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Bacterial genetics and applications


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Bacterial genetics and applications

Bacterial genetics and applications

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  • 1. Bacterial Genetics and Applications Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  • 2. Gene Transfer Processes for Bacteria and Their Viruses 1. Conjugation 2. Transformation 3. Transduction 4. Infection with bacteriophage Dr.T.V.Rao MD 2
  • 3. Conjugation Lederberg - Tatum • A process by which a Donor cell or male cell makes contact with another cell, the recipient or Female cell. • DNA is directly transferable • Plasmid Carry genetic information necessary for conjugation to occur. • Only cell that contain such plasmids can act as donor. the cell lacking a corresponding plasmid act as recipient. • Requires direct contact between donor and Dr.T.V.Rao MD 3 recipient
  • 4. Conjugation - Transferring genes with plasmids • Plasmids mediating conjugation carry genes coding for properties, of 1-2 microns long protein appendage termed Pilus on the Donor cell Dr.T.V.Rao MD 4
  • 5. Mechanism of Transfer I: Conjugation Dr.T.V.Rao MD 5
  • 6. Conjugation Dr.T.V.Rao MD 6
  • 7. Simple Conjugation Dr.T.V.Rao MD 7
  • 8. Conjugation Dr.T.V.Rao MD 8
  • 9. Pilus helps Conjugation • Different types of Pilus are specified by different types of plasmids and can help in aid of plasmid classification. • Only one strand of circular DNA of the plasmid nicked upon at a specific site and passed into a recipient. • Spread to all other cells. Dr.T.V.Rao MD 9
  • 10. F factor • Transfer factor that contains the genetic information necessary for synthesis of Sex Pilus and for self transfer without any other identifiable genetic materials such as drug resistance Dr.T.V.Rao MD T.V.Rao MD 10
  • 11. F factor helps transformation • F+ called as Donor bacteria can transform F- into F+ cell Can be Episomes able to exist in some cells in the integrated state in the donor cell chromosome Can transform chromosomal genes to recruitment with high frequency are known as Hfr cells Conversion of F+ cells into Hfr state is reversible. F factor incorporates some chromosomal genes and is called as F’ Sexduction The process of transfer of host genes Dr.T.V.Rao MD through F’ factor 11
  • 12. DNA transfer through Bacteriophages • When the Phage particle infects another bacteria DNA transfer is effected and the recipient cell acquires new characters coded by donor DNA Dr.T.V.Rao MD 12
  • 13. Types of DNA transfer through Bacteriophages Dr.T.V.Rao MD 13
  • 14. Colicinogenic ( Col ) Factor • Coliform Bacteria produce Colicins • Colicins are lethal to other Enterobacteriaceae • Pyocins produce by Pseudomonas • Diptherocins produced by C.diptheria • Plasmid transmits col factor leads to self transfer of chromosomal segments T.V.Rao MD Dr.T.V.Rao MD 14
  • 15. Resistance Transfer Factor RTF • Plasmids – helps to spread multiple drug resistance • Discovered in 1959 Japan • Infections caused due to Shigella spread resistance to following Antibiotics Sulphonamides Streptomycin Chloramphenicol, Tetracycline Dr.T.V.Rao MD 15
  • 16. RTF • Shigella + E.coli excreted in the stool resistant to several drugs in vivo and vitro • Plasmid mediated – transmitted by Conjugation • Episomes spread the resistance Dr.T.V.Rao MD 16
  • 17. Bacterial Conjugation: High Frequency Transfer (Her) Cells Dr.T.V.Rao MD 17
  • 18. Hfr Conjugation Dr.T.V.Rao MD 18
  • 19. Sequence of RTF transmission Dr.T.V.Rao MD 19
  • 20. Hfr cell conjugating a Normal cell Dr.T.V.Rao MD 20
  • 21. Composition of RTF • Plasmid consists of two components • A transfer factor RT, helps conjugational transfer and resistant determinants ( r ) to each of the several drugs • RTF + r determinants are known as R factor Dr.T.V.Rao MD 21
  • 22. R factor • R factor can contain several determinants as many as 8 or > 8 drugs • Guide the cell for production of Enterotoxins too • But R factors can be inhibited by Bile salts R factors can be transferred to animals Dr.T.V.Rao MD 22
  • 23. Genesis of R factors • In discriminate use of Antibiotics in vet nary Medicine has increased the spread of R factors to Human • Addition of Antibiotics to Animal feeds to be prohibited. Dr.T.V.Rao MD 23
  • 24. Genetic Mechanisms of Drug Resistance • Bacteria acquire drug resistance through several Mechanisms • Mutations • Genetic transfer Transformation, Transduction Conjugation Several Biochemical Mechanisms Decreasing permeability of drugs, Attaining alternative pathways Produce enzymes and inactivate drugs Dr.T.V.Rao MD 24
  • 25. Genetic Mechanisms in Bacteria helps to Spread the Infectious diseases Dr.T.V.Rao MD 25
  • 26. Mutations • Mutilations can be 1 Stepwise mutation as in Penicillin use 2 One step mutation Streptomycin use May show low resistance or High resistance If tuberculosis is treated with sole drug as of Only Streptomycin some resistant mutants appear and replaces sensitive bacteria in due course so the occurrence of MDR - TB Dr.T.V.Rao MD 26
  • 27. Other Mechanisms • Use of Penicillin created resistant Staphylococcus by transduction • R factors created resistance to several drugs, caused increased virulence • Spread to several humans and animals Best option- To restrict use of Antibiotics Dr.T.V.Rao MD 27
