Evolution, Variation, and Excision of Developmentally Regulated DNA Elements in the Heterocystous Cyanobacteria
In some cyanobacteria, heterocyst differentiation is accompanied by developmentally regulated DNA rearrangements that occur within the nifD, fdxN, and hupL genes, referred to as the nifD, fdxN, and hupL elements. These elements are excised from the genome by site-specific recombination during the latter stages of heterocyst differentiation. In this dissertation, two major questions are addressed: 1) what is the evolutionary history of the nifD and hupL elements and 2) how is the nifD element excised? To answer the first question, full length nifD and hupL element sequences were characterized and compared; and xisA and xisC sequences (which encode the recombinases that excise the nifD and hupL elements, respectively) were phylogenetically analyzed. Results indicated extensive structural and compositional variation within the nifD and hupL elements. The data suggests that the nifD and hupL elements are of viral origin and that they have variable patterns of evolution in the cyanobacteria. To answer the second question, a recombination system was devised where the ability of XisA to excise or recombine variants of the nifD element (substrate plasmids) was tested. Using PCR directed mutagenesis, specific nucleotides within the flanking regions of the nifD element were altered and the effects on recombination determined. Results indicate that nucleotides within and outside of the direct repeats are involved in excision, and that not all nucleotides within the direct repeats are required. In certain nucleotide positions, the presence of a purine versus a pyrimidine greatly affected recombination. Although excision was inhibited when certain nucleotides were mutated, PCR analyses revealed that excision occurred at a low level. The data also indicate that the site of excision occurs within the direct repeats. The results presented here suggest that the elements may be variable in size, composition, and excision.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:cyanobacteria nifd element hupl heterocyst differentiation developmentally regulated dna rearrangement
Date of Publication:01/01/2005