GENE TRANSFER MECHANISM GUIDED BY :- Dr. S.T. GIRISHA Dept.of Microbiology Presented by:- JEEVITHA.H.A II Sem, MSc.

CONTENTS Definition of gene Gene transfer mechanism Types Bacterial transformation Transduction Conjugation Mechanism Application References

GEnE …??? A gene is the molecular unit of heredity of a living organism.

GENE TRANSFER MECHANISM???? It is the mechanism where a particular gene is transferred from donor cell to recipient cell….

It is the insertion of copies of a gene into living cells. The desired gene may be microinjected directly into the cell or it may be inserted into the core of a virus by gene splicing and the virus allowed to infect the cell for replication of the gene in the cell's DNA.

TYPES Bacterial Transformation Transduction Conjugation Host cell restriction Complementation Transfection

TRANSFORMATION The process by which a donor DNA molecule is taken up from the external environment and incorporated into the genome of a recipient cell….

History First discovered by Frederick Griffith in 1928 in Diplococcus pneumoniae. In 1944,Avery and co-workers showed that DNA was responsible for transformation.

KEY POINTS Energy requiring process. Proteins and enzymes are required. Maximum frequency of competent cells during late log phase. Naturally occuring transformation.

Types NATURAL TRANSFORMATION It is a bacterial adaptation for DNA transfer that depends on the expression of numerous bacterial genes. Competent development in Bacillus subtilis require about 40 genes. Usually requires bacteria of same species.

ADAPTATION FOR DNA REPAIR Competence is induced by DNA damaging conditions. Mitomycin C and fluoroquinoline. UV light

MECHANISM

STEP-1 A competent cell binds a large double stranded fragment of DNA at specific receptor sites on the surface of the bacterium. One of the DNA strands is hydrolyzed by a membrane bound exonuclease, providing energy to help DNA transport across the membrane.

STEP-2 Induced proteins binds to single stranded donor fragments.

STEP-3 (A) The single stranded fragment of DNA is coated with DNA –binding protein and recombination proteins search for the same or very similar DNA sequences in the chromosome of the recipient cell.

STEP-3 (B) Remainder of the ssDNA fragment is degraded.

APPLICATIONS    Transformation is one of the many ways of today to create recombinant DNA, DNA in which genes from two different sources-many times different species- are combined together and placed into the same molecule or organism.  This manipulation of genes is called genetic engineering and has many practical applications in the world today.

This is a very basic technique that is used on a daily basis in a molecular biological laboratory. The purpose of this technique is to introduce a foreign plasmid into a bacteria and to use that bacteria to amplify the plasmid in order to make large quantities of it. This is based on the natural function of a plasmid: to transfer genetic information vital to the survival of the bacteria.

TRANSDUCTION The process by which DNA transferred from one bacterial cell to another by bacteriophage.

KEY POINTS VIRULENT PHAGES - are those that immediately lyse and kill the host. TEMPERATE PHAGES – can remain within the host cell for a period without killing it.

TYPES GENERALISED TRANSDUCTION SPECIALISED TRANSDUCTION

Generalized Transduction In generalized transduction, a small piece of the host cell’s degraded DNA is packaged within a capsid, rather than the phage genome. When this phages attaches to another bacterium, it will inject this foreign DNA into its new host.

Specialized Transduction Occurs via a temperate phage. When the prophage viral genome is cut from the host chromosome, it sometimes takes with it a small region of the host bacterial DNA. These bacterial genes are injected along with the phage’s genome into the next host cell. Specialized transduction only transfers those genes near the prophage site on the bacterial chromosome.

Specialized transduction occurs via a temperate (can in corporate its genome in to the bacterialcell) phage.

The DNA of lambda is inserted into the host DNA at the site adjacent to the galactose genes On induction, Under rare conditions, the phage genome is excised incorrectly A portion of host DNA is exchanged for phage DNA, called lambda dgal ( dgal means "defective galactose“ ) Phage synthesis is completed Cell lyses and releases defective phage capable of transducing galactose genes

APPLICATIONS Resistance to anti-biotic drugs. Correcting genetic diseases by direct modification of genetic errors.

CONJUGATION Conjugation is a process in which bacteria makes contact with each other through pilus formation and genetic material is transferred unidirectionally from donor to recipient cells. F factor directs conjugation. F+ cells F- cells Hfr F’ factor

Mating types in bacteria Donor F factor (Fertility factor F (sex) pilus Recipient Lacks an F factor Donor Recipient

Physiological States of F Factor Autonomous (F+) Characteristics of F+ x F- crosses F- becomes F+ while F+ remains F+ Low transfer of donor chromosomal genes F+

Characteristics of Hfr x F- crosses Integrated (Hfr) Characteristics of Hfr x F- crosses F- rarely becomes Hfr while Hfr remains Hfr High transfer of certain donor chromosomal genes. F+ Hfr

Autonomous with donor genes (F’) Characteristics of F’ x F- crosses F- becomes F’ while F’ remains F’ High transfer of donor genes on F’ and low transfer of other donor chromosomal genes Hfr F’

Mechanism of F+ x F- Crosses Pair formation Conjugation bridge F+ F- DNA transfer Origin of transfer Rolling circle replication

Mechanism of Hfr x F- Crosses Pair formation Conjugation bridge Hfr F- DNA transfer Origin of transfer Rolling circle replication Homologous recombination

Mechanism of F’ x F- Crosses Pair formation Conjugation bridge F’ F- DNA transfer Origin of transfer Rolling circle replication

Lederberg and Tatum Showed that genetic information could be transferred from one bacterium to another. Davis Showed Conjugation requires direct, physical contact between cells.

Hayes CONJUGATION is polar. Requires the F+ plasmid. Recipient becomes F+ once conjugation is complete. Noted that only rarely did auxotrophs become prototroph’s after conjugation.

Consequences of Hfr conjugation Can transfer bacterial genes other than the F+ genes Many different genes could be transferred. Recombination or Genetic variability occurs.

Schematic drawing of bacterial conjugation Schematic drawing of bacterial conjugation. Conjugation diagram 1- Donor cell produces pilus. 2- Pilus attaches to recipient cell and brings the two cells together. 3- The mobile plasmid is nicked and a single strand of DNA is then transferred to the recipient cell. 4- Both cells synthesize a complementary strand to produce a double stranded circular plasmid and also reproduce pili; both cells are now viable donors.

APPLICATIONS Conjugation is a convenient means for transferring genetic material to a variety of targets. In laboratories, successful transfers have been reported from bacteria to yeast, plants, mammalian cells and isolated mammalian mitochondria. Conjugation has advantages over other forms of genetic transfer including minimal disruption of the target's cellular envelope and the ability to transfer relatively large amounts of genetic material.

REFERENCES General Microbiology ( 5th edition ) Roger.Y.Stanier Prescott’s Microbiology ( 8th edition ) Joanne.M.Willey Linda.M.Sherwood Christopher.J.Woolverton ‘BROCK’-Biology of Microbiology ( 11th edition ) Michael.T.Madigan John.M.Martinko Principles of Genetics D.Peter Snustad Michael.J.Simmons John.B.Jenkins Microbial Physiology and Microbial Genetics B.D.Singh R.P.Singh

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