cloning: 1 single gene is inserted into
a vector and replicated as the vector is
replicated
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Marker
Genes
Beta-galactosidase
Restriction
Enzyme Sites
Ampicillin
Resistance
Vector Name
Origin of Replication
for Independent
Replication
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Beta-galactosidase
inactivated
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Agar with Ampicillin and
X-gal (substrate for
beta-galactosidase)
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DNA can be inserted
into a cell by:
 Transformation (naked
DNA in solution)
 Transduction (via virus)
 Electroporation
 Gene gun
▪ DNA coated gold bullets
 Microinjection
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DNA fingerprinting
PCR reaction
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Identical DNA will generate
identical DNA fragments
when subjected to
restriction enzyme
digestion
Subject DNA to agarose gel
electrophoresis and
compare DNA fragment
pattern (restriction
fragment length pattern)
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To quickly specifically amplify small samples
of DNA
From 1 copy to 1 billion copies within hours
 25 to 35 reaction cycles
 High specificity
 High sensitivity
 Not a functional assay
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Original DNA (purified or cDNA made from
RNA via reverse transcription)
 DNA polymerase
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 taq polymerase
▪ From thermophile bacterium Thermus aquaticus
▪ Heat stable, functions at ~ 72C
Primers (complementary short nucleotide
sequences matching the beginning/end of DNA
of interest)
 Nucleotides
 Appropriate buffer
 Thermocycler
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1.
Denaturing by heat
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2.
Separate DNA strands
at ~ 95C
Annealing
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3.
Primers attach at
~50– 60C
Extension
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Polymerase extends
DNA strand at ~72C
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In clinical diagnostics
 Organism is hard or not to culture
 Very low numbers of organism are present
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In research
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Subunit vaccines against infectious diseases
▪ HPV (virus coat)
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Gene therapy
 Introducing functional genes into defective
genome
 Gene silencing via inhibitory RNA (short
interfering RNA, double stranded)
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Virus speci
PCR results of patient samples
1: bp size ladder; 2:negative control;
3-8: patient samples
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Recombinant DNA technology
 Artificial DNA recombination between
unrelated species
 Insertion of new genes into cells
 Typically requires restriction enzymes and
vectors
 Cloning: to amplify a gene in another cell
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PCR (polymerase chain reaction)
 To specifically detect and amplify small
samples of DNA
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The method of using RFLPs to identify
bacterial or viral pathogens is called
a. Proteomics
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b. DNA fingerprinting
c. Genetic screening
d. DNA sequencing
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The use of an antibiotic resistance gene on a
plasmid used in genetic engineering makes
 Direct selection possible.
 The recombinant cell dangerous.
 Replica plating possible
 The recombinant cell unable to survive