Bacterialgenetics pptteaching-121228195540-phpapp01


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Bacterialgenetics pptteaching-121228195540-phpapp01

  1. 1. Bacterial Genetics Learning the Basics Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 2. Genetics Guide Life Dr.T.V.Rao MD 2
  3. 3. Genes are Eternal Run In Progeny Dr.T.V.Rao MD 3
  4. 4. Understanding Genetics We resemble and differ because of Genetic configurations Parents - Son - Daughter, how they resemble each other. They breed true from Generation to Generation But vary in small proportions in progeny. Bacteria too obey the laws of Genetics Dr.T.V.Rao MD 4
  5. 5. Watson - Crick Discovery of DNA Dr.T.V.Rao MD 5
  6. 6. Beginning of Bacterial Genetics • The principles of Genetics were applied to bacteria and viruses • Advances in Genetic process also of lead to fundamental advances in Biology and Biochemistry. A Birth of New Branch of Science Molecular Biology Dr.T.V.Rao MD 6
  7. 7. DNA A Complex Structure Makes Life Dr.T.V.Rao MD 7
  8. 8. DNA ( Deoxyribonucleic Acid ) • DNA is composed of Many Units of Adenine – Thymine A – T Guanine – Cytosine G - C A+ T G+C proportion differ for each species DNA replicates first unwinding at one end to form a fork Each strand of fork acting as template for the synthesis of complementary strand Dr.T.V.Rao MD 8
  9. 9. Dr.T.V.Rao MD 9
  10. 10. Structure of DNA Dr.T.V.Rao MD 10
  11. 11. DNA • A DNA molecule is composed of two chains of Nucleotides wound together in the form of a Double Helix • Each chain has back bone of Deoxyribose and Phosphates residues arranged alternatively Dr.T.V.Rao MD 11
  12. 12. Structure of DNA • Attached to each Deoxyribose and phosphate residues arranged alternatively • Attached to each Deoxyribose are of four nitrogen bases • Purines - Adenine, Guanine • Pyramidine Thymidine and Cytosine Dr.T.V.Rao MD 12
  13. 13. How RNA differs from DNA • RNA contains - Sugar Ribose instead of Deoxyribose • Uracil is present instead of Thymine • Types of RNA Messenger RNA mRNA Ribosomal RNA rRNA Transfer RNA tRNA Dr.T.V.Rao MD 13
  14. 14. DNA - RNA Dr.T.V.Rao MD 14
  15. 15. Knowledge on DNA lead to advances in Molecular Biology • Central dogma of Life – Deoxyribonucleic acid • DNA carries the Genetic information • DNA is transcribed to RNA – Polypeptides Cell Function depends upon specific polypeptides – Proteins – Enzymes DNA is a store house of Protein synthesis DNA acts a Template for synthesis of mRNA Virus differs from other as they contains either DNA or RNA Dr.T.V.Rao MD 15
  16. 16. What is a Code in Genetics • Code is a unit consists of sequence of three Bases • Code is triplet A-T- C • A code can make single Amino acid • More than one code present for making similar sequence of Amino acid • AGA make Arginine • AGC, CGU, CGG, also code for similar Amino acid • Some Codons UAA Dr.T.V.Rao MD don't code for any Amino acid called as Nonsense codon 16
  17. 17. What is a Gene • Gene is a sequence of DNA carrying codons specifying for particular polypeptide. • DNA contains many Genes( A combinations of hundreds and thousands of Nucleotides ) Dr.T.V.Rao MD 17
  18. 18. Bacterial Chromosome • Contains a Double stranded molecules of DNA arranged in circular form. • Length 1,ooo microns. • Bacterial DNA contains about 4,000kilobases • I kb = 1000 base pairs ( A-T ) ( G-C) • Humans have about 3,000 kb pairs. Dr.T.V.Rao MD 18
  19. 19. How bacterial Genome differs from Higher forms of Life • Several stretches of DNA don't appear to function as codons, occurs between the coding sequences of Gene. called as INTRONS. • Coded are called as EXONS • In transcription introns are excised when form RNA before translated by ribosomal proteins. Dr.T.V.Rao MD 19
  20. 20. Extra chromosomal Genetic Elements Bacteria posses Extra chromosomal genetic elements Not Essential for survival of Bacteria But makes the Bacteria Resistant to antibiotics, and makes them survive Able to produce toxins Dr.T.V.Rao MD 20
