Bacterial Genetics and Applications

Dr.T.V.Rao MD

Dr.T.V.Rao MD

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Gene Transfer Processes for Bacteria and Their Viruses
1. Conjugation
2. Transformation

3. Transduction 4. Infection with bacteriophage

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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

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
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Mechanism of Transfer I: Conjugation

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Conjugation

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Simple Conjugation

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Conjugation

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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.
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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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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

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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
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Types of DNA transfer through Bacteriophages

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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
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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
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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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Bacterial Conjugation: High Frequency Transfer (Her) Cells

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Hfr Conjugation

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Sequence of RTF transmission

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Hfr cell conjugating a Normal cell

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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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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
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Genetic Mechanisms in Bacteria helps to Spread the Infectious diseases

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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

Best option- To restrict use of Antibiotics
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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.
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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
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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 await many surprises?
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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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Genetic Engineering changing the Diagnostic and Therapeutic Protocols in MEDICINE

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Research on Gene transfer shapes the future of Science

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Genetically Engineered Products
• Can prepare desired protein in pure form in economic way Somatostatin • Commercial preparations pdf Cloned Human Insulin Interferons

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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Restriction Endonucleases Made the advances in Genetic Engineering

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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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Polymerase chain reaction Kary B Mullis 1983
• Rapid • Automatic amplification of specific DNA sequences • Nobel prize winning Technology 1993

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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.
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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.
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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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Newer Understanding on GENES

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Human Genome Project

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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
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Genetics are Complex - Leading the birth of BIOINFORMATICS

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Genes Evolved and made us Men What NEXT ?

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Understanding of human Genome is Changing the Future of Medicine

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• The Programme Created by Dr.T.V.Rao MD for Medical Students in the Developing World

• Email
• doctortvrao@gmail.com

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