GENE TRANSFER

Transformation, Transduction and Conjugation

Renz L. Salumbre, M.Sc.

Transfer of genetic material

Gene transfer is the movement of genetic information between organisms

Eukaryotes Essential part of the life cycle Sexual reproduction Gametes fuse to form zygote Each parent produces genetically
different gametes

Several genetic combinations

transferred to offspring

Recombination is the combination of DNA from two different cells

In Bacteria, not an essential part of the

life cycle

Some genes of the donor cell are

transferred to the recipient cell

Resulting cell is called a recombinant

Rec proteins are essential to bacterial recombination

Mutant genes: recA, recB, recC and recD Reduced recombinations RecA protein RecBCD protein - enzyme consisting of a
polypeptide subunits encoded by other rec genes

Vertical Gene Transfer

Parents to offspring Plants and animals In bacteria, asexual reproduction by

binary fission

Bacteria pass genes to other microbes of the same generation

Horizontal / Lateral Gene Transfer Transformation Transduction Conjugation

Significance of Gene Transfer

Increase genetic diversity Mutations may account for some

genetic diversity

Environmental pressures lead to

evolutionary changes

Transformation

Frederick Griffith (1928) Pneumococcal infections in mice Natural transformation observed in

Acinetobacter, Bacillus, Haemophilus, Neisseria, and Staphylococcus

Also found in Saccharomyces cerevisiae

Mechanism of Transformation

Naked DNA DNA released from an organism after Organisms take up a maximum of
about 10 fragments

the cell is lysed and the DNA no longer incorporated into chromosomes or other structures

 High cell density and depletion of nutrients Uptake of DNA Competence factor released into the
medium

Mechanism of Transformation

Other factors Modifications of the cell wall Formation of specific receptor sites on
the plasma membrane

Protein that facilitates entry of DNA

DNA transport proteins

DNA exonuclease

Mechanism of Transformation

DNA reaches the entry sites Endonucleases cut dsDNA into units of
7000-10000 nucleotides the cell

Strand separates and one strand enters ssDNA vulnerable to nucleases Nucleases must be inactivated ssDNA base pairs immediately with a
portion of the recipient chromosome

Mechanism of Transformation

Donor ssDNA is positioned alongside the

recipient DNA

Identical loci are next to one another Enzymes in the recipient cell excise a
portion of the recipients DNA and recombine it with the donor DNA chromosome

Permanently part of the recipients Leftover DNA is broken down Number of nucleotides in the cells
DNA remains constant

Mechanism of Transformation

Naturally transformable bacteria take up DNA from any source

With a few exceptions

Neisseria gonorrhoeae

Haemophilus influenzae

Transduction

DNA is carried by bacteriophage (phage) Discovered in Salmonella by Joshua

Lederberg and Norton Zinder (1952)

Properties of Bacteriophages

Composed of a core of
on the cell wall of the bacterium

nucleic acid covered by a protein coat

Attaches to a receptor site Phage enzyme weakens

cell wall allowing the passage of phage DNA

Type of pathway taken depends on type of bacteriophage Virulent phage causes

destruction and death of a bacterial cell synthesize phage-specific nucleic acids and proteins

Phage genes direct the cell to Destroy host DNA Other proteins and nucleic

acids form phages eventually filling the cell up with it

Phage enzymes rupture the

Type of pathway taken depends on type of bacteriophage

Temperate phage does not cause a disruptive

infection

Phage DNA is incorporated into a

bacteriums DNA and is replicated with it prevents destruction of bacterial DNA phage particles

Produces a repressor substance that Replicate either as a prophage in a

Phage DNA does not direct synthesis of

bacterial chromosome or by assembling into new phages

Lysogenic cycle

Prophage - phage DNA incorporated into

host bacteriums DNA

Lysogeny - persistence of a prophage

enter lytic cycle

without phage replication and destruction

Known mechanisms to induce cells to

Bacteriophage Life Cycle

Transduction happens when some bacterial DNA is packed into the heads of phages

Generalized transduction Any bacterial gene can be transferred

by the phage

Specialized transduction Only specific genes are transferred

Lysogenic phages usually carry out specialized transduction

Lambda () phage in E. coli Inserts into specific locations

during integration with a chromosome

gal gene - galactose use bio gene - biotin synthesis

Specialized Transduction

Cells containing lambda phage are

induced to enter the lytic cycle

Phage genes form a loop and are excised

from the bacterial chromosome

phage directs the synthesis and

assembly of new phage particles and the cell lyses contain only phage genes; rarely does the phage contain one or more bacterial genes

New phage particles released usually

Specialized Transduction by Phage in E. coli

Generalized Transduction

Bacterial cell with phage DNA enter lytic

cycle

Phage enzymes break host cell DNA into

many small segments new phage particles

Phage directs synthesis and assembly of DNA packaged by the headful Bacterial DNA occasionally incorporated
into phage particle; plasmids and DNA from other viruses may be incorporated

Generalized Transduction

Significance of Transduction

Prophage DNA and host DNA demonstrate close

evolutionary relationship

Regions of similar base sequence Suggest viral origin of cancer Prophage can exist in a cell for long periods of
time

Malignant changes Animal viruses may have brought along genes

from their previous hosts chromosome mapping

Provides a way to study gene linkage and

Conjugation differs from transformation and transduction

Requires contact between donor and

recipient cells

Transfers much larger quantities of DNA

(occasionally, whole chromosomes)

Discovered by Joshua Lederberg (1946)

Conjugation

Plasmids are extrachromosomal DNA

molecules

Bacterial cells contain several different Conjugation involves Transfer of F plasmids High frequency recombinations (Hfr) Transfer of F plasmids

plasmids that carry genetic information for non-essential cell functions

Characteristics of Plasmids

Most are circular, double stranded

extrachromosomal DNA

Self-replicating F plasmid was first discovered Promiscuous cells Self-transmissible plasmids Conjugation with other species than
their own kind

Functions of Plasmids

F plasmids - synthesis of proteins that will

assemble into conjugation pili

Resistance (R) plasmids - genes that Plasmids that direct the synthesis of
bacteriocins

provide resistance to various antibiotics and to heavy metals

Virulence plasmids that cause diseases Tumor-inducing (Ti) plasmids causing

tumor formation in plants

Transfer of Fertility plasmids

F+ and F- were found to exist in any population of E. coli capable of conjugating F+ cells contain Fertility plasmids F- lack F plasmids

F plasmids carry

information for the synthesis of F pilus (sex / conjugation pilus)

Transfer of Fertility plasmids

DNA is transferred as a single strand via a

conjugation bridge (mating channel) the passage of ssDNA

Sex pilus contains a hole that may permit Evidences suggest that mating cells
temporarily fuse during DNA transfer

Transfer of Fertility plasmids

Pilus makes contact with a

receptor site on surface of the Fcell forming a pore Inside the F- cell, pilus is pulled in and dismantled DNA from F+ cell enters F- cell complementary strand of DNA Both cells will become F+

Each cell synthesizes the

High-frequency recombinations F strain that could induce 1000x more than

+

the F+ x F- conjugations (L.L. Cavalli-Sforza) plasmid is incorporated into the bacterial chromosome

Hfr strains arise from F+ strains when F

Hfr cell is a donor in conjugation F plasmid initiates transfer of chromosomal

DNA

Only part of the F plasmid is transferred

(initiating segment)

Transfer of DNA occurs in a linear fashion

with a precise time schedule (Wollman & Jacob)

Recipient cell does not become F+

High-frequency recombinations

Transfer of F plasmids

Process of incorporating an F plasmid into a

bacterial chromosome is reversible

DNA incorporated into a chromosome can Imprecise - can carry fragments of the
chromosome

separate from it and become an F plasmid

F conjugate with F Whole F plasmid is transferred Recipient cells have 2 of some

chromosomal genes

Resistance Plasmids

AKA R factors Formation of R plasmids are not due to

antibiotics

Use of antibiotics contribute to the

be selected to survive

survival of strains that contain R plasmids

Organisms with R plasmids are said to Rapid process Large numbers of previously non-resistant
organisms can become resistant quickly

R plasmids have two components Resistance Transfer Factor (RTF) DNA similar to F plasmids Implements transfer by conjugation of the
whole R plasmid

Essential for the transfer of resistance to

another organism

Resistant (R) genes One or more may be present Carries information that confers resistance Synthesis of an enzyme that inactivates the
antibiotic

Transposition

R genes can move from one plasmid to

sequence

another in a cell or even become inserted in the chromosome

Transposable elements - mobile genetic Insertion sequence contains gene that

codes for an enzyme needed to transpose the insertion sequence

Flanked by inverted repeats Replicate only when in plasmids or in a

chromosome

Transposition

Insertion sequence is copied by the

transposase and cellular enzymes chromosome or another plasmid mutations)

Copy randomly inserted into bacterial May cause mutations (spontaneous Transposons - transposable elements that
contain genes for transposition

Genes for toxin production or R genes