Lecture 4 microbial genetics biotechnology and recombinant dna edith porter m d
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Lecture 4:Microbial genetics, biotechnology, and recombinant DNA Edith Porter, M.D. MICR 201 Microbiology for Health Related Sciences. Lecture Outline. Microbial genetics Genotype and phenotype DNA and chromosomes Flow of genetic information DNA replication, RNA and Protein synthesis

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Lecture 4 microbial genetics biotechnology and recombinant dna edith porter m d

Lecture 4:Microbial genetics, biotechnology, and recombinant DNA

Edith Porter, M.D.

MICR 201 Microbiology for Health Related Sciences


Lecture outline
Lecture Outline

  • Microbial genetics

    • 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



Genetics
Genetics

  • 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


Genes are composed of dna
Genes are composed of DNA

  • A gene is a specific sequence of nucleotides along the DNA strand

  • Consists of a promotor, coding and terminator region

  • 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)

Promoter

Coding region

Terminator

Binds RNA-polymerase Indicates end of gene


What is dna
What is DNA?

  • 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


Supercoiling is required to package the entire dna into a cell
Supercoiling is required to package the entire DNA into a cell

  • 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)

  • Proteins to stabilize

    • Histonesin eukaryotes

    • Histone-like proteins in prokaryotes

Ciprofloxacin:

Gyrase inhibitor



Important enzymes for dna replication and repair
Important enzymes for DNA replication and repair


Dna replication
DNA replication

  • 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

    • In eukaryotes: uni-directional

    • In prokaryotes: circular genome and bi-directional replication


Bidirectional dna replication in prokaryotes
Bidirectional DNA replication in prokaryotes

Origin may be attached to the cell membrane


Transcription
Transcription

  • 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' 3' direction

  • Transcription stops when it reaches theterminator sequence


Translation
Translation

The universal (degenerative) genetic code

  • 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


Protein synthesis 1
Protein synthesis (1)

In bacteria, first amino acid is always formylmethionine


Protein synthesis 2
Protein synthesis (2)

Elongation is target for many bacterial toxins and antibiotics!


Protein synthesis 3
Protein synthesis (3)

  • Usually a number of ribosomes are attached to one mRNA molecule

  • Multiple protein copies from one mRNA molecule


Differences between eukaryotic and prokaryotic transcription and translation
Differences between eukaryotic and prokaryotic transcription and translation

  • 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 3

Gene 2


Bacterial gene regulation
Bacterial gene regulation and translation

  • 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

  • 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

Promoter

Coding region

Terminator

Binds RNA-polymerase Indicates end of gene


Structure of a bacterial operon
Structure of and translationa bacterial operon

  • An operon consists of promoter, operator and the associated structural genes that need to be regulated


Gene induction
Gene induction and translation

  • 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


Inducible gene regulation lactose operon
Inducible and translationgene regulation: lactose operon


Gene repression
Gene repression and translation

  • 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


Repressible gene regulation tryptophan operon
Repressible and translationgene regulation: tryptophan operon


Change in the genetic material
Change in the genetic material and translation

  • Mutations

  • Gene transfer and recombination


Mutations
Mutations and translation

  • 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

  • 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

  • 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


Types of mutations
Types of mutations and translation


Consequence of mutations in the microbial world
Consequence of mutations in the microbial world and translation

  • Increased antibiotic resistance or loss of antibiotic resistance

  • Increased pathogenicity or loss of pathogenicity


Mutagens
Mutagens and translation

  • 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 (Aspergillusflavus toxin)

  • Physical

    • UV- radiation

      • Thymine dimerization


Use of bacteria to detect m utagens
Use of and translationbacteria to detect mutagens

  • 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

  • Assay performed with addition of liver extract

    • Some mutagens are only formed after metabolisation by liver


Auxotrophic mutants
Auxotrophic and translation mutants


The ames test
The Ames Test and translation


Genetic transfer and recombination
Genetic transfer and recombination and translation

  • 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


Different types of horizontal dna uptake
Different and translationtypes of horizontal DNA uptake

