CHAPTER 8: GENETIC ANALYSIS AND MAPPING IN BACTERIA AND BACTERIOPHAGES8.1 Bacteria Mutate Spontaneously and Are Easily Cultured- The Adaptation Hypothesis proposes that interaction of bacteriophage and bacterium is essential to the bacterium’s acquisition of immunity to the phage- Exposure to the phage induces resistance in the bacteria- A prototrophs a bacteria that can synthesize all essential organic compounds and therefore can be grown on minimal mediumo A minimal medium is one that is composed of an inorganic carbon source and a variety of ions (Na+,K+,Mg2+)- Through mutation, an auxotroph has lost the ability to synthesize one or more essential compounds; It must be provided with them in the medium if itis to growo Typically result from loss-of-function type mutationso Multiple Auxotroph’s means multiple mutations- Bacteria mutate spontaneously and grow at an exponential rate8.2 Genetic Recombination Occurs in Bacteria- Genetic information can be transferred from one bacterium toanother, resulting in an altered genotype- Three processes result in this transfer of genetic informationo Conjugation- bacterial sex, F (fertility) factor-mediated Genetic information istransferred from onebacterium to another It recombines with the secondbacterium’s DNAo Transformation- Direct uptake ofnaked DNA by 1 cello Transduction- Viral transfer ofgenetic material between bacteria - Vertical Gene Transfer -When transfer ofgenetic information occurs betweenmembers of the same species - Horizontal Gene transfer - When transferoccurs between members of related, butdistinct bacterial species- Cells serving as donors of parts of theirchromosomes are designated F+ cells (F forfertility)Fig- Genetic recombination of twoauxotrophic strains producing prototrophs.- F- cells -Recipient bacteria receive the donor DNA from the F+ cells and recombine it with part of their own chromosome - If there is no direct contact, auxotrophs (F+ and F- cells) do not exchange genetic material and no prototrophs are formedo Physical contact is the first step in conjugation which is established bythe F pilus o Bernard David showed this using the Davis U-tube for growing F+ andF- cells- F+ cells contain fertility factor (F factor) thatconfers the ability to donate part of theirchromosome during conjugationo F factor is mobile and consists of acircular, double-stranded DNA moleculecontaining 19 geneso One strand of the double helix moves intorecipient cell via the sex pilus, and theother one remains. Both re-form theirdouble helix and become F+ - Figure - How an F- cell is converted to an F+cell during conjugation- F+ X F- produces all F+ cells - E. coli may or may not contain the F factoro When the F factor is present, the cell isable to form a sex pilus and serve as adonor of genetic informationo During conjugation, a copy of the F factor is transferred from the F+ cell to the F- recipient, converting it to the F+ state- The F factor is an autosomous genetic unit called a plasmid- Hfr (high-frequency recombination) cells behave as donors and are a special class of F+ cells- An Hfr strain can donate genetic information to an F- cell but the recipient does not become F+o F+ X F- recipient becomes F+ (low rate recombination)o Hfr X F- recipient remains F- (high rate of recombination)- Interrupted matings technique demonstrated that the genes of a given Hfr strain are transferred and recombined sooner than others - Figure – Wollman and Jacob demonstrated an order oftransfer of genes that correlates with the length of timeconjugation proceeded.- An ordered linear transfer of genes is correlated withthe length of time conjugation proceededo The gene order and distance between genescould be predictedo Basis for first genetic map in bacteria- E. Coli chrmosome is circular and the point of origin (O) varies from strain to strain.o The F factor integrates into the chromosome at different points and itsposition determines the O site. The direction of transfer is determinedby the orientation of the F factor as it integrates- When F++ and F- cells are mixed, conjugationoccurs readily, and each F- cell involved inconjugation with an F++ cell receives a copy ofthe F factor, but no genetic recombinationoccurs.- At an extremely low frequency in a populationof F+ cells, the F factor integratesspontaneously from the cytoplasm to a randompoint in the bacterial chromosome, convertingthe F++ cells to the Hfr state as in the Figure>>- In F++ ´ F - mating’s, the low frequency ofgenetic recombination is attributed to the rare,newly formed Hfr cells which undergoconjugation with F - cells.- Because the point of integration of the F factoris random, The gene or genes that aretransferred by any newly formed Hfr donorwill also appear to be random the larger F+/F-populationo The recipient bacteria is a recombinantbut will remain F -, upon conjugation with an F++ cell will become F+- In some cases, the F factor is removed fromthe chromosome of an Hfr strain and revertsto the F++ state. FIGURE>>>- This condition is labeled F’ to distinguish itfrom F+ and Hfr. F’ is a conversion from Hfrwhereas Hfr is a conversion from F+- Transfer of an F' to an F– cell results in apartially diploid cell called a merozygote8.3 Rec Proteins are Essential to Bacterial Recombination- RecA protein plays an important role in recombination involving either a single-stranded DNA molecule or the linear end of a double-stranded molecule that has unwound.- Single-stranded displacement is a common form of recombination in manybacterial species.- Double-stranded DNA entering a recipient cell undergoes one-strand degradation.o The remaining strand joins the homologous region along the host chromosome and once it does, RecA protein facilitates recombination.- The RecBCD protein is important for unwinding a double-stranded DNA molecule that serves as the source for genetic recombination.o RecBCD unwinds the helix facilitating recombination that involves RecA.o This model of recombination based on the rec discoveries also applies to eukaryotes.o Rec A homologs in eukaryotes include meiosis-specific enzymes that carryout the recombination associated with crossovers8.4 The F Factor is an Example of a Plasmid- Plasmids are composed of a double-stranded closed circle of DNA o Exist in multiple copies in the cytoplasmo May contain one or more genes o Use the same replication enzymes as hosto Are distributed to daughter cells o Replicate
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