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Transfer & Recombin
| Question | Answer |
|---|---|
| Recombination: | Genetic recombination in prokaryotes occurs when donor DNA is transferred via transformation, transduction, or conjugation and integrates into the recipient genome through homologous recombination. |
| Transformation: | the process where a cell takes up foreign DNA from its environment, incorporating it into its own genetic material. A Competent cell is a cell that is able to take up a molecule of DNA and be transformed. |
| Transduction: | transfer of host genes from one cell to another mediated by a virus (phage). |
| Conjugation: | transfer of genes from one prokaryotic cell (donor) to another (recipient) by a mechanism involving cell to cell contact and a conjugative plasmid. |
| Homologous Recombination: | exchange of similar DNA between two sources, mediated by RecA. DNA is nicked and unwound, single strands are coated with SSB, then RecA replaces SSB to drive strand invasion, forming heteroduplexes that are resolved into patches or splices. |
| Transformation: | genetic transfer process involving uptake of circular or linear DNA from the environment outside the cell and maintenance of the DNA in the recipient cell in a heritable form. |
| Competence: | state in which the cells are able to take up free DNA released by other organisms. |
| Natural Competence: | Natural competence is a physiological state where cells can take up free DNA from the environment. It depends on growth stage, stress, and the expression of specific competence proteins. Not all bacteria are naturally competent. |
| Artificially Induced Competence: | cells are treated with calcium ions/cold exposure to make their membrane permeable. |
| Electroporation (Competence): | cells are exposed to pulsed electrical fields to open small pores in their membranes. |
| Steps of Transformation: | DNA binds to surface receptors → becomes single-stranded → enters the cell → integrates into the chromosome via RecA. |
| Naturally Competent Bacteria Example: | Bacillus subtilis |
| Bacteriophage (Phage): | Virus that infects bacteria. Has two major life cycles. |
| Lytic Cycle (Bacteriophage Life Cycle): | propagate by infecting the bacteria, replicating itself, and then lysing the cell to leave. |
| Lysogenic Stage: | some phages can enter lysogenic stage where they insert themselves into the bacterial genome and not harm the host. Later, they remove themselves and go through a lytic cycle. The integrated phase is called a propage and the host is called a lysogen. |
| Temperate Phages: | can propagate a lytic or lysogenic cycle. |
| Transduction: | transfer of DNA from one cell to another by a bacteriophage. |
| General Transduction: | DNA from any portion of the host genome is packaged inside the virion so virtually any gene can be transferred to the recipient. |
| How Does General Transduction Occur? | Transduction occurs when host DNA is mistakenly packaged into a phage, creating a defective particle that cannot cause lytic infection. When released, these particles can transfer DNA to recipients, which may integrate it through recombination. |
| Efficiency of General Transduction: | 10^6 - 10^8 cells transduced, very low efficiency |
| Specialized Transduction: | DNA from a specific region of the host chromosome is integrated directly into the virus genome. Transfer frequency can be very high. |
| How Does Specialized Transduction Occur? | Specialized transduction occurs when DNA near a temperate phage’s insertion site is accidentally excised and packaged into the phage. In a new host, this DNA can integrate via lysogeny or homologous recombination. |
| Example of Specialized Transduction: | Phage Lambda integrates into the E.coli genome next to the galactose utilization genes. |
| Conjugation (mating): | horizontal gene transfer that requires cell-to-cell contact. Normally plasmid-encoded. Other genetic elements (other plasmids or chromosomes) may be mobilized and transferred during conjugation. |
| Donor Cell (Conjugation): | contains conjugative plasmid |
| Recipient Cell (Conjugation): | will receive the plasmid |
| F “fertility” plasmid of E.coli (Conjugation): | model system for conjugation. Contains transposable elements for plasmid integration into host chromosome, contains tra genes that encode transfer functions, encodes pili that allow specific pairing through receptor contact, pulling cells together. |
| Tra Genes (Conjugation): | found in the F plasmid, encodes transfer functions → synthesis of sex pilus and type IV secretion system for DNA transfer. |
| Cells with the F plasmid are called… | F+ (Donor cell). |
| Cells without the F plasmid are called… | F- (Recipient cell). |
