Microbiology Procedure

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Integration Of Donor DNA

	Integration Of Donor DNA If the DNA strand entering the recipient cell is to be maintained within the cell and express its genetic information, it must recombine with host DNA. Theoretically, there are two models possible to explain recombination of donor and recipient DNA during conjugation. According to one model the single stranded donor DNA entering the recipient cell would be first converted into a DNA duplex before recombination with the recipient duplex. The recombinant chromosome would, therefore, be homozygous in sequence with both strands carrying donor information in the inserted region
According to the second model a single DNA donor strand would be directly integrated into the recipient chromosome, where it would replace a homologous segment. Differential radio-active labelling experiments of donor and recipient DNA indicate that a single donorstrand is inserted into the recipient chromosome Oppenheim and Riley( 1966) labelled donor DNA with 3H-thymidine and recipient DNA with 15N heavy isotope to follow the fate of donor DNA. They found that covalent linkage between recipient and donor DNA does not take place up to nearly three hours after mating
After dilution of the culture to stimulate division, the donor and recipient DNAs were found to be covalently linked. This indicated that the final stages of recombination occur only under conditions permitting cell growth, i. e. during replication. Siddiqui and Fox (1973) using both radioactive and density labels confirmed that a single donor strand was inserted into the DNA, and that covalent linkage between the two molecules took place only during replication
There are two models representing the extremes of conditions of integration of single-stranded donor DNA into the recipient chromosome . According to the first model, pairing between donor and recipient DNA takes place at one site, and one recipient strand is degraded and replaced. Initial pairing is extended along the chromosome, and the donor strand replaces one recipient strand, which undergoes degradation. The recipient strand is nicked at a point where it is to be replaced, and pairing takes place at this site. One recipient strand is degraded exonucleotically, thus freeing the bases of the other recipient strand for complementary pairing with the donor strand. The insertion usually extends for less than 5 x 105 bases.
	This is indicated by the observation that genetic markers located more than 10 minutes apart on the chromosome are not linked in conjugation. Repair synthesis fills in the gaps between donor and recipient strand9 and covalent bonds are finally formed at the breaks. In this model only one strand of the recipient DNA is replaced by donor DNA. The model closely resembles the models for the formation of hybrid DNA which bring about recombination between DNA duplexes. According to the second model, pairing takes place at two sites and both recipient strands are degraded and replaced by donor strands

In this model, proposed by Curtiss (1969), both recipient strands are degraded and removed between the sites of pairing. The donor strand forms a single stranded region in the recipient chromosome. Repair synthesis then produces a complementary strand on the inserted donor segment, and fills in the remaining gaps. Covalent bonds are finally formed at the breaks, and the gaps sealed by polynucleotide ligase. The model proposing that the donor DNA forms a duplex before recombination is similar to the Curtiss model in that the recombinant chromosome would be homozygous.

Hybrid DNA formed by recombination will be heterozygous if the two parental molecules differ at the same site. The DNA may become homozygous if a correction is brought about by an excision repair system. If repair does not take place before replication of DNA, then each strand will synthesize a complementary strand. The two daughter DNA molecules will differ from each other. Each will be homozygous for the information of one parent. This will give rise to mixed clones from a mating

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