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 Dr.T.V.Rao MD 2
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
Dr.T.V.Rao MD 4
Mechanism of Transfer I: Conjugation Dr.T.V.Rao MD 5
Conjugation Dr.T.V.Rao MD 6
Simple Conjugation Dr.T.V.Rao MD 7
Conjugation Dr.T.V.Rao MD 8
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
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
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
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
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Types of DNA transfer through Bacteriophages Dr.T.V.Rao MD
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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 T.V.Rao
MD Dr.T.V.Rao MD 14
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
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
Bacterial Conjugation: High Frequency Transfer (Her) Cells
Dr.T.V.Rao MD 17
Hfr Conjugation Dr.T.V.Rao MD 18
Sequence of RTF transmission Dr.T.V.Rao MD 19
Hfr cell conjugating a Normal cell Dr.T.V.Rao MD 20
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
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
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
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
Genetic Mechanisms in Bacteria helps to Spread the Infectious
diseases Dr.T.V.Rao MD 25
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
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
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
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
Molecular Genetics Analysis and manipulation of DNA using
Biochemical and Microbiological techniques Dr.T.V.Rao MD 30
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
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
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
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
Genetic Engineering changing the Diagnostic and Therapeutic
Protocols in MEDICINE Dr.T.V.Rao MD 35
Research on Gene transfer shapes the future of Science
Dr.T.V.Rao MD 36
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
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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 Dr.T.V.Rao MD 38
Restriction Endonucleases Made the advances in Genetic
Engineering Dr.T.V.Rao MD 39
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
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
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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)
Dr.T.V.Rao MD 42
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
Western Blot Dr.T.V.Rao MD 44
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
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
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
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
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
Genetic Mapping Genetic sequences for Bacteriophages and virus
Genetic mapping is done most of the Human Genes Dr.T.V.Rao MD
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Newer Understanding on GENES Dr.T.V.Rao MD 51
Human Genome Project Dr.T.V.Rao MD 52
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
Genetics are Complex - Leading the birth of BIOINFORMATICS
Dr.T.V.Rao MD 54
Genes Evolved and made us Men What NEXT ? Dr.T.V.Rao MD 55
Understanding of human Genome is Changing the Future of
Medicine Dr.T.V.Rao MD 56
The Programme Created by Dr.T.V.Rao MD for Medical Students in
the Developing World Email [email protected] Dr.T.V.Rao MD
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