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Lecture 4:Microbial genetics, biotechnology, and recombinant DNA Edith Porter, M.D. 1 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 2 3 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 4 A gene is a specific sequence of nucleotides along the DNA strand Consists of a promotor, coding and terminator region Promoter Binds RNA-polymerase 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) 5 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 6 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 Histones in eukaryotes Histone-like proteins in prokaryotes Ciprofloxacin: Gyrase inhibitor 7 8 9 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 10 Origin may be attached to the cell membrane 11 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 the terminator sequence 12 Protein synthesis Nucleotide language encoded within mRNA is translated into amino acid language mRNA is translated in codons 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 13 In bacteria, first amino acid is always formyl methionine 14 Elongation is target for many bacterial toxins and antibiotics! 15 Usually a number of ribosomes are attached to one mRNA molecule Multiple protein copies from one mRNA molecule 16 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 17 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 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 18 An operon consists of promoter, operator and the associated structural genes that need to be regulated 19 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 20 21 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 22 23 Mutations Gene transfer and recombination 24 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 25 26 Increased antibiotic resistance or loss of antibiotic resistance Increased pathogenicity or loss of pathogenicity 27 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) Physical UV- radiation ▪ Thymine dimerization 28 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 29 30 31 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 32 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 33 34 35 36 37 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) 38 39 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 40 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 41 42 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 43 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 44 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 45 46 Marker Genes Beta-galactosidase Restriction Enzyme Sites Ampicillin Resistance Vector Name Origin of Replication for Independent Replication 47 Beta-galactosidase inactivated 48 Agar with Ampicillin and X-gal (substrate for beta-galactosidase) 49 DNA can be inserted into a cell by: Transformation (naked DNA in solution) Transduction (via virus) Electroporation Gene gun ▪ DNA coated gold bullets Microinjection 50 DNA fingerprinting PCR reaction 51 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) 52 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 53 Original DNA (purified or cDNA made from RNA via reverse transcription) DNA polymerase taq polymerase ▪ From thermophile bacterium Thermus aquaticus ▪ Heat stable, functions at ~ 72C Primers (complementary short nucleotide sequences matching the beginning/end of DNA of interest) Nucleotides Appropriate buffer Thermocycler 54 1. Denaturing by heat 2. Separate DNA strands at ~ 95C Annealing 3. Primers attach at ~50– 60C Extension Polymerase extends DNA strand at ~72C 55 In clinical diagnostics Organism is hard or not to culture Very low numbers of organism are present In research 56 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) 57 58 Virus speci PCR results of patient samples 1: bp size ladder; 2:negative control; 3-8: patient samples 59 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 60 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