No description available.

Transformation by human adenovirus type 5 requires the cooperation of gene products from both the E1a and E1B early transcription units. Our major goal was to better understand the individual roles that the E1B proteins play in the transformation process. In order to determine the specific contribution made by the two major E1B proteins, 19K and 58K, mutants were constructed which were defective in the synthesis of each protein. Analysis with these mutants suggested that 58K appeared to be necessary for efficient plaque formation on human HeLa cells whereas 19K was not required. Mutants which failed to produce 19K or made a truncated 19K product displayed the cyt/deg phenotype characterized by production of large plaques and degradation of DNA These properties were not apparent with point mutants at methionine 120 or serine 164 of 19K or with mutants defective for 58K production. All E1B mutants produce E1A at levels comparable to wild type adenovirus 5, suggesting that neither E1B protein affects the regulation of E1A expression. Of interest was the observation that in combination with E1A, both 19K and 58K were able to induce transformation of baby rat kidney cells. However, the efficiency of transformation was greatly increased if both these E1B products were present. It seems likely that the mechanism of transformation involving each of these E1B proteins utilizes different pathways, but these pathways appear to be additive. / Thesis / Master of Science (MS)

Background: Genome-wide association studies (GWAS) prove to be a powerful approach to identify the genetic basis of various human[1] diseases. Here we take advantage of existing GWAS data and attempt to build a framework to understand the complex relationships among diseases. Specifically, we examined 49 diseases from all available GWAS with a cascade approach by exploiting network analysis to study the single nucleotide polymorphisms (SNP) effect on the similarity between different diseases. Proteins within perturbation subnetwork are considered to be connection points between the disease similarity networks. Results: shared disease subnetwork proteins are consistent, accurate and sensitive to measure genetic similarity between diseases. Clustering result shows the evidence of phenome similarity. Conclusion: our results prove the usefulness of genetic profiles for evaluating disease similarity and constructing disease relationship networks. / Master of Science

No description available.

Digitized by Kansas Correctional Industries

The ability of mutations to change the fold of a protein provides evolutionary pathways to new structures. To study hypothetical pathways for protein fold evolution, we designed intermediate sequences between Xfaso1 and Pfl6, two homologous Cro proteins that have 40% sequence identity but adopt all–α and α+β folds, respectively. The designed hybrid sequences XPH1 and XPH2 have 70% sequence identity to each other. XPH1 is more similar in sequence to Xfaso1 (86% sequence identity) while XPH2 is more similar to Pfl6 (80% sequence identity). NMR solution ensembles show that XPH1 and XPH2 have structures intermediate between Xfaso1 and Pfl6. Specifically, XPH1 loses α-helices 5 and 6 of Xfaso1 and incorporates a small amount of β-sheet structure; XPH2 preserves most of the β-sheet of Pfl6 but gains a structure comparable to helix 6 of Xfaso1. These findings illustrate that the sequence space between two natural protein folds may encode a range of topologies, which may allow a protein to change its fold extensively through gradual, multistep mechanisms. Evolving a new fold may have consequences, such as a strained conformation. Here we show that Pfl6 represents an early, strained form of the α+β Cro fold resulting from an ancestral remnant of the all-α Cro proteins retained after the fold switch. This nascent fold can be stabilized through deletion mutations in evolution, which can relieve the strain but may also negatively affect DNA-binding function. Compensatory mutations that increase dimerization appear to offset these effects to maintain function. These findings suggest that new folds can undergo mutational editing through evolution, which may occur in parallel pathways with slightly different outcomes.

A wide variety of proteins which are synthesised in the cytoplasm of E. coli are subsequently directed either to non-cytoplasmic compartments or transported to the extracellular medium. Proteins which are exported from the cytoplasm are thought to interact with a complex cellular machinery and a number of mutations affecting this secretion machinery have been isolated. In this study, the export of the outer membrane protein TonA was used as a model system to examine the effect on protein translocation of two temperature sensitive secretion mutants, secA and secY. Initial analysis of the effect of secAts mutations on bulk envelope protein synthesis confirmed the key role of SecA in protein transport, including many proteins assembled into the inner membrane. Analysis of the rate of processing of preTonA, pulse-labelled at the restrictive temperature and chased at the permissive temperature revealed differences between SecA and SecY mutants. In particular these data indicate that SecA and SecY may interact sequentially to promote protein export and that SecA may be required to maintain preTonA in a translocationally competent form prior to interaction with SecY. In order to investigate the nature of a specific "export" signal within a protein to be exported, the possibility of using the novel secretion signal at the C-terminus of E. coli haemolysin to direct chimeric protein into the medium was also investigated. The C-terminal signal was successfully fused to a hybrid protein containing a few residues of ss-galactosidase and the majority of E. coli outer membrane protein OmpF lacking its own NH2-terminal signal sequence. The chimeric protein is specifically translocated across the inner and outer membranes and is released into the medium. Consistent with a transport system which bypasses the periplasm, other studies indicated that haemolysin transport is secA independent but may involve secY. Finally, the localisation of haemolysin and several outer membrane proteins synthesised in spheroplasts was also examined in the hope of gaining some further insight into the route taken by proteins which reach the outer membrane or the external medium.

The majority of outer membrane, periplasmic and some inner membrane proteins of Escherichia coli are synthesised with signal sequences which initiate the translocation process. It has been suggested that other polypeptide sequences within the mature protein carry additional information which determines the final localisation of the product. The aim of this project was to investigate the assembly into the outer membrane of the E. coli ferrichrome receptor protein, TonA. The tonA gene was subcloned onto pBR325 in order to maximise expression of this normally minor outer membrane protein. A study of the kinetics of assembly of TonA in a strain harbouring a multicopy plasmid carrying tonA revealed the occurrence of a processed assembly intermediate which separated with the soluble (cytoplasmic plus periplasmic) fraction of sonicated cells. The position and direction of transcription of tonA was deduced by Tn1000 mutagenesis followed by analysis of the resultant truncated TonA' polypeptides synthesised in vitro and in maxicells. All the TonA' polypeptides thus produced, even those with apparently small C-terminal deletions, fractionated with the sarkosyl soluble envelope material in maxicells (wild type TonA is sarkosyl insoluble), suggesting an important role for the C-terminus in assembly. A similar result was obtained when the tonA gene was truncated using an "oligo-stop translation" sequence. This eliminated the possibility that complete assembly of the TonA' polypeptides truncated by Tn1000 insertion was prevented by Tn1000 encoded sequences at their C-termini. Synthesis of the hybrid MalE-LacZ protein, 72-47, was demonstrated to inhibit the processing of TonA and several inner membrane proteins. Since this hybrid was already known to block the assembly and processing of periplasmic and outer membrane proteins, this result suggests that all three classes of exported protein share common steps in their assembly.

Various results have suggested that in Escherichia coli murein assembly may involve a protein complex(es) which could include low molecular mass penicillin-binding prpteins (PBPs). These proteins include PBP4, PBP5 and PBP6 which are penicillin sensitive enzymes associated with the periplasmic face of the inter membrane. The levels of these associations have been linked to enzymic activity and elucidation of the mechanism(s) involved in these associations may help identify and understand the regulation of this putative protein complex. It is currently accepted that the membrane associations of PBP5 and PBPÔ involve C-terminal amphiphilic cz-helices and such helices are ubiquitously employed in the lipid associations of membrane interactive protein molecules. Whether such helical structure features in the membrane associations of PBP4 or indeed if this protein is membrane bound or soluble, are, as yet, open questions. The focus of this research has been to investigate the lipid and membrane interactions of PBP4, PBP5 and PBP6 and in particular, to investigate the role played by these interactions of the C-terminal region of these proteins. Haemolytic analysis has shown that peptide homologues of the PBP5 and PBP6 C-terminal regions, P5 and P6, are active at the membrane interface and CD analysis has shown that these peptides possess a capacity for a-helix formation. CD and pressure - area isotherm analysis of monolayers formed from PS and P6 have shown that these peptides are able to adopt a-helical structure at an air - water interface. Monolayer studies have shown that P5 and P6 are able to interact with lipids and that these interactions are characterised by minor requirements for anionic lipid and the involvement of predominantly hydrophobic forces which are enhanced by low pH. Similar characteristics were revealed when perturbant washes and Western blotting were used to investigate the interactions of PBP5 with membranes derived from a mutant E. coli strain, HDL 11, in which the level of anionic lipid can be controlled. Overall, these results strongly support the hypothesis that C-terminal amphiphilic a-helices feature in PBP5 and PBP6 membrane anchoring. Molecular area determinations have implied that a peptide homologue of the PBP4 C-terminal region, P4 is able to adopt a-helical structure and this was confirmed by CD analysis. P4 showed no haemolytic activity but the peptide was found to interact generally with lipid monolayers. These monolayer interactions were characterised by a requirement for anionic lipid and involved predominantly electrostatic forces, which were enhanced by low pH. Similar characteristics but with no detectable requirement for anionic lipid were revealed when perturbant washes and chemiluminesence were used to investigate the interactions of PBP4 with membranes of the overproducing strain HB 10 I/pBK4 and those of HDL1 1. It is suggested that the PBP4 C-terminal region may play a role in PBP4 - membrane anchoring. Using chemiluminesence, no soluble form of PBP4 could be detected in the wild type E. coli, MRE600, suggesting that in wild type strains, PBP4 is exclusively membrane bound. It is suggested that PBP4 - membrane anchoring occurs at a specific binding site and overall, may differ fundamentally from that of PBP5 and PBP6.

No description available.