  • 28. Transposable Genetic Elements Structurally / Genetically – Discrete sequence of DNA – Move around in a cut and paste manner between Chromosomal and Extra chromosomal DNA molecules within cells. Called as Transposons _ Jumping Genes Genetic transfer due to Transposition Small Transposons 1 – 2 Kb Not self replicating and depend on Plasmid or Chromosome for replication. A chunk of DNA is added by Transposons. Dr.T.V.Rao MD 28
  • 29. Transposons and R factor • R forms may have evolved as a collection of Transposons • Each carrying Genes that confers resistance to one or several Antibiotics • Seen in Plasmids, Microorganisms Animals Laboratory Manipulations are called as Genetic Engineering Dr.T.V.Rao MD 29
  • 30. Molecular Genetics • Analysis and manipulation of DNA using Biochemical and Microbiological techniques Dr.T.V.Rao MD 30
  • 31. Genetic Engineering • Under standing Molecular genetics in Biochemistry fuels genetic Engineering • Recombinant DNA (renal) techniques changed the ideals of Medicine • Genetic Engineering await many surprises? Dr.T.V.Rao MD 31
  • 32. Genetic Engineering Genetic Engineering Was Born from Genetic Recombination •Genetic engineering involves changing the genetic material in an organism to alter its traits or products •A recombinant DNA molecule contains DNA fragments spliced together from 2 or more organisms Dr.T.V.Rao MD 32
  • 33. Modern applications • Pharmaceutical production –Insulin, interferon, hormones, vaccines etc. • Genetically engineered plants • Animal gene alterations • Gene probes • DNA fingerprinting • The human genome initiative Dr.T.V.Rao MD 33
  • 34. Genetic Engineering • Isolation of Genes coding for any desired protein from Microorganism or from cell of higher life forms including human beings and their introduction into a suitable microorganism in which genes would function directing the production of specific proteins Dr.T.V.Rao MD 34
  • 35. Genetic Engineering changing the Diagnostic and Therapeutic Protocols in MEDICINE Dr.T.V.Rao MD 35
  • 36. Research on Gene transfer shapes the future of Science Dr.T.V.Rao MD 36
  • 37. Genetically Engineered Products • Can prepare desired protein in pure form in economic way Somatostatin • Commercial preparations pdf Cloned Human Insulin Interferons Hepatitis B vaccine Dr.T.V.Rao MD 37
  • 38. Restriction Endonucleases • A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i.e., each strand) of the double helix without damaging the nitrogenous bases They work with cutting up foreign DNA, a process called Dr.T.V.Rao MD 38
  • 39. Restriction Endonucleases Made the advances in Genetic Engineering Dr.T.V.Rao MD 39
  • 40. DNA Probes • There are Radioactive Biotinylated otherwise labeled copies united single stranded DNA Contains 20 -25 nucleotides Helps detection of Homology DNA by Hybridization. Helps Diagnosis of Infectious Diseases Minute quantities of DNA can be detected. Dr.T.V.Rao MD 40
  • 41. Blotting Techniques • Drug fragments obtained by restriction enzyme digestion on separation Gel can be transferred to Nitrocellulose or nylon membranes • Several methods 1 Southern blotting 2 Northern Blotting 3 Western blotting Dr.T.V.Rao MD 41
  • 42. western blot • The western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions) Dr.T.V.Rao MD 42
  • 43. Western Blotting • In Western Blot Protein ( Antigen ) mixture is separated by SDS ( Sodium dodecyl sulfate – polyacrylamide gel electrophoresis ) Blotted on to Nitro cellulose strips and identified by radio labeled or enzyme labeled antibodies as probes Dr.T.V.Rao MD 43
  • 44. Western Blot Dr.T.V.Rao MD 44
  • 45. Western Blot to confirm HIV Infections made land mark Diagnostic tool • Western Blot testing is confirmatory test for diagnosis of HIV/AIDS • Identifies antibodies directed against different antigens in pathogen Surface, Core Dr.T.V.Rao MD RT antigen 45
  • 46. Polymerase chain reaction Kary B Mullis 1983 • Rapid • Automatic amplification of specific DNA sequences • Nobel prize winning Technology 1993 Dr.T.V.Rao MD 46
  • 47. Polymerase chain reaction (PCR) • The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Dr.T.V.Rao MD 47
  • 48. Polymerase chain reaction (PCR) • Developed in 1983 by Kary Mullis,PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR. Dr.T.V.Rao MD 48
  • 49. PCR -Sequences • PCR consists of several cycles of sequential DNA replication where the products of first cycle becomes the template for the Next • It makes available abundant quantities of specific DNA sequences starting Dr.T.V.Rao MD 49
  • 50. Genetic Mapping • Genetic sequences for Bacteriophages and virus • Genetic mapping is done most of the Human Genes Dr.T.V.Rao MD 50
  • 51. Newer Understanding on GENES Dr.T.V.Rao MD 51
  • 52. Human Genome Project Dr.T.V.Rao MD 52
  • 53. Human Genome Project • Completed in 2003, the Human Genome Project (HGP) was a 13-year project coordinated by the U.S. Department of Energy and the National Institutes of Health. During the early years of the HGP, the Welcome Trust (U.K.) became a major partner; additional contributions came from Japan, France, Germany, China, and others Dr.T.V.Rao MD 53
  • 54. Genetics are Complex - Leading the birth of BIOINFORMATICS Dr.T.V.Rao MD 54
  • 55. Genes Evolved and made us Men What NEXT ? Dr.T.V.Rao MD 55
  • 56. Understanding of human Genome is Changing the Future of Medicine Dr.T.V.Rao MD 56
  • 57. • The Programme Created by Dr.T.V.Rao MD for Medical Students in the Developing World • Email • Dr.T.V.Rao MD 57