GTP binding proteins (G proteins) are molecular switches that regulate many aspects of physiology in all cells. DRG1 and DRG2 (Developmentally Regulated GTP-binding proteins) have been found in all eukaryotes examined. Amino acid identity across eukaryotes is about 65% within an orthologous group and 55% between paralogs of a single species. Although DRGs are likely involved in some aspect of translation regulation, details of their precise cellular functions are not known.
In addition to DRGs there are other genes/proteins that comprise the “DRG pathway.” DFRPs (DRG Family Regulatory Proteins) have been shown to physically and genetically interact with DRGs. Previous work elsewhere and at NIU using the model plant Arabidopsis demonstrated that DFRP and DRG proteins mutually prevent the degradation of their binding partners (DRG1 with DFRP1, and DRG2 with DFRP2).
Current work is focused on the DRG pathway genes in the model moss Physcomitrella patens. Mosses offers several benefits to gene research: 1) most of the moss lifecycle is spent in the haploid phase, which greatly simplifies genetic analyses; 2) mosses can be kept in this phase indefinitely; 3) individual living cells are easy to study with various types of microscopy; 4) unlike virtually all other plants, homologous recombination is very efficient in P. patens, allowing knockout mutants to be generated quite readily; and 5) the Physcomitrella genome sequence and other molecular tools are available.
Physcomitrella contains 7 genes in the DRG pathway. The focus of this dissertation is the four Dfrp genes (1a, 1b, 2a and 2b). This required production of an extensive set of knockout mutants: 4 singles, 5 doubles, 2 triples and a quadruple mutant (all four Dfrp genes removed). Analysis of the different genotypes included detailed phenotype studies. The mRNA and protein levels of all 7 genes were studied using qRT-PCR and Western blotting, respectively. These techniques gave more details about the level of gene/protein regulation in the gene pathway. Some of the genes were expected to take part in the amino acid starvation response. That hypothesis was tested by blocking amino acid production with glyphosate in all knockouts to examine if any of these genes alter the response. The various analyses of all knockouts have led to a better understanding of the gene functions in the DRG pathway.
|Advisor:||Stafstrom, Joel P., Bode, Barrie|
|Commitee:||Bujarski, Jozef, Grayburn, Scott, Johns, Mitrick|
|School:||Northern Illinois University|
|School Location:||United States -- Illinois|
|Source:||DAI-B 81/7(E), Dissertation Abstracts International|
|Keywords:||DRG, Gene knockout, Physcomitrella patens|
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