Date of Award
Doctor of Philosophy (PhD)
Radhey S. Gupta
The 70-kDa heat shock protein (HSP70) is one of the most conserved proteins known to date. The HSP70 genes from Mycoplasma capricolum, Giardia lamblia, Rhizobium meliloti, and Pseudomonas cepacia were cloned and sequenced. Sequence comparison of HSP70 shows that homologs from gram-positive bacteria and archaebacteria share a number of sequence signatures that are not found in other prokaryotic or eukaryotic species. Other sequence features were found to be specific to eukaryotes and gram-negative bacteria. Phylogenetic analyses of HSP70 sequence strongly support a specific evolutionary relationship between gram-positive bacteria and archaebacteria on the one hand, and gram-negative bacteria and eukaryotes on the other. The mycoplasma homologs of HSP70 contain all sequence signatures that are characteristic of gram- positive bacteria and archaebacteria. In the HSP70 tree, mycoplasma branch with the low-G+C-content gram-positive group of bacteria indicating their close evolutionary relationship to this group. The G. lamblia HSP70 homologs (from the cytosol and endoplasmic reticulum), similar to other eukaryotic homologs, were found to contain sequence signatures specific to gram-negative species. Phylogenetic analyses show that G. lamblia homologs are the deepest homologs in the eukaryotic tree. The cytosolic and endoplasmic reticulum homologs in eukaryotes, including those of G. lamblia, are similar and form two distinct subfamilies indicating that they evolved from a common eukaryotic ancestor by gene duplication early in the evolution of eukaryotic cells. The early divergence of G. lamblia and its evolutionary linkage to gram-negative bacteria indicate that eukaryotes evolved from a gram-negative bacterium. The cloning of the endoplasmic reticulum-HSP70 gene from G. lamblia suggested the existence of endoplasmic reticulum in this ancient eukaryote. The existence of endoplasmic reticulum in G. lamblia has been investigated using the endoplasmic reticulum-HSP70 protein as a molecular marker. The endoplasmic reticulum-HSP70 gene of G. lamblia was expressed in Escherichia coli and a polyclonal antibody was raised to the purified protein. Using cryoelectron microscopy and antibody to endoplasmic reticulum-HSP70, we demonstrated the presence of endoplasmic reticulum in G. lamblia. The presence of endoplasmic reticulum in G. lamblia, which is one of the earliest diverging eukaryotic species lacking mitochondria, supports the view that both the nucleus and endoplasmic reticulum co-evolved in a common ancestor of eukaryotic cells. We have used the HSP70 sequences from Rhizobium meliloti, and Pseudomonas cepacia to study the evolution of mitochondria. Rhizobium meliloti, and Pseudomonas cepacia belong to the $\alpha$- and $\beta$-subdivisions of gram-negative proteobacteria, respectively. On the basis of sequence alignment, several sequence signatures have been identified that are distinctive to mitochondrial homologs and gram-negative proteobacteria. Phylogenetic analyses of HSP70 sequences suggest the endosymbiotic origin of mitochondria from a member of the α-proteobacteria and that all mitochondrial homologs are monophyletic in origin.
Falah, Mizied, "Evolutionary studies based on the 70-kDa heat shock family of protein sequences" (1997). Open Access Dissertations and Theses. Paper 3352.