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BACTERIAL GENETICS Learning objectives: At the end of the lecture the student should be able to: • Enlist the methods of DNA transfer in microorganisms. • State the significance of DNA transfer in drug resistance. • Enlist & describe the types of mutations in bacteria. • Describe the process of lysogeny. • Describe the role of mutations in drug resistance in infectious diseases Understanding Genetics We resemble and differ because of Genetic configurations Parents - Son - Daughter, how they resemble each other. They breed true from Generation to Generation But vary in small proportions in progeny. Bacteria too obey the laws of Genetics DNA A Complex Structure Makes Life Knowledge on DNA lead to advances in Molecular Biology Central dogma of Life – Deoxyribonucleic acid DNA carries the genetic information DNA is transcribed to RNA – Polypeptides Cell Function depends upon specific polypeptides – Proteins – Enzymes DNA is a store house of Protein synthesis DNA acts a Template for synthesis of mRNA Virus differs from other as they contains either DNA or RNA The Bacterial Genome Chromosomes Contains a Double stranded molecules of DNA arranged in circular form. Length 1,000 microns. Bacterial DNA contains about 4,000 kilobases Bacteria are haploid: they have a single chromosome and therefore a single copy of each gene. In haploid cells, any gene that has mutated – and is therefore not expressed – results in a cell that has lost that trait. Plasmids Plasmids are circular DNA molecules present in the cytoplasm of the Bacteria Capable of Autonomous replication Can transfer genes from one cell to other Act as vectors in Genetic engineering. Can also present in Yeasts Plasmids Plasmid seem to be ubiquitous in bacteria, May encode genetic information for properties Resistance to Antibiotics Bacteriocins production Enterotoxin productions Enhanced pathogencity Reduced Sensitivity to mutagens Degrade complex organic molecules Mutations A mutation is a change in the base sequence of DNA that usually results in insertion of a different amino acid into a protein and the appearance of an altered phenotype. Mutations result from three types of molecular changes: 1) Base substitution 2) Frameshift mutation 3) Transposons or insertion sequences Mutations can be caused by chemical, radiations or viruses Mutation is a Random, Undirected, Heritable variation Transfer of DNA within Bacterial cells Transposons Programmed rearrangement These gene rearrangements account for many of the antigenic changes seen in Neisseria gonorrhoea and Borrelia recurrentis the cause of relapsing fever. A programmed rearrangement consist of movement of a gene from a silent storage site where the gene is not expressed to an active site where transcription and translation occur. The insertion of a new gene into the active site in a sequential repeated programmed manner is the source of the consistent antigenic variation. These movements have the effect of allowing the organism to evade the immune response. TRANSPOSONS Mobile genetic elements The arrangement of genes in the genome of bacteria and probably all organisms is not entirely static. Certain DNA segments called transposons have the ability to move place to place on the chromosome and into and out of plasmids. Mobile genetic elements are probably responsible for most of the genetic variability in natural bacterial populations, and for the spread of antibiotic resistance genes. Transformation of Genetic material(Gene Transfer) Occur by three methods: 1) Conjugation 2) Transduction 3) Transformation From a medical point of view the most important consequence of a DNA transfer is that antibiotic resistance genes are spread from one bacterium to another by these processes CONJUGATION Conjugation is the process by which the bacteria transfer genes from one cell to another by cell-to-cell contact. The process requires the presence on the donor cells F+ of hair like projections called sex pili that make contact with specific receptor sites on the surface of recipient cells High frequency recombinant Conjugation • • • When it exists as a free plasmid, the F plasmid can only transfer itself. This isn’t all that useful for genetics. However, sometimes the F plasmid can become incorporated into the bacterial chromosome, by a crossover between the F plasmid and the chromosome. The resulting bacterial cell is called an “Hfr”, which stands for “High frequency of recombination”. Hfr bacteria conjugate just like F+ do, but they drag a copy of the entire chromosome into the F- cell. TRANSDUCTION Transduction is defined as transfer of portion of DNA from one bacterium to another by Bacteriophages. During the growth of virus within the cell a piece of bacterial DNA is incorporated into the virus particle and is carried into the recipient cell at the time of infection. Within the recipient cell the phage DNA can integrate into the cell DNA and the cell can acquire a new trait, a process called lysogenic conversion This process can change a non pathogenic organism into a pathogenic one. Bacteriophages Are viruses that parasitize bacteria and consists of nucleic acid core and a protein coat A phage particle may have at its core besides its own nucleic acid and a segment of the Host DNA Specialized transduction Only certain bacterial genes located in the bacterial chromosome in close proximity to the prophage insertion site of the transducing phage are transduced. Generalised transduction A random fragment of bacterial DNA resulting from phage induced cleavage of bacterial chromosome, is accidentally in a phage protein coat in place of the phage DNA. When this rare phage particle infects a cell it injects the bacterial DNA fragment into the cell, it becomes integrated into the recipient chromosome by recombination. Transformation Is the transfer of DNA itself from one cell to another. In nature, dying bacteria may release their DNA which may be taken up by recipient cells. In the laboratory an investigator may extract DNA from one type of bacteria and inject it into genetically different bacteria. When purified DNA is injected into the nucleus of a eukaryotic cell, the process is called transfection. Transfection is frequently used in genetic engineering procedures Griffith Phenomenon Recombination Once the DNA is transferred from the donor to the recipient cell it can integrate into the host cell chromosome by recombination. 1) Homologous recombination: in which two pieces of DNA that has extensive homologous regions pair up and exchange pieces by the process of breakage and reunion 2) Non homologous recombination in which little if any homology is necessary What is Gene Therapy? Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, such as a hereditary disease in which a deleterious mutant allele is replaced with a functional one. Although the technology is still in its infancy, it has been used with some success. Vivo to Vitro What Gene therapy can achieve? Replacing a mutated gene that causes disease with a healthy copy of the gene. Inactivating, or “knocking out,” a mutated gene that is functioning improperly. Introducing a new gene into the body to help fight a disease. Genetically Engineered Products Can prepare desired protein in pure form in economic way Somatostatin Commercial preparations of Cloned Human Insulin Interferons Hepatitis B vaccine 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 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 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 RT antigen 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 Genetic Mapping Genetic sequences for Bacteriophages and virus Genetic mapping is done most of the Human Genes References Lippincott’s Illustrated Reviews, Biochemistry 5th edition Ch. 33 Biotechnology & Human Disease Pgs. # 465-487