Using maize Heat Shock Factor Binding Protein paralogues to study heat shock response, development and protein-protein interaction specificity determination
MetadataShow full item record
Mutator (Mu) transposon insertion induced mutants are a great genetic resource to clone genes and study molecular mechanisms of cellular and developmental processes. The research described in this dissertation exemplifies the significance of this approach. The first part of this dissertation describes the characterization of a Mu induced, recessive, embryo lethal maize mutant, empty pericarp 2 (emp2) and the cloning of the emp2 locus. It was found that emp2 mutant phenotypes co-segregated with a Mu transposon insertion in the first intron of the emp2 locus. The transposon insertion was correlated with 5’-end truncation of the emp2 transcripts and loss of EMP2 protein accumulation. EMP2 protein was found to be homologous to human HEAT SHOCK TRANSCRIPTION FACTOR BINDING PROTEIN 1 (HSBP1), a negative regulator of heat shock response (HSR). The un-attenuated HSR in emp2 loss of function mutant embryos indicates EMP2 is a functional homologue of HSBP1 in maize, and EMP2 represents the first protein found in plants to be essential for HSR attenuation. Emp2 gene was found to be constitutively expressed throughout maize development, which prompted me to study post-embryonic functions of EMP2. In order to overcome the embryo lethality of emp2 mutants, clonal mosaics of emp2 null mutant tissues were induced by X-ray irradiation. As reported in the second part of this dissertation, emp2 null mosaics were associated with diverse developmental defects, and the mutant phenotypes are not affected by heat stress treatment. Therefore, the clonal mosaic analyses with emp2 mutant tissues elegantly demonstrated the separation of EMP2 functions in regulating HSR and normal development. Hsbp genes were found to be duplicated in all monocot grass species. Consequently, the paralogue of emp2, hsbp2 was cloned from maize. The maize hsbp paralogues exhibit differential expression patterns in response to heat stress and during normal development. In addition, yeast two-hybrid studies revealed that EMP2 and HSBP2 interact with different proteins through their coiled-coil domain. Comprehensive mutagenesis analyses identified that many residues within the coiled-coil domain of HSBP2 contributed to the hetero-oligomerization of HSBP2 and the maize HEAT SHOCK FACTOR, HSFA4b. More importantly, it was found that the specificity of HSBP2 and HSFA4b interaction is determined by non-coiled-coil peptide sequences that flank the coiled-coil domain of HSBP2. Thus, comparative study of the different protein-protein interaction selectivities of the maize paralogues not only provided novel insights about molecular determinants of protein interaction specificity, but also revealed important evolutionary mechanisms that lead to functional divergence of paralogous proteins.
Showing items related by title, author, creator and subject.
Liu, Shangbin (uga, 2008-12)The main goal of analytical proteomics is the complete and quantitative proteome analysis of species, cells, and/or tissues. Although the great success has been achieved via incremental improvements in Mass Spectrometry ...
Canseco, Miguel Angel (uga, 2002-08)Two experiments were conducted to determine the value of specific protein supplements in lactating dairy cows fed high fat diets. Experiment 1, six feedstuffs (wheat silage, corn, soybean meal, soybean hulls, whole cotton ...
Analysis of protein-DNA interactions within the transcription preinitiation complex of Pyrococcus furiosus Renfrow, Matthew Blain (uga, 2002-12)Initiation of transcription requires the assembly of a multi-protein preinitiation complex (PIC) on promoter DNA. The protein-DNA interactions within the transcription PIC of the archaeal hyperthermophile, Pyrococcus ...