MICR 201 Microbiology for Health Related Sciences

1. Lecture 4:Microbial genetics, biotechnology, and recombinant DNA Edith Porter, M.D. MICR 201 Microbiology for Health Related Sciences

2. 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

3. Microbial Genetics

4. 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

5. 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

6. 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

7. 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

8. The flow of genetic information

9. Important enzymes for DNA replication and repair

10. 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

11. Bidirectional DNA replication in prokaryotes Origin may be attached to the cell membrane

12. 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

13. 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

14. Protein synthesis (1) In bacteria, first amino acid is always formylmethionine

15. Protein synthesis (2) Elongation is target for many bacterial toxins and antibiotics!

16. Protein synthesis (3) • Usually a number of ribosomes are attached to one mRNA molecule • Multiple protein copies from one mRNA molecule

17. 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

18. Bacterial gene regulation • 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

19. Structure of a bacterial operon • An operon consists of promoter, operator and the associated structural genes that need to be regulated

20. Gene induction • 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

21. Inducible gene regulation: lactose operon

22. Gene repression • 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

23. Repressible gene regulation: tryptophan operon

24. Change in the genetic material • Mutations • Gene transfer and recombination

25. Mutations • 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

26. Types of mutations

27. Consequence of mutations in the microbial world • Increased antibiotic resistance or loss of antibiotic resistance • Increased pathogenicity or loss of pathogenicity

28. Mutagens • 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

29. Use of bacteria 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

30. Auxotrophic mutants

31. The Ames Test

32. Genetic transfer and recombination • 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

33. Different types 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

34. Transformation: first description by Griffith in 1928

35. Transformation

36. Conjugation

37. Transduction

38. Important to Remember • 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)

39. Biotechnology and recombinant DNA

40. Biotechnology and Recombinant DNA • 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

41. Recombinant DNA Technology • 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

42. A Typical Genetic Modification Procedure (1)

43. Important tools 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

44. Vectors in recombinant 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

45. Cloning • 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

46. A Closer Look at Restriction Enzymes

47. pUC19: Plasmid Vector used for Cloning in E. coli Beta-galactosidase Marker Genes Restriction Enzyme Sites Ampicillin Resistance Vector Name Origin of Replication for Independent Replication

48. Introduction of foreign DNA into uPC19 Beta-galactosidase inactivated

49. Detection of recombinant clones Agar with Ampicillin and X-gal (substrate for beta-galactosidase)

50. Inserting Foreign DNA into Cells • DNA can be inserted into a cell by: • Transformation (naked DNA in solution) • Transduction (via virus) • Electroporation • Gene gun • DNA coated gold bullets • Microinjection