  21. 21. Plasmids • Plasmids are circular DNA molecules present in the cytoplasm of the Bacteria • Capable of Autonomous replication • Can transfer genes from one cell to other • Act as vectors in Genetic engineering. • Can also present in Yeasts Dr.T.V.Rao MD 21
  22. 22. Plasmid ( Blue ) Dr.T.V.Rao MD 22
  23. 23. Plasmids • Plasmid seem to be ubiquitous in bacteria, May encode genetic information for properties 1 Resistance to Antibiotics 2 Bacteriocins production 3 Enterotoxin production 4 Enhanced pathogen city 5 Reduced Sensitivity to mutagens 6 Degrade complex organic molecules Dr.T.V.Rao MD 23
  24. 24. R plasmid R: drug resistance RTF: transfer of R plasmid Dr.T.V.Rao MD 24
  25. 25. Plasmids • Can be integrated with Chromosomal DNA • Episomes Integrated form of plasmid with DNA Dr.T.V.Rao MD 25
  26. 26. Potentials of Plasmids • Plasmids can be self transmissible and Non transmissible • Transfers the Sex and Drug resistance with the help of restriction end nucleases Dr.T.V.Rao MD 26
  27. 27. Classification of Plasmids • Incompatibility typing • Don't accommodate others which are similar • Other methods of Classification Centrifugation Electrophoresis Genetic methods Dr.T.V.Rao MD 27
  28. 28. Genotypic and Phenotypic variation • Genome – Sum total of Gene that make up the genetic apparatus of cell established as Genotype. • Hereditary constitution of cell this transmitted to its progeny • Phenotype – is the physical expression in a environment. Change according to environment. Dr.T.V.Rao MD 28
  29. 29. What is Phenotypic expression • • • • Exhibit – different phenotypes Appearance differs in different situations. Eg Typhoid bacilli flagellated normally But grown in Phenol agar don't grow flagella So flagella are lost physical variation • Lactose fermentation in E.coli dependent on Beta Galactosidase When lactose present - test is positive When lactose is absent - test turns negative Dr.T.V.Rao MD 29
  30. 30. Different Enzymes Guided by Genomic configurations • Inducer enzyme acts in the presence of substrate • Constitutive enzyme acts irrespective of presence or absence of enzyme. • Phenotypic variations influenced by environment limited in range by genes • Genotypic variations are stable not influenced by environment. Dr.T.V.Rao MD 30
  31. 31. Principles of Genotypic variations • Mutations • Genotypic by transfer of genes Transformation Transduction (Lysogenic conversion) Conjugation Dr.T.V.Rao MD 31
  32. 32. Replica Plating, pt. 2 Dr.T.V.Rao MD 32
  33. 33. Mutations in Bacteria • Bacteria Multiply by asexual binary fission • Altered Nucleotide sequence in expresses new or altered characteristics • Selective value to the organism • Evolutionary value • Acquires Antibiotic resistance grows in body without inhibition • Become a prominent organism • Phenotypic variation occurs when genes changes in response to the environment but reversible. T.V.Rao MD Dr.T.V.Rao MD 33
  34. 34. Mutations • Mutation is a Random, Undirected, Heritable variation • Caused by alteration in the Nucleotide sequence at some point of DNA which can occur due to Addition Deletion Substitution of one or more bases Dr.T.V.Rao MD 34
  35. 35. Mutation Type Frameshift (deletion) (leu) (ser) (arg) Normal AAT AGT GCC (leu) (val) (pro) Mutant AAT AGT GCC A Dr.T.V.Rao MD 35
  36. 36. Mutation Type Frameshift (insertion) (leu) (ser) (arg) Normal AAT AGT GCC (leu) (glut) (cyst) Mutant AAT CAGT GCC Dr.T.V.Rao MD 36
  37. 37. Dr.T.V.Rao MD 37
  38. 38. Mutations can occur in any sequence,inveitable, useful for Survival Dr.T.V.Rao MD 38
  39. 39. Multiple Mutations • Causes extensive chromosomal rearrangement • Missense mutation -Triplet code is acted so as to specify an Aminoacid different from that normally located at particular position in the protein • Nonsense mutation - Deletion of nucleotide within a gene may cause premature polypeptide chain termination by nonsense codon • Tran version is Substitution of purine for pyramidine or vice versa in the base pairing Dr.T.V.Rao MD 39
