Lecture 4:Microbial genetics, biotechnology, and recombinant DNA
Edith Porter, M.D.
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Microbial genetics
 Genotype and phenotype
 DNA and chromosomes
 Flow of genetic information
 DNA replication, RNA and Protein synthesis
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Bacterial gene regulation
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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 Coding region Terminator
Binds RNA-polymerase 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 m m, the average cell ~
2-4 m m
Eukaryotic DNA ~ 1.8 m (= 1,800,000 m m), the average cell ~ 15-30 m m
Supercoiling
Requires special enzymes to
 Supercoil
 Relax supercoiling (topoisomerases; e.g. gyrase in prokaryotes)
 Unwind (helicases)
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/ 10 10 bases!
Direction
 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
Transcription begins when RNA polymerase binds to the promotor sequence
Transcription proceeds in the 5'
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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
 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
The universal (degenerative) genetic code
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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)
In prokaryotes exons only
 Exceptions: archaea and cyanobacteria
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 Coding region Terminator
Binds RNA-polymerase Indicates end of gene
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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
When needed
 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
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 1/10 9 replicated base pairs
Lead to permanent changes in genotype
 If coupled to changes in proteins with altered function: changes in phenotype
Base substitutions (point mutations) can lead to
 Missense: one amino acid change with major consequences
▪ A
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T leads to glutamic acid
 valine in hemoglobin: sickle cell disease
 Nonsense: can lead to stop of transcription
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
10 9 replicated base pairs (or in 10 6 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)
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
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 Can grow in the absence of this nutrient
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
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
Conjugation
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Plasmid uptake through Sex-Pili
 Requires cell to cell contact and two mating types
Transduction
Uptake of foreign DNA through a bacteriophage
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DNA replication DNA  DNA
 In bacteria, bi-directional
Transcription: DNA  RNA
Translation: RNA  Protein
 In bacteria, transcription and translation occur simultaneously
Bacterial gene regulation utilizes operons
 Inducible genes
 Repressible genes
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)
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
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 Foods, antibiotics, vitamins, enzymes
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
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”
Ligases to join the DNA fragments
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Self replicating DNA
Must not be destroyed by recipient cell
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Circular DNA like plasmids
 Virus which is rapidly integrated into host genome
Vectors contain marker genes
Tag to identify vector
 Often antibiotic resistance genes or enzyme carrying out easily identifiable reactions
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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Ampicillin
Resistance
Marker
Genes
Beta-galactosidase
Restriction
Enzyme Sites
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
 taq polymerase
▪ From thermophile bacterium Thermus aquaticus
▪ Heat stable, functions at ~ 72
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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
 Separate DNA strands at ~ 95
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C
2.
Annealing
 Primers attach at
~50– 60
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C
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Extension
 Polymerase extends
DNA strand at ~72
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C
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In clinical diagnostics
 Organism is hard or not to culture
 Very low numbers of organism are present
In research
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Subunit vaccines against infectious diseases
▪ HPV (virus coat)
Gene therapy
 Introducing functional genes into defective genome
 Gene silencing via inhibitory RNA (short interfering RNA, double stranded)
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Virus specific
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
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
 b. DNA fingerprinting
 c. Genetic screening
 d. DNA sequencing

 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