Bacterial Genetics Basics
Bacterial Genetics Basics
Dr.T.V.Rao MD
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Dr.T.V.Rao MD
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
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Molecular Biology
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Structure of DNA
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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
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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
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DNA - RNA
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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 )
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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.
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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
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Plasmid ( Blue )
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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
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R plasmid
R: drug resistance
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Plasmids
Can be integrated with Chromosomal DNA Episomes Integrated form of plasmid with DNA
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Potentials of Plasmids
Plasmids can be self transmissible and Non transmissible Transfers the Sex and Drug resistance with the help of restriction end nucleases
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Classification of Plasmids
Incompatibility typing Don't accommodate others which are similar Other methods of Classification Centrifugation Electrophoresis Genetic methods
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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.
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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
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Mutation Type
Frameshift (deletion)
(leu) (ser) (arg)
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Mutation Type
Frameshift (insertion)
(leu) (ser) (arg)
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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
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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
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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
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Mutagenic Agents
U V rays Alkyl ting agents Arcidine Dyes
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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.
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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
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Griffith Phenomenon
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Demonstration of transformation
Avery, MacLeod, and McCarty (1944)
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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
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Transduction
Transduction is defined as transfer of portion of DNA from one bacteria to another by Bacteriophages, is known as Transduction
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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
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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
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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
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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 dont have an F plasmid are called
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Conjugation
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Simple Conjugation
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Conjugation
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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
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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
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Hfr Conjugation
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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
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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
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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.
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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
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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
Molecular Genetics
Analysis and manipulation of DNA using Biochemical and Microbiological techniques
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Genetic Engineering
Under standing Molecular genetics in Biochemistry fuels genetic Engineering Recombinant DNA (renal) techniques changed the ideals of Medicine
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
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Modern applications
Pharmaceutical production
Insulin, interferon, hormones, vaccines etc.
Genetically engineered plants Animal gene alterations Gene probes DNA fingerprinting The human genome initiative
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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
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Hepatitis B vaccine
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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
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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.
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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
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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)
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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
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Western Blot
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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
Western Blot to confirm HIV Infections made land mark Diagnostic tool
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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
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Genetic Mapping
Genetic sequences for Bacteriophages and virus Genetic mapping is done most of the Human Genes
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Programmed Created by Dr.T.V.Rao MD for Undergraduate Medical Students in the Developing World Email [email protected]
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