  40. 40. Mutations • Suppressor Mutation is reversal of mutant phenotype by another mutation at a point of DNA distant from that of original mutation. • All genes are susceptible for mutations, but all mutations are not expressed • Lethal mutation is harmful destroy the vital functions Dr.T.V.Rao MD 40
  41. 41. Mutations • Conditional Lethal mutant may be Live under certain conditions • Common example is temperature ( its ) mutant • Temp sensitive ( ts) mutant lives at 350c but not at 390c • Each gene undergoes mutation at a fixed frequency. Bacteria undergo mutations at 10-4 - to 10-10 • Tutomerisim T – A is replaced by G - A Dr.T.V.Rao MD 41
  42. 42. Mutagenic Agents • U V rays • Alkyl ting agents • Arcidine Dyes Dr.T.V.Rao MD 42
  43. 43. GENE TRANSFER Occurs by Complex Mechanisms Dr.T.V.Rao MD 43
  44. 44. The three bacterial sexual processes – 1. Conjugation: direct transfer of DNA from one bacterial cell to another. –2. Transduction: use of a Bacteriophages (bacterial virus) to transfer DNA between cells. –3. Transformation: naked DNA is taken up from the environment by bacterial cells. Dr.T.V.Rao MD 44
  45. 45. Transformation of Genetic material ( Gene Transfer ) • Different Mechanisms Transformation Transduction Conjugation Dr.T.V.Rao MD 45
  46. 46. Gene Transfer Processes for Bacteria and Their Viruses 1. Conjugation 2. Transformation 3. Transduction 4. Infection with bacteriophage Dr.T.V.Rao MD 46
  47. 47. What is Transformation • Transformation is defined as transfer of Genetic information through the activity of DNA • Griffith experiment Mice injected with Live non capsulated ( R ) Pneumococci with heat killed capsulated (S) Pneumococci Lead to death of Mice with isolation of Live capsulated Pneumococci It means that some factor from Dead pneumococci transferred to live non pathogenic Pneumococci Dr.T.V.Rao MD Avery, McCleod, Mc Cartny in 1944 identified to be DNA 47
  48. 48. Griffith Phenomenon Dr.T.V.Rao MD 48
  49. 49. Demonstration of transformation Avery, MacLeod, and McCarty (1944) Dr.T.V.Rao MD 49
  50. 50. Transduction • Generalized involve any segment of DNA • Restricted when specific Bacteriophages traduces only a particular genetic trait. • Transduction effects Plasmids ,and Episomes • Plasmid transfer induces Penicillin resistance in Staphylococcus • Helps Genetic mapping, also in eukaryotic cell • Helps Genetic Engineering T.V.Rao MD Dr.T.V.Rao MD 50
  51. 51. Transduction • Transduction is defined as transfer of portion of DNA from one bacteria to another by Bacteriophages, is known as Transduction Dr.T.V.Rao MD 51
  52. 52. 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 52
  53. 53. Bacteriophages • Are viruses that parasitize bacteria and consists of Nucleic acid core and a protein coat • A phage particle may have at its core besides its own nucleic acid and a segment of the Host Dr.T.V.Rao MD DNA 53
  54. 54. Transduction Types • Two types of Transduction • 1 Lytic and 2 Lysogenic • 1 Virulent or Lytic cycle after large number of progeny are built up inside the host bacterium ruptures and phages are released Dr.T.V.Rao MD 54
  55. 55. Lysogenicity creates new characters • Eg - Lysogenic conversion in Diphtheria bacilli which acquires toxigenicity by lysogenization with phage beta • Elimination of phage for toxigenic strain renders nontoxigenic Dr.T.V.Rao MD 55
  56. 56. 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 56 recipient
  57. 57. Conjugation • The ability to conjugate is conferred by the F plasmid. A plasmid is a small circle of DNA that replicates independently of the chromosome. Bacterial cells that contain an F plasmid are called “F+”. Bacteria that don’t have an F plasmid are called “ Dr.T.V.Rao MD 57
  58. 58. 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 58
  59. 59. Mechanism of Transfer I: Conjugation Dr.T.V.Rao MD 59
  60. 60. Conjugation Dr.T.V.Rao MD 60
  61. 61. Simple Conjugation Dr.T.V.Rao MD 61
  62. 62. Conjugation Dr.T.V.Rao MD 62
  63. 63. 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 63
  64. 64. 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 64
  65. 65. 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 through F’ factor Dr.T.V.Rao MD 65
  66. 66. 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 66
  67. 67. Types of DNA transfer through Bacteriophages Dr.T.V.Rao MD 67
  68. 68. Colicinogenic ( Col ) Factor • Coli form 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 68
  69. 69. 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 69
  70. 70. 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 70
  71. 71. Bacterial Conjugation: High Frequency Transfer (Her) Cells Dr.T.V.Rao MD 71
  72. 72. Hfr Conjugation Dr.T.V.Rao MD 72
  73. 73. Sequence of RTF transmission Dr.T.V.Rao MD 73
  74. 74. Hfr cell conjugating a Normal cell Dr.T.V.Rao MD 74
  75. 75. 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 75
  76. 76. 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 76
  77. 77. 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 77
  78. 78. 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 78
  79. 79. Genetic Mechanisms in Bacteria helps to Spread the Infectious diseases Dr.T.V.Rao MD 79
  80. 80. 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 80
  81. 81. 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 81
  82. 82. 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 82
  83. 83. 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 83
  84. 84. Molecular Genetics • Analysis and manipulation of DNA using Biochemical and Microbiological techniques Dr.T.V.Rao MD 84
  85. 85. 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 85
  86. 86. 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 86
  87. 87. 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 87
  88. 88. 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 88
  89. 89. Genetic Engineering changing the Diagnostic and Therapeutic Protocols in MEDICINE Dr.T.V.Rao MD 89
  90. 90. Research on Gene transfer shapes the future of Science Dr.T.V.Rao MD 90
  91. 91. 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 91
  92. 92. 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 92
  93. 93. Restriction Endonucleases Made the advances in Genetic Engineering Dr.T.V.Rao MD 93
  94. 94. 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 94
  95. 95. 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 95
  96. 96. 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 96
  97. 97. 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 97
  98. 98. Western Blot Dr.T.V.Rao MD 98
  99. 99. 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 99
  100. 100. Polymerase chain reaction Kary B Mullis 1983 • Rapid • Automatic amplification of specific DNA sequences • Nobel prize winning Technology 1993 Dr.T.V.Rao MD 100
  101. 101. 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 101
  102. 102. Genetic Mapping • Genetic sequences for Bacteriophages and virus • Genetic mapping is done most of the Human Genes Dr.T.V.Rao MD 102
  103. 103. Newer Understanding on GENES Dr.T.V.Rao MD 103
  104. 104. Human Genome Project Dr.T.V.Rao MD 104
  105. 105. 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 105
  106. 106. Genetics are Complex - Leading the birth of BIOINFORMATICS Dr.T.V.Rao MD 106
  107. 107. Genes Evolved and made us Men What NEXT ? Dr.T.V.Rao MD 107
  108. 108. Are We Playing with Genes in the Right Direction ? Dr.T.V.Rao MD 108
  109. 109. Understanding of human Genome is Changing the Future of Medicine Dr.T.V.Rao MD 109
  110. 110. • Programmed Created by Dr.T.V.Rao MD for Undergraduate Medical Students in the Developing World • Email • Dr.T.V.Rao MD 110