LNX1 belongs to a family of multi-PDZ domain containing RING-type E3 ligases. Several interactions have been mapped to its PDZ domains, but the role of each domain in LNX function has not yet been determined. To study individual PDZ domain function in the context of full length protein I generated point mutations in peptide binding sites of each of PDZ domain, and in a putative phosphoinositide binding site of LNX1 PDZ4. Peptide binding was successfully disrupted by an arginine or lysine to alanine mutation in the peptide binding cleft. A LNX1 PDZ4 mutant with lysine residues in a putative phosphoinositide binding site mutated to glutamate displayed decreased membrane localization. The impact of each PDZ mutation on cell morphology and substrate ubiquitination was also investigated. I identified a potential role for PDZ binding in auto-inhibition of RING function. Additionally, novel interactions between LNX1 and Frizzled family members were identified and characterized.

LNX1 belongs to a family of multi-PDZ domain containing RING-type E3 ligases. Several interactions have been mapped to its PDZ domains, but the role of each domain in LNX function has not yet been determined. To study individual PDZ domain function in the context of full length protein I generated point mutations in peptide binding sites of each of PDZ domain, and in a putative phosphoinositide binding site of LNX1 PDZ4. Peptide binding was successfully disrupted by an arginine or lysine to alanine mutation in the peptide binding cleft. A LNX1 PDZ4 mutant with lysine residues in a putative phosphoinositide binding site mutated to glutamate displayed decreased membrane localization. The impact of each PDZ mutation on cell morphology and substrate ubiquitination was also investigated. I identified a potential role for PDZ binding in auto-inhibition of RING function. Additionally, novel interactions between LNX1 and Frizzled family members were identified and characterized.

No description available.

In this study, self-catalytic maturation of heterologously expressed pro-galactose oxidase was analysed in E.coli by altering some amino acids which were supposed to play a crucial role in pro-peptide removal. Galactose oxidase (GOase / EC 1.1.3.9) from Fusarium graminearum / having a molecular mass of 68kDa, is a monomeric, copper containing enzyme with an unusual thioether bond. The enzyme is produced as a precursor with an additional 8 amino acid pre- and a 17- amino acid pro-sequence at the N terminus. Previous work has shown that the pre-peptide is removed possibly by a protease during secretion, whereas the 17 amino acid pro-peptide is removed autocatalytically by the aerobic addition of Cu2+ to the precursor, preceding the formation of the thioether bond at the active site. The pro-gao gene was on ProGON1 and ProGOMN1 constructs which were previously established on pET101/D/lacZ vector in England by directed evolution. ProGON1 contains silent mutations at the N-terminus different from native galactose oxidase whereas ProGOMN1 has six further mutations within the mature enzyme, providing high expression. The cleavage site mutations R-1P/A1P, R-1X/A1X, S2A, and the H522A mutation just against the cleavage site in the three dimensional configuration, were carried out by site-directed mutagenesis. Those and some extra mutations were confirmed by DNA sequence analysis. Next, mutant galactose oxidases were expressed in E. coli BL21 Star (DE3), and were purified by Strep-Tactin&reg / Sepharose&reg / column, operating on the basis of affinity chromatography. Subsequently, SDS-PAGE was performed to analyze self-processing by detecting molecular mass difference of protein bands resulting from pro-sequence removal or existence. When the bands obtained in SDS-PAGE were compared, it was seen that the products of original recombinant plasmids, i.e. ProGON1, ProGOMN1 / and the mutational variants showed no difference in band size, all slightly above 70kDa / indicating pro-sequence presence on all constructs. Non-mutants and some of the mutants showed galactose oxidase activity, signifying proper active site construction by thioether bond formation. ProGOMN1 was submitted for N-terminal amino acid sequencing to be able to assert that a size above 70kDa is not solely due to the existence of a 1 kDa Strep-tag II at C-terminus. Sequencing data affirmed the presence of both the pre-peptide and the pro-preptide showing that processing has not occurred at the N-terminus. Accordingly, in this study, it was shown for the first time that the existence of a pre-pro-peptide at the N-terminus of galactose oxidase does not prevent thioether bond formation at the active site. Furthermore, since the pro-peptide is cleaved autocatalytically, the lack of removal of the pre-peptide in E.coli in the presence of Cu 2+ and oxygen is very likely to be the cause of lack of pro-peptide cleavage. In future studies the region corresponding to the pre-peptide will be deleted to prove this hypothesis.

The G-protein coupled receptors (GPCRs) comprise the largest family of receptors in humans and other vertebrates. They are embedded in the cell membrane and are activated by many different signaling molecules. Activation modulates cellular signal transduction pathways and influences many physiological processes. Therefore the GPCRs are important as targets for numerous drugs. The receptors for NPY (neuropeptide Y) belong to GPCRs of Class A (rhodopsin-like). NPY and its related peptides PYY and PP are involved in the regulation of appetite, blood pressure and many other processes. They share a common structure and interact with the receptors Y1, Y2, Y4 and Y5 in mammals, and, in addition, Y7 and Y8 in amphibians and bony fishes. This thesis is focused on the human Y2 receptor, known to reduce appetite, by investigating the importance of thirteen amino acid residues for ligand binding. Mutagenesis followed by functional expression and receptor binding was conducted. During the course of this work several new GPCR crystal structures have been resolved, thereby improving the receptor modeling in papers I-III. The major finding is that even though the Y1 and Y2 receptors have evolved from a common ancestor, their points of ligand interaction differ and have thus changed during evolution. In general, the positions investigated resulted in milder changes in the ligands’ affinities for Y2 compared to Y1. These findings were incorporated in the design of new Y1 and Y2 receptor models, leading to improved understanding of how such divergent receptors, sharing only 30 percent sequence identity, can still interact with the same ligands. Notably, several of the mutations introduced in Y2 resulted in increased affinity. A novel NPY receptor gene named Y2-2 was identified in the genomes of zebrafish and medaka. This brings the number of zebrafish NPY receptors to seven. The binding characteristics of zebrafish Y2-2 differed from zebrafish Y2 mainly in the interaction with NPY13-36 and the antagonist BIIE0246. In conclusion, these results increase our understanding of ligand interactions with GPCRs and will be useful for refinement of ligand-receptor models for future development of receptor subtype-selective drugs.

Errata sheet pasted opposite Table of Contents. / Includes bibliographies. / 142, [171] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis investigates the UV-induced inhibition of oriC initiation function and the nature of the phenotype of the mutant origin of replication of Escherichia coli, oriCc. The specific aims of this study are to delineate the source of the UV-induced trans-acting inhibition of oriC function, utilizing the phage vector [lambda]poriCc, and to demonstrate the oriCc allele as an enhanced DNA replication initiator from oriC. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1997?

Bibliography : leaves i-xxiii. / vi, 95, xxiii, [170] leaves, [4] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1991

The epigenetic contribution to phenotype is now well established. Studies over the past decade have shown that proteins that are able to establish and propagate epigenetic modifications are essential for mammalian development. Some of the genes involved in these processes have been identified, but the roles of many remain unknown. The mutagenesis screens for modifiers of position effect variegation in Drosophila suggest that there are over 200 genes that are able to modify epigenetic variegation. We emulated this screen in the mouse to identify mammalian modifiers of a variegating transgene. The screen aimed to identify novel genes involved in epigenetic reprogramming, and to generate mouse models to study the impact of disruption to the epigenome. Inbred male mice carrying a variegating GFP transgene expressed in erythrocytes were mutagenised with ENU. Offspring were screened by flow cytometry and in the initial rounds of mutagenesis, 11 dominant mutant lines were identified. These lines were called MommeDs (Modifiers of murine metastable epialleles, dominant). This thesis describes the mapping and phenotypic characterisation of three Momme lines: MommeD7, MommeD8 and MommeD9. The MommeD9 mutation enhances variegation and was mapped to a 3.4 Mb interval on Chromosome 7. A mutation in a 5? splice site was found in the Trim28 gene. Analysis of Trim28 mRNA and protein in heterozygotes showed that the mutant allele was null. Homozygotes die before mid-gestation. Heterozygotes are viable but display variable and complex phenotypes, including infertility, obesity, behavioural abnormalities and premature death. Obese MommeD9 mice have liver steatosis, impaired glucose tolerance and other indicators of metabolic syndrome. This phenotype has not previously been reported for mice haploinsufficient for Trim28. There is considerable variability of phenotypes among inbred MommeD9 heterozygotes, which suggests a role for epigenetics in phenotypic noise or “intangible variation”. MommeD8 is a semi-dominant enhancer of variegation. Some homozygotes are viable but some die around birth. Viable homozygotes weigh less than wildtype littermates and have increased CpG methylation at the GFP transgene enhancer element. The mutation was mapped to a 4 Mb interval on chromosome 4. Extensive candidate gene sequencing failed to find a mutation and so DNA from mutant and wildtype individuals were sequenced across the entire linked interval by 454 Sequencing technology. MommeD8 individuals carry two point mutations, one is intergenic and the other lies in an intron of the Ppie gene. Analysis of Ppie mRNA in heterozygotes and homozygotes shows that mutants have reduced transcript levels, suggesting that a deficiency in Ppie causes the increased silencing of GFP. The Ppie gene has not been reported to be involved in epigenetic reprogramming and little is known about its function. Mice heterozygous for MommeD7 have a marked increase in expression of GFP. Heterozygotes have a range of hematopoietic abnormalities including splenomegaly, anaemia and reticulocytosis. Homozygotes die at birth and appear pale. The increased GFP in the peripheral blood appears to be the consequence of an increase in reticulocytes. The mutation is linked to a 1.5 Mb interval on Chromosome 7. MommeD7 mice appear to have hematopoietic abnormalities that affect the expression of the erythroid-specific GFP reporter transgene. MommeD7 mice serve as a reminder that, as well as discovering bona fide modifiers of epigenetic reprogramming, the ENU screen can also identify hematopoietic mutants.

Thesis (Ph.D.)--McMaster University (Canada), 1996. / Source: Dissertation Abstracts International, Volume: 58-06, Section: B, page: 3006. Adviser: B. E. McCarry.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 25, 2009). Includes bibliographical references.