  • Transformation

    • Uptake of naked DNA

  • Conjugation

    • Plasmid uptake through Sex-Pili

    • Requires cell to cell contact and two mating types

  • Transduction

    • Uptake of foreign DNA through a bacteriophage


Transformation first description by griffith in 1928
Transformation: first description by and translationGriffith in 1928


Transformation
Transformation and translation


Conjugation
Conjugation and translation


Transduction
Transduction and translation


Important to remember
Important to Remember and translation

  • 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)



Biotechnology and recombinant dna1
Biotechnology and Recombinant DNA and translation

  • Biotechnology: the use of microorganisms, cells, or cell components to make a product that is not naturally produced

    • Foods, antibiotics, vitamins, enzymes

  • Recombinant DNA technology: insertion or modification of genes to produce desired proteins


Recombinant dna technology
Recombinant DNA Technology and translation

  • 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



Important tools to generate r ecombinant dna
Important and translationtools to generate recombinant DNA

  • 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


Vectors in recombinant dna technology
Vectors in and translationrecombinant DNA technology

  • Self replicating DNA

  • Must not be destroyed by recipient cell

    • 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


Cloning
Cloning and translation

  • 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



Puc19 plasmid vector used for cloning in e coli
pUC19: Plasmid Vector used for Cloning in and translationE. coli

Beta-galactosidase

Marker

Genes

Restriction

Enzyme Sites

Ampicillin

Resistance

Vector Name

Origin of Replication for Independent Replication


Introduction of foreign dna into upc19
Introduction of and translationforeign DNA into uPC19

Beta-galactosidase

inactivated


Detection and translation of recombinant clones

Agar with Ampicillin and X-gal (substrate for beta-galactosidase)


Inserting foreign dna into cells
Inserting Foreign DNA into Cells and translation

  • DNA can be inserted into a cell by:

    • Transformation (naked DNA in solution)

    • Transduction (via virus)

    • Electroporation

    • Gene gun

      • DNA coated gold bullets

    • Microinjection


Technical applications of biotechnology
Technical and translationapplications of biotechnology

  • DNA fingerprinting

  • PCR reaction


Dna fingerprinting
DNA Fingerprinting and translation

  • 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)


The polymerase chain r eaction pcr
The and translationpolymerase chain reaction (PCR)

  • 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


The players in a pcr reaction
The and translationplayers in a PCRreaction

  • Original DNA (purified or cDNA made from RNA via reverse transcription)

  • DNA polymerase

    • taq polymerase

      • From thermophile bacterium Thermusaquaticus

      • Heat stable, functions at ~ 72C

  • Primers (complementary short nucleotide sequences matching the beginning/end of DNA of interest)

  • Nucleotides

  • Appropriate buffer

  • Thermocycler


Simplified pcr reaction
Simplified and translationPCRreaction

  • Denaturing by heat

    • Separate DNA strands at ~ 95C

  • Annealing

    • Primers attach at ~50– 60C

  • Extension

    • Polymerase extends DNA strand at ~72C


Applications of pcr
Applications of PCR and translation

  • In clinical diagnostics

    • Organism is hard or not to culture

    • Very low numbers of organism are present

  • In research


Therapeutic applications of biotechnology
Therapeutic and translationapplications of biotechnology

  • 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)


  • Mode of action of sirna
    Mode of Action of siRNA and translation


    Case study norovirus outbreak
    Case study: and translationNorovirus outbreak

    Virus specific

    PCR results of patient samples

    1: bp size ladder; 2:negative control;

    3-8: patient samples


    Important to remember1
    Important to Remember and translation

    • 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


    Topic 5 products of genetic engineering
    Topic 5: Products of Genetic and translationEngineering

    • The method of using RFLPs to identify

    • bacterial or viral pathogens is called

    • a. Proteomics

      • b. DNA fingerprinting

      • c. Genetic screening

      • d. DNA sequencing


    Topic 4 applications of rdna
    Topic 4: and translationApplications of rDNA

    • 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


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