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Chem*4570 Applied Biochemistry Lecture 11 Conjugation and Recombination Stryer pp. 819-828. Lehninger pp. 949-967 Bacterial conjugation allows for the transfer of genetic characters within a species, from genomic DNA to genomic DNA in the gene’s normal context. Alleles - alternative variants of a gene, e.g. wild type versus mutant, normal versus Thr supersensitive etc. In contrast vectors are agents that allow transfer of genes between organisms, not necessarily the same species, but the transferred genes do not usually end up in their normal genomic context. Plasmids are small autonomously replicating DNA circles that lie outside the main chromosome, placing genes in an episomal context. Plasmids can be set up to express the genes they carry but represent an extra cost to their host so can be lost. Genes can also be integrated into the genome via phage such as phage λ and its derivatives, but will not be in their normal genetic context. Recombination plays a major role in role certain kinds of gene transfer. Bacterial conjugation E. coli K12 exists in several different mating types. F+ cells contain the F plasmid or fertility factor, and can transfer it by conjugation to F– cells which lack the factor. Recipients of the F factor then become F+. F+ cells may spontaneously lose the F factor and revert to F– . The F plasmid includes the genes required for formation of the sex pilus, responsible for attaching to F– cells, which express an appropriate receptor. The F plasmid may incorporate a section of the bacterial genome, allowing transfer of genes to another cell. Recombination events may then integrate the genes into the recipient’s genome. F plasmid may also itself become incorporated into the genome, in which case tranfer of the F factor to the recipient can carry the entire genome into the recipient. Since this creates far more opportunities for recombination, strains with genomic F factor are designated Hfr cells, for high frequency of recombination. Integration occurs spontaneously at a frequency of about 10–5 . Reversion of Hfr to F+ occurs at a similar frequency. In reverting to F+, the F plasmid may acquire a small portion of the host genome, and will pass this on in future conjugation events. Conjugation in yeasts. Wild type yeast, like many eukaryotes, is a diploid (two copies of each chromosome) although lab strains for convenience are maintained as haploids (single chromosomes). Yeast exists in two mating types a and α which are heritable, i.e. progeny of simple cell fission have the mating type of their parent. The mating type is maintained by an expressible copy of one of two separate silent genes, one for each mating type. Cells can undergo mating type switch, e.g. induced by starvation, in which one of the silent genes places a new copy of itself in the expressed locus. This drop-in replacement is known as a gene cassette. Mixed mating types will then undergo conjugation, allowing exchange of genetic characters. The mated cells undergo meiosis and then sporulate, forming a tetrad containing the haploid progeny of the conjugation. After conjugation, selection and screening techniques are used to recover cells which have acquired the desired characters. Conjugation allows strains selected for one mutation, e.g. Hom– to be combined with genes from another strain e.g. AECR (and also to replace the lexA– with a normal regulated SOS repair system). Recombination Recombination is the process whereby sequences from one DNA molecule can exchange with sequences in another molecule. Homologous recombination may occur where where there are regions of sequnce match between the incoming and the target DNA. Incoming DNA may be DNA transferred by conjugation, by transformation with a plasmid, or bare DNA inserted by a technique such as electroporation. Site specific recombination occurs as a mechanism to integrate external DNA into a host genome. This requires the existence of specific target sequence in the host. For example the attB locus in E.coli acts as the attchment site for insertion of phage λ DNA into the host genome. Homologous recombination involves several proteins. The existence of such an elaborate process is probably a consequence of recombination being an important DNA repair mechanism.. RecBCD then provides the helicase/nuclease complex, or unwindase that generates single stranded DNA. A a sequence called the chi site, GCTGGTGG, the nuclease cuts near its 3’, end, releasing a single strand of DNA with a 3’ end. Chi sites are widely distributed in E.coli DNA, about 4 kbp apart. RecA binds the single stranded DNA, and promotes invasion of the single strand into homologous double stranded DNA, displacing a loop. The displaced loop can also be nicked, leaving another 3’ end, and it too will invade the opposite strand, creating a crossover or Holliday junction. The junction then migrates along the DNA, mediated by the proteins RuvA and RuvB in an ATP dependent process. Thus the final crossover site need not be closely associated with the original chi site. Cleavage of the Holliday junction is followed by religation so that two independent duplex strands form once more. Depending on the plane of cleavage, this may result in a minimal transfer of DNA between strands, or alternatively opposite strands may religate, so that a cross-over event has occurred. These crossover events allow transfer of sequences from the incoming DNA to the genomic DNA of the cell. Nick and strand displacement RecB, RecC RecA binds ssDNA RecA promotes strand invasion into homologous duplex second nick and displacement of strand on 2nd duplex junction formed by ligation branch migration, RuvA, RuvB plane of cleavage determines extent of recombination