Transcript Slayt 1
What is genetic material? Griffith experiment 1928 DNA Watson-Crick model 1953 DNA polymerase I and III DNA ligases Primase DNA replication is semiconservative! Meselson-Stahl experiment 1958 oriC and dnaA Boxes Cis acting sites: on this side of (acting only on the DNA, they made of Trans acting (proteins) : on the other side of (acting on any DNA) dnaA, B, C, G, Termination Bacterial genetic information: On bacterial DNA On plasmids On bacteriphages On transposons BActerial Genome -Usually 1 chromosome Circular or linear No histon proteins In circular bacterial DNA the replication begins at the ori locus Ends at ter locus Plasmids: ds DNA; circular Various copy number 800-300 000 bp long Carry genes providing advantages for the bacterium Transposons (IS seequences) Can couple their replication to the cell division - Their propagation depends on the integration with the bacterial replicon - The insertion sites are not spesific Bacteriophages Viruses of the bacteria Ds/ss DNA,ds/ss RNA Lytic or temperate phages (prophage) Different propagation strategies Gene transfer among bacteria Vertical transfer Lateral or horizontal transfer - conjugation - transduction - transformation Conjugation Most frequently plasmids are transferred Tra gene products are needed F+ E. Coli Sex pilus Hfr R plasmids Interrupted Mating Chromosome transfer from the Hfr into the F- is slow: it takes about 100 minutes to transfer the entire chromosome. The conjugation process can be interrupted using a kitchen blender. By interrupting the mating at various times you can determine the proportion of Fcells that have received a given marker. This technique can be used to make a map of the circular E. coli chromosome. Transduction General Phage Life Cycle 1. Phage attaches to the cell and injects its DNA. 2. Phage DNA replicates, and is transcribed into RNA, then translated into new phage proteins. 3. New phage particles are assembled. 4. Cell is lysed, releasing about 200 new phage particles. Total time = about 15 minutes. Generalized Transduction Some phages, such as phage P1, break up the bacterial chromosome into small pieces, and then package it into some phage particles instead of their own DNA. These chromosomal pieces are quite small: about 1 1/2 minutes of the E. coli chromosome, which has a total length of 100 minutes. A phage containing E. coli DNA can infect a fresh host, because the binding to the cell surface and injection of DNA is caused by the phage proteins. After infection by such a phage, the cell contains an exogenote (linear DNA injected by the phage) and an endogenote (circular DNA that is the host’s chromosome). A double crossover event puts the exogenote’s genes onto the chromosome, allowing them to be propagated. Transduction Mapping Only a small amount of chromosome, a few genes, can be transferred by transduction. The closer 2 genes are to each other, the more likely they are to be transduced by the same phage. Thus, “co-transduction frequency” is the key parameter used in mapping genes by transduction. Transduction mapping is for fine-scale mapping only. Conjugation mapping is used for mapping the major features of the entire chromosome. Specialized Transduction Some phages can transfer only particular genes to other bacteria. Phage lambda (λ) has this property. To understand specialized transduction, we need to examine the phage lambda life cycle. lambda has 2 distinct phases of its life cycle. The “lytic” phase is the same as we saw with the general phage life cycle: the phage infects the cell, makes more copies of itself, then lyses the cell to release the new phage. Lysogenic Phase The “lysogenic” phase of the lambda life cycle starts the same way: the lambda phage binds to the bacterial cell and injects its DNA. Once inside the cell, the lambda DNA circularizes, then incorporates into the bacterial chromosome by a crossover, similar to the conversion of an F plasmid into an Hfr. Once incorporated into the chromosome, the lambda DNA becomes quiescent: its genes are not expressed and it remains a passive element on the chromosome, being replicated along with the rest of the chromosome. The lambda DNA in this condition is called the “prophage”. After many generations of the cell, conditions might get harsh. For lambda, bad conditions are signaled when DNA damage occurs. When the lambda prophage receives the DNA damage signal, it loops out and has a crossover, removing itself from the chromosome. Then the lambda genes become active and it goes into the lytic phase, reproducing itself, then lysing the cell. Transduction Phage mediated recombination Transformation Competent cells Artificially (forced) with CaCl or temperature shock Genetıc Mapping Sequencing of the gene Cloning the gene Gene labeled Hybridization Localization of the gene on the bacterial genome Nucleic - - acid amplification PCR and other technologies Real time PCR