Genotypic Variations in Microbiology & Immunology: Lecture No. 6

1. NAJRAN UNIVERSITY College of Medicine Microbiology &Immunology Course Lecture No. 6 By Dr. Ahmed MoradAsaad Associate Professor of Microbiology

2. II- GenotypicVariations : These are permanent (irreversible) variations, which are heritable, i.e. they will be transmitted among generations. They may be due to: 1- mutation 2- gene transfer

3. Genetic transfer Bacterial DNA may be transferred within or between bacterial cells. A. Transfer of DNA within bacterial cells: Transposition: Transposons or jumping genes can move from one site in a DNA molecule to other target chromosomal or plasmid sites in, the same or a different DNA molecule. This process is termed transposition and it results in insertion or deletion mutations.

4. B. Transfer of DNA between bacterial cells: Bacterial DNA may be transferred between bacterial cells by three mechanisms: transformation, transduction, and conjugation.

5. Transformation: • The transfer of DNA from one cell to another by either of 2 mechanisms: • 1- In nature, dying bacteria may release their DNA which may be taken by another cells • 2- In the lab, DNA may be extracted from one bacterial cell into another one

6. Transduction: • Transfer of bacterial DNA by means of a bacterial virus (bacteriophage). • 2 types of transduction: generalized and specialized • Generalized transduction: • This occurs when the bacterial virus carries a segment from any part of the bacterial chromosome. This occurs because cell DNA is fragmented after phage infection and a piece of DNA is incorporated into the virus.

7. Specialized transduction: • This occurs when the bacterial virus that has integrated into the cell DNA is excised and carries with it an adjacent part of the cell DNA

8. Conjugation: • 1- It is the mating of 2 bacterial cells during which DNA is transferred from the donor to recipient cell. • 2- The mating process is controlled by F (fertility) plasmid (F factor) which carries genes for synthesis of pilin and form sex pilus. • 3- Mating begins when the sex pilus of donor male bacteria carrying F factor (F+) attach to to recipient female bacteria (F-). • 4- Cleavage of F factor DNA: one strand to recipient cell. • The process is completed by synthesis of a complementary strand in each cell

9. Genetic engineering • *- A method to isolate genes coding for certain properties and join them together to form new combinations. • *- Also called genetic recombination, recombinant DNA technology, DNA cloning. • *- Major 3 steps (It requires): • 1- Separation of required gene (by restriction endonuclease). • 2- Carrying this gene by a vector • 3- Introducing the gene into a host cell (by transformation)

10. Restriction endonucleases: • *- Enzymes from bacteria and fungi that can recognize and cut DNA fragments (genes) at specific sites • Vectors: • 1- Plasmids • 2- Bacteriophage • 3- Cosmids: circular double-stranded DNA molecule constructed from plasmid DNA+phage DNA. They carry large genes • 4- Retroviruses and adenoviruses

11. Recombinant DNA technique: • 1- Chromosomal DNA is extracted and cleaved by Restriction endonuclease which cut at specific sites to separate the required fragment containing the required gene (insert) • 2- The vector (e.g., plasmid) is cleaved by the same step • 3- The insert + vector are mixed under certain conditions. This results in recombinant plasmid = plasmid+insert • 4- By transformation, the recombinant plasmid is introduced into a suitable host (bacteria or yeast cell) which can replicate autonomousely

12. Applications of recombinant DNA technology: • 1- Extensive chromosomal and genes studies • 2- Preparation of probes for diagnostic purposes • 3- Production of proteins of medical importance (large amount + low coast) • 4- production of recombinant vaccines • 5- Gene therapy (virus vectors)