| What Mechanism Does Plasmid DNA Transfer Use During Conjugation? | A rolling circle mechanism. One strand is transferred to the recipient and DNA replication synthesizes a second strand in both recipient and donor. 5’ end goes into recipient first and 3’ end stays behind to serve as the primer for the mechanism. |
| Efficiency of Plasmid Transfer & Rolling Circle Mechanism: | efficient and rapid; under favorable conditions nearly 100 percent of recipients acquire plasmids. F plasmid transfer takes about five minutes! |
| Episome: | The F plasmid is an episome, meaning it can integrate into the host chromosome. All episomes are plasmids, but not all plasmids are episomes. |
| Cells possessing a nonintegrated F plasmid are called… | F+. |
| Cells possessing an integrated F plasmid are called… | HFR (high frequency of recombination) for high rates of genetic recombination between genes on the donor (Hfr) and recipient (F-) chromosomes during conjugation (transfer chromosomal genes at high frequency). |
| Integration: | provides the mechanism for mobilizing a genome. Integration is accomplished through recombination of the plasmid insertion sequences (IS elements) with corresponding chromosome insertion sequences (IS elements) through homologous recombination. |
| Hfr Conjugation Process: | Begins at the oriT site, leading end of F plasmid enters recipient first. The mating bridge often breaks before transfer finishes, so only part of the donor chromosome is transferred and cannot replicate. Integration requires homologous recombination. |
| Result of Hfr: | recipient usually remains F- (doesn’t receive all of F plasmid), may acquired donor chromosomal markers, high frequency of recombinants. |
| Why is Recombination Required During Hfr? | Because of the breakage of DNA during transfer, only a part of the donor chromosome is transferred. Since this partial chromosome cannot replicate in the recipient cell, donor genes will become part of the recipient only through recombination. |
| Formation of Different HFR Strains: | Form when F plasmid integrates into different insertion sites on chromosome. Each Hfr strain transfers genes adjacent to its insertion point, and the direction of transfer (clockwise or counterclockwise) depends on the plasmid’s orientation. |
| Chromosome Mapping in Bacteria using HFR: | When an Hfr strain (His⁺ Arg⁺ Rif⁺ Trp⁺) donates DNA to F⁻ strain (His⁻ Arg⁻ Rif⁻ Trp⁻), genes are transferred in a specific sequence. By checking which genes appear in recombinants, scientists can map gene order along the circular chromosome. |
| Explanation of Graphs on Slide 94: | Left shows the frequency of recombinants versus minutes, revealing the transfer order: hisG → argH → rif → trpA. Right shows where these genes are located on the E. coli chromosome, with the Hfr insertion site marking the transfer’s starting point. |
| Major Takeaway of Chromosome Mapping Hfr: | Genes near the Hfr insertion site transfer first and recombine more often, while distant genes enter later and are less likely to transfer fully before mating ends—greater oriT distance means lower recombination frequency. |
| Compare and Contrast Transformation, Transduction, and Conjugation: | Transformation takes up free DNA, transduction transfers DNA via bacteriophage, and conjugation moves DNA through cell contact. In conjugation, plasmid transfer moves only plasmids, while Hfr transfer passes chromosomal genes sequentially. |
| Transposable Elements: | discrete segments of DNA that move as a unit from one location to another within DNA molecules. Bounded by inverted repeats on either end. |
| Transposition: | the process of moving from one location to another. |
| Transposase: | the enzyme that moves them from one location to another (recognizes the inverted repeat). |
| Insertion Sequences: | encode only the ability to move new locations (excise and insert into the new location, called IS). Very small, about 1,000 bp. |
| Composite Transposons: | a mobile genetic element consisting of two insertion sequences (ISs) flanking a segment of cargo DNA often containing antibiotic resistance (AR) genes - called Tn |
| Noncomposite Transposons: | a type of genetic element that lacks flanking insertion sequences (IS elements) but has its own terminal inverted repeats, which are necessary for its movement. (called Tn). |
| Transposition: | the process where a transposable element (or “jumping gene”) moves into a new location on the chromosome. When it inserts, it’s flanked by inverted repeats (IRs) and causes a duplication of the target DNA sequence at the insertion site. |
| Conservative Transposition: | transposon is excised from one location and reinserted at a second location. Typical insertion sequences and composition transposons (NO REPLICATION OF INSERTION SEQUENCES). |
| Replicative Transposition: | a new copy of the transposon is produced and inserted at a second location. Typically noncompositive transposons. (REPLICATION OF INSERTION SEQUENCES). |
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smurtab