Gene transfer and their techniques by sivasangari Shanmugam.

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GENE TRANSFER
Transfer of genetic material such as DNA from one bacterium to another bacterium that process
is called gene transfer. There are three types of gene transfer. They are,
1. Conjugation (One bacterium to another bacterium by conjugation tube)
2. Transformation ( Direct uptake of DNA)
3. Transduction (One bacterium to another by virus)
CONJUGATION
 Conjugation is the transfer of genetic material between the bacterial cells by directs
contact/by a bridge like connection between the two.
 Discovered by Joshua Lederberg & Edward Tatum in 1946.
 The evidence for cell to cell contact was provided by Bernard Davis in 1950.
 Bacterial conjugation is also known as type IV secretion system.
 Conjugation occurs in and between many species of bacteria, including gram negative,
gram positive bacteria, and even occurs between bacteria and plants.
1. F+ & F- Conjugation:
 F+ is fertile which acts as a donor.
 F- is unfertile which acts as a recipient.
 F+ cell contain both chromosomal DNA and Plasmid DNA and F- contain lack of
plasmid DNA.
Mechanism of F+ & F- conjugation:
1. F plasmid contains tra locus, which includes the pilin. This gene, along with some
regulatory proteins results in the formation of pili on the F+cell surface.
2. Pilus attaches to recipient cell and brings the two cells together.
3. Only single standard of plasmid is then transferred to the recipient cell.

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4. The traD enzyme, located at the base of the pilus, initiates membrane fusion.
5. Once the conjugation is initiated, enzyme relaxase creates a nick in the conjugative
plasmid at the oriT.
6. The nicked strand then unwinds and is transferred to the recipient cell in the 5’-3’
direction.
7. Bothe the cells synthesis a complementary strands to produce double stranded circular
plasmid and also produce pilus.
8. Both cells are now viable donor.
2. Hfr Conjugation:
 High frequency recombination (Hfr).
 In the 1950’s, Luca Cavalli-Sforza discovered a strain of E.coli that was very efficient at
transferring chromosomal DNA.

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 He designated this strain as Hfr.
 Hfr strain is derived from F+ strains.
Mechanism of Hfr conjugation:
1. When F plasmid integrated with chromosomal DNA such bacteria is known as Hfr
bacteria.
2. In the conjugation between Hfr cells and F- cells, Hfr is very high but frequency of
transfer of whole F- factor is very low.
3. An Hfr cell is donor and F- cell act as recipient.
4. F factor makes sex pilus that joins donor and recipient.
5. F factor opens as replication origin then one strand is cut down.
6. Now 5’ end of the strand enters into recipient cell through conjugation tube (Pilus).
7. 5’ end enters first into recipient cell but the portion situated at 3’ end enters only when
whole chromosomal DNA enters into the recipient cell.
8. To transfer whole chromosomal DNA, it takes 100mns in E.coli.
9. In most of the cases, sex pilus breaks before transfer of whole chromosomal DNA takes
place. So, frequency of transfer of whole F- factor is very low.
10. After the cross between Hfr cell and F- cell, recipient cell remains recipient.
11. In this conjugation, chromosomal DNA is always transferring from donor to recipient cell
together with portion of F- factor. So, frequency of recombination is high.

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TRANSFORMATION
 In molecular biology, transformation is genetic alteration of a cell resulting from the
direct uptake, incorporation and expression of exogenous genetic material from its
surroundings and taken up through the cell membrane.
 This process doesn’t require a living donor cell and only requires free DNA in the
environment.
 The recipient that successfully propagates the new DNA is called the transformant.
 During extreme environmental conditions, some bacterial genera spontaneously release
DNA from the cells into the environment free to be taken up by the competent cells.
Griffith’s Experiments

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Avery & Macleod Experiment
Hershey & Chase Experiment

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 Two types of transformation. They are,
1. Natural Transformation
2. Artificial Transformation
Natural Transformation:
 In natural transformation, bacteria naturally have the ability to incorporate DNA from the
environment directly.
Artificial Transformation:
 In the case of artificial transformation, the competence of the host cell has to be
developed artificially through different techniques.
 Competence- The ability of cells to uptake foreign DNA.
 Competent cell- The cell which uptakes the foreign DNA by producing competent
factors including small protein essentials for transformation.
 Competent cell preparation
1. Heat-shock transformation: Competent cells are chemically prepared by
incubating the cells in calcium chloride to make the cell membrane more
permeable.
2. Electroporation: It is another way to make hole on the bacterial cell walls by
briefly shocking them with an electric field of 10-20kV/cm. Plasmid DNA can
enter through these holes. After the shock hole is closed by natural membrane
repair mechanism.
Mechanism of Transformation:
1. A long double stranded DNA molecule binds to the surface with the aid of a DNA
binding protein.
2. It is nicked and by endonucleases into ds DNA fragments of 5-15kb.

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3. One strand is degraded by the endonucleases.
4. The undegraded strand associates with a competence specific protein.
5. The single strand enters the cell and integrated into the host chromosomal DNA.

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TRANSDUCTION
 DNA fragment is transferred from one bacterium to another bacterium by a
bacteriophage.
 Discovered on 1951 by Lederberg & Zinder in Salmonella & P22.
 There are two types of transduction. They are,
1. Generalized Transduction
2. Specialized Transduction
Mechanism of Transduction:
Specialized Transduction:
 In specialized transduction, the phage undergoes lysogeny usually at specific locations in
the bacterial genome called attachment sites.
 During this process, the phage genome usually integrates into the bacterial chromosome
as virus replication is repressed during lysogeny.
 The phage genome then excises from the bacterial genome and, due to imprecise excision
and recombination, adjacent bacterial genes are also excised.
 During the subsequent infection, the newly acquired gene is inserted into the bacterial
genome along with phage DNA to form a new round of lysogeny.
 Specialized transduction is independent of host homologous recombination and recA but
requires phage integrase.
 Specialized transduction is instrumental in the isolation of the genes in molecular
biology, and in the discovery of insertion elements, which often serve as attachment sites
for phage DNA integration.

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Generalized transduction:
 In generalized transduction, phage mistakenly packages bacterial DNA instead of their
own phage DNA during phage assembly.
 This results in an infectious virus particle containing bacterial DNA, but one that can no
longer replicate in the bacterium due to the loss of all of the phage DNA.
 The phage particle then attaches to a bacterial cell surface receptor and injects the
packaged DNA into the cytoplasm of the bacterium.
 If the bacterial DNA in the phage is from the bacterial chromosome, the DNA
recombines with the homologous DNA of the bacterial recipient to generate stable
transductants. This process requires a host recombinase, recA.
 However, studies have indicated that the majority of transduced DNA is not stably
integrated into the bacterial genome but rather remains extra chromosomal.

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 Generalized transduction is used for mapping genes, mutagenesis, transferring plasmids
and transposons, and determining whether different genera of bacteria have homologous
genes.

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F- GENETIC CROSSES
1. F+ × F- genetic crosses
2. Hfr × F- genetic crosses
3. F’ × F- genetic crosses
F+ × F- genetic crosses

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Hfr × F- genetic crosses

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F’ × F- genetic crosses

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CONJUGATION DISCOVERY
 Discovered by Joshua Lederberg & Edward Tatum in 1946.
 They experimented with two auxotrophic strains of E.Coli K12.
 Denoted by Strain 1 & Strain 2.
 The evidence for cell to cell contact was provided by Bernard Davis in 1950.
1. LEDERBERG AND TATUM EXPIREMENTS (1946)
 Discovered by Joshua Lederberg & Edward Tatum in 1946.
 They experimented with two auxotrophic strains of E.Coli K12.
 Denoted by Strain 1 & Strain 2.
 Strain A and Strain B were plated on minimal medium and incubated at over night, no
growth observed.
 Also Strain A and Strain B were mixed together and when plated on minimal medium
resulted in prototrophs.

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2. DAVIS “U” TUBE EXPIREMENTS (1950):
 The evidence for cell to cell contact was provided by Bernard Davis in 1950.
 The arms of the U tube are separated by a filter.
 On the right side is medium containing auxotrophic strain A while on the left side is
medium containing auxotrophic strain B.
 The filter allows only the medium but not allows the cell on either side.
 When culture was plated from both sides on minimal medium, no prototrophs growth
was observed as in Lederberg and Tatum’s experiments.

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TRANSPOSONS
Transposable elements (TEs), also known as "jumping genes," are DNA sequences
that move from one location on the genome to another. These elements were first identified
by geneticist Barbara McClintock.
There are two distinct types:
1. Class II transposons. These consist of DNA that moves directly from place to place.
2. Class I transposons. These are retrotransposons that first transcribe the DNA into
RNA and then use reverse transcriptase to make a DNA copy of the RNA to insert in a
new location. The central block of the composite transposable elements consist a gene for
antibiotic resistance gene.