Structural and Functional Analysis of the Rabbit Erythroid-Specific Lipoxygenase Gene Promoter

The rabbit erythroid-specific lipoxygenase (RBC 15-LOX) is a member of a family of enzymes which catalyse the addition of molecular oxygen to certain polyunsaturated fatty acids. It functions in the inactivation and degradation of mitochondria during the final stages of erythroid maturation, following extrusion of the nucleus. The protein has been purified to homogeneity and extensively characterised (Rapoport et al, 1979), and both cDNA and genomic clones have recently been obtained (Thiele et al, 1987). RBC 15-LOX expression shows several interesting features, including an erythroid-specific accumulation of its mRNA (Thiele et al, 1987), and translational inactivation of the messenger (Thiele et al, 1982) . Data presented in this thesis includes the sequencing of the recently-cloned RBC 15-LOX gene in the region surrounding the ATG translation initiation codon, and mapping of the transcription initiation site to a position 28nt upstream of the ATG codon by a combination of primer extension and S1 nuclease protection analyses. No transcripts arising from the available 2.7kb of sequences upstream of this point have been detected by Northern blot, primer extension or SI nuclease protection analyses. A unique mRNA sequence, matching the genomic sequence immediately downstream of the putative transcription initiation site, has been obtained by primer extension mRNA sequencing. Functional analysis of 5' flanking sequences reveals two regions which affect transient expression of a cis-linked bacterial chloramphenicol acetyItransferase (CAT) reporter gene in established murine erythroleukaemia (MEL) and murine fibroblast cell lines. A proximal region within 150nt of the transcription initiation site functions as a promoter in both erythroid and non-erythroid cell lines, elevating CAT enzyme activity 7- to 8-fold above background, and can respond to a heterologous Friend Murine Leukaemia Virus enhancer. A distal region between 1.0 and 2.7kb upstream of the transcription initiation site confers a 6- to 8-fold cell type-specific effect upon CAT expression from the RBC 15-LOX promoter in the same assay system. The proximal region contains several sequence motifs which are also seen in the 5' flanking sequences of a variety of other genes, including: a TATA-like sequence; two CACCC motifs; a GGGCGG element; and a sequence resembling the cognate binding site for the CTF/NF-1 family of transcription factors. In vitro DNasel footprinting of this proximal region using nuclear protein extracts from erythroid and non-erythroid murine cells shows protection of both the proximal CACCC sequence and the CTF/NF-l-like element. Future experimental strategies for investigating a possible role for the regulation of transcription in the erythroid-specific accumulation of RBC 15-LOX mRNA are discussed. Also considered are aspects of the data presented here which, in conjunction with the work of others, may have implications for the translational inactivation of RBC 15-LOX transcripts, and for the evolution and structure-function relationships of the lipoxygenase enzyme family

Studies on ovine tumour necrosis factor-alpha

Tumour necrosis factor alpha (TNFa), a mediator of inflammatory responses and pathologies of a wide variety of diseases, has been extensively studied in humans and mice. However, little has previously been known about this cytokine in the sheep, a species of value not only to the agricultural industry, but also as a laboratory animal.In this work, the cDNA encoding ovine TNFa has been amplified, cloned, sequenced and used to express recombinant ovine TNFa (rovTNFa). The latter has been partially purified, characterised and used to raise both poly- and mono-clonal antibodies.The sequence of ovine TNFa shows a high degree of homology to those of other species. Certain regions, which are known to be structurally or functionally important to the mRNA and/or protein, are particularly well conserved. Consequently, rovTNFa displays several biological activities previously noted for TNF'sa of other species, including cytotoxicity, enhancement of thymocyte and fibroblast proliferation and cartilage-degrading and anti-viral activities. However, whilst rovTNFa is active in assays on ovine cells at concentrations comparable to those observed in similar assays for other species, it is 1000 fold less active than recombinant human TNFa (rhTNFa) in cyto¬ toxicity assays on TNF-sensitive murine (L929) cells, whose general lack of species specificity allows their use in detecting TNF'sa from many sources.A monoclonal antibody raised to rovTNFa detects a glycoprotein of appropriate size for mature ovine TNFa in the supernatants of stimulated ovine cell cultures. As in other species both ovine TNFa mRNA and protein are rapidly inducible. Such supernatants repeatedly have no activity in cytotoxicity assays (sensitive to 30pg rhTNFa/ml) on L929 cells, in spite of many containing >lng ovine TNFa/ml. At least one of these supernatants displays biological activity attributable to TNFa (through its neutralisation by a polyclonal antiserum raised to rovTNFa) in an assay on ovine cells. By comparing the amino acid sequences of TNF'sa from many species and using knowledge gained from structure/function studies on human TNFa, possible explanations for the apparent species specificity of ovine TNFa are proposed.Finally, preliminary investigations have been performed to examine the role that TNFa plays in Maedi-Visna (MV) disease, a chronic lentivirally-induced ovine disease. RovTNFa can differentially regulate MV viral expression in different cell types in vitro, whilst the native protein is produced in MV-infected cultures of adherent ovine lung cells. Ovine TNFa may therefore play a complex role in MV disease, both by contributing to its pathogenesis and influencing the viral life-cycle

The Distribution of Interferon-Alpha in Normal Human Tissues

The presence of interferon-alpha (IFN-alpha) in human tissues has been described extensively in viral infections. In the last decade many workers have also shown the presence of low levels of IFN-alpha in conditions other than viral infections. While the precise origin of the synthesis of low levels of IFN-alpha in these physiological conditions has not been clearly defined, some evidence has suggested that macrophages may be involved. In an attempt to find the likely source of IFