The D153del mutation in GNB3 gene causes tissue specific signalling patterns and an abnormal renal morphology in rge chickens

Background The GNB3 gene is expressed in cone but not rod photoreceptors of vertebrates, where it acts as the β transducin subunit in the colour visual transduction process. A naturally occurring mutation ‘D153del’ in the GNB3 gene causes the recessively inherited blinding phenotype retinopathy globe enlarged (rge) disease in chickens. GNB3 is however also expressed in most other vertebrate tissues suggesting that the D153del mutation may exert pathological effects that outlie from eye. Principal Findings Recombinant studies in COS-7 cells that were transfected with normal and mutant recombinant GNB3 constructs and subjected to cycloheximide chase showed that the mutant GNB3d protein had a much shorter half life compared to normal GNB3. GNB3 codes for the Gβ3 protein subunit that, together with different Gγ and Gα subunits, activates and regulates phosphorylation cascades in different tissues. As expected, the relative levels of cGMP and cAMP secondary messengers and their activated kinases such as MAPK, AKT and GRK2 were also found to be altered significantly in a tissue specific manner in rge chickens. Histochemical analysis on kidney tissue sections, from rge homozygous affected chickens, showed the chickens had enlargement of the glomerular capsule, causing glomerulomegaly and tubulointerstitial inflammation whereas other tissues (brain, heart, liver, pancreas) were unaffected. Significance These findings confirm that the D153del mutation in GNB3 gene targets GNB3 protein to early degradation. Lack of GNB3 signalling causes reduced phosphorylation activity of ERK2 and AKT leading to severe pathological phenotypes such as blindness and renal abnormalities in rge chickens

Identification and Characterization of Novel Collagen Chains

Collagen VI and collagen XXVIII are two extracellular matrix proteins that belong to the superfamily of von Willebrand Factor A (VWA) domain containing molecules. Earlier studies on collagen VI indicated that this widely distributed protein is composed of α1, α2 and α3 polypeptide chains, which form a microfibrillar network in close association with basement membranes in muscle and other tissues. In contrast, an initial study on collagen XXVIII reported that it forms a homotrimer and has a very restricted localization at specific basement membranes of peripheral nerves. In this dissertation, the identification and characterization of three novel collagen VI chains, α4, α5 and α6, that show similarity to the collagen VI α3 chain is described. The genes coding for the new chains are arranged in tandem on mouse chromosome 9. The proteins contain seven N-terminal VWA domains followed by a collagenous domain, two C-terminal VWA domains and a unique domain. In addition the collagen VI α4 chain carries a Kunitz domain at the C-terminus whereas the collagen VI α5 chain contains an additional VWA domain and unique domain. The lengths of the collagenous domains and the positions of the structurally important cysteine residues are identical in the collagen VI α3, α4, α5 and α6 chains. In mouse, the new chains show a very restricted and differential expression mainly associated with basement membranes. They are sometimes detected in regions where the collagen VI α3 chain is not expressed, suggesting that the α3 chain is not required for their assembly. Analysis of the collagen VI α1 chain deficient mouse strain, confirmed that the new chains require the α1 chain and may substitute for the α3 chain, probably forming α1α2α4, α1α2α5 and α1α2α6 heterotrimers. In humans, only the genes coding for the collagen VI α5 and α6 chains are preserved. The COL6A4 gene has been inactivated due to large pericentric inversion on chromosome 3 that split the gene in two pieces and transformed it into two non-processed pseudogenes. In humans, the collagen VI α5 and α6 chains are present in close association with the basement membranes of skeletal muscle and skin. Ullrich Congenital Muscular Dystrophy (UCMD) and Bethlem Myopathy (BM) patients carrying mutations in COL6A1, COL6A2 and COL6A3 show also skin phenotypes like keloid scarring or keratosis pilaris. Immunohistochemical analysis of the new chains in the skin of UCMD and BM patients showed a disturbed staining pattern only when the COL6A1 or COL6A2 genes are affected. This indicates that the new chains may substitute for the collagen VI α3 chain forming α1α2α5 and α1α2α6 heterotrimers. However the exact role of new chains for the development of skin phenotypes in myopathy patients remains to be elucidated. The functional role of collagen XXVIII is not known. Therefore, the inactivation of the Col28a1 gene in mouse was initiated. A targeting vector disrupting the exon 2 of Col28a1 was generated in vitro, followed by ES cell targeting in vivo. Positive ES clones were injected into blastocysts and transferred to surrogate mothers, which resulted in a chimeric mice carrying both the wild type and the targeted allele. However, the targeted allele did so far not enter the germline

Buschke-Ollendorff Syndrome Associated with Elevated Elastin Production by Affected Skin Fibroblasts in Culture

Buschke-Ollendorff syndrome (BOS; McKusick 16670) is an autosomal dominant connective-tissue disorder characterized by uneven osseous formation in bone (osteopoikilosis) and fibrous skin papules (dermatofibrosis lenticularis disseminata). We describe two patients in whom BOS occurred in an autosomal dominant inheritance pattern. The connective tissue of the skin lesions showed both collagen and elastin abnormalities by electron microscopy. Cultured fibroblasts from both patients produced 2–8 times more tropoelastin than normal skin fibroblasts in the presence of 10% calf serum. Involved skin flbroblasts of one patient produced up to eight times normal levels, whereas apparently uninvolved skin was also elevated more than threefold. In a second patient, whose involvement was nearly complete, elastin production was high in involved areas and less so in completely involved skin. Transforming growth factor-β1 (TGFβ1), a powerful stimulus for elastin production, brought about similar relative increases in normal and BOS strains. Basic fibroblast growth factor, an antagonist of TGFβ1-stimulated elastin production, was able to reduce elastin production in basal and TGFβ1 stimulated BOS strains. Elastin mRNA levels were elevated in all patient strains, suggesting that Buschke-Ollendorff syndrome may result, at least in part, from abnormal regulation of extracellular matrix metabolism that leads to increased steady-state levels of elastin mRNA and elastin accumulation in the dermis

CK2 kinase activity but not its binding to CK2 promoter regions is implicated in the regulation of CK2α and CK2β gene expressions

Protein kinase CK2, a ubiquitous serine/threonine kinase in control of a variety of crucial cellular functions, is composed of catalytic a- and a0-subunits and non-catalytic b-subunits which form holoenzymes such as CK2(ab)2, CK2aa\u27b2, or CK2(a\u27b)2. In addition, there is sample evidence for the occurrence of the individual subunits beside the holoenzyme. While the CK2 subunits are well analyzed on the protein level, only little is known about the regulation of their transcription. The existence of multiple forms of CK2 subunits raised the question about a mutual regulation of their expression. Here we defined two 50-upstream regions of the CK2alpha and the CK2beta genes, respectively, as sequences with promoter activities. We found that CK2alpah and CK2alpha\u27 stimulated the expression of the reporter constructs whereas, CK2beta was inactive. Using chromatin immunoprecipitation assays, we were unable to detect binding of endogenous CK2 subunits to these promoter sequences in vivo. However, it turned out that inhibition of the kinase activity of CK2 attenuated the promoter activity indicating that CK2alpha and CK2alpha\u27 might regulate their gene expression indirectly by phosphorylation reactions. Thus, we have shown here (i) that under normal physiological conditions CK2 does not bind to CK2 promoter regions and (ii) that the CK2 kinase activity is implicated in the regulation of its own expression

The identification and characterization of novel haemolysin genes from Clostridium difficile

Clostridium difficile is a Gram positive spore forming bacteria that is the leading cause of antibiotic associated diarrhoea and pseudomembranous colitis. C. difficile infection is a significant health care burden in hospital settings and in the community. One of the main factors that lead to C. difficile infection is the prolonged use of antimicrobial agents that disturb the microflora of the gut. This allows it to colonize and produce toxins which disrupt the integrity of the intestinal epithelium. Other than toxin production, C. difficile has a number of virulence factors including surface proteins and enzymes that aid in bacterial attachment and spread of infection. Another possible virulence factor that was described in clostridia and other bacteria is the production of haemolysins. These are proteins (sometimes lipids) produced by the bacterium and cause cytolysis of red blood and eukaryotic cells for example epithelial cells, leukocytes, lymphocytes, and macrophages. This work is the first report of C. difficile showing haemolytic activity and the aim of this thesis is to identify genes responsible for haemolysis in C. difficile. A CD630 genomic library was constructed in E. coli and six genes were found to confer haemolysis. One clone contained a gene (designated atlf) encoding a peptide homologous to an anthrax toxin lethal factor which exhibited large zones of haemolysis. Clostron and CRISPR-Cas9 based CD630 mutants were constructed with insertion and deletion in atlf respectively. The haemolysis of the mutants was tested on agar and by a quantitative assay, which revealed no change in the haemolytic phenotype compared to the wild type. This work suggests that haemolysis is a multifactorial phenomenon in C. difficile and may involve several genes. The CRISPR-Cas9 mutagenesis system constructed in this work will aid in many mutagenic studies to understand haemolysis and other virulence mechanisms in C. difficile

An investigation into the molecular basis of familial forms of osteoarthropathy in South Africa

Generalised osteoarthritis (OA) is a common disorder of the joints which can lead to pain and disability. Identification of the determinant gene(s) is limited in part by the lack of Mendelian inheritance in most forms of the disorder, the combination of genetic and environmental influences and the late development of the condition. An approach to the investigation of the aetiology of OA would be to take advantage of the monogenic basis of inherited skeletal dysplasias in which OA is a major component. For this reason, the molecular genetic basis of the epiphyseal dysplasias, which encompass a spectrum of phenotypes ranging from mild to severe skeletal involvement, is addressed in this thesis. Familial skeletal disorders in South Africa in which OA is a major feature were identified and investigated using intragenic and closely linked microsatellite markers in order to determine linkage to candidate genes. Mutational analysis was undertaken to identify the genetic defect

Developmental gene expression profile of Vmo1 in the mouse auditory system

Hearing loss (HL) is a sensory disorder that affects an estimated 250 million people worldwide and can greatly affect quality of life. In New Zealand, more than 10% of the population is affected by HL with the Māori population being overrepresented among all age groups. Therefore, understanding the mechanism of HL is extremely important for the development of new pharmaceuticals for the prevention or treatment of HL disorders. The main aim of the research undertaken in this thesis was to characterise the function of the Mus musculus (mouse) vitelline membrane outer layer one (Vmo1) gene. This gene is considered an excellent candidate for being involved in human HL and/or balance disorders. Our hypothesis is based on its restricted gene localisation within the mouse inner ear and the postulated function of Reissner’s membrane. Two methods were used to address this aim. Firstly, comparative genomics was used to determine the level of nucleotide and amino acid conservation of VMO1 across mammalian species, and to search for DNA motifs that may imply a biological function. Secondly, molecular biology and histochemical techniques were used to DNA sequence the Vmo1 gene, detect the expression of 22 kDa VMO1 protein within mouse tissues, and to localise the expression of VMO1 protein within the mouse inner ear. Comparative genomics results showed VMO1 to be highly conserved across 36 species. An in-depth analysis of the differences and similiarites between the mouse, human and chicken indicated a high level of gene conservation with an even greater degree of identity and similarity seen at a proteomic level. In addition, a high level of conservation across amino acids involved in the formation and stabilisation of the three dimensional structure. Thus, results suggest an important function for the VMO1 protein. Two commerical VMO1 antibodies were purchased to determine the localisation of the mouse VMO1 protein. They were validated for specificity using western blot analysis of protein lysates dissected from postnatal day 28 mice (P28). VMO1 was identified within the inner ear protein lysate and tear gland protein lysate of an expected molecular weight size of 20-37kDa with additional binding observed in the ear sample at 250kDa. Immunohistochemistry detected high concentrations of VMO1 protein within the tectorial membrane (TM) and inner pillar cells (IPC) in inner ear sections from the mouse at P5. In agreement with the comparative genomics analysis, VMO1 is a secreted protein. The movement of the hair cells (HC) relative to the TM is is essential for the transduction of sound into electrical signals. The IPC act as supporting cells for the hair cells, and help to couple movement of the basilar membrane to the HC. In conclusion, the importance of the TM and IPC in hearing function, and the localisation of the VMO1 protein within these structures implies an important role for VMO1 in hearing function. We recommend further studies to examine the specificity of the VMO1 antibody, and the development of a Vmo1 knockout mouse to support the functional analysis of Vmo1 in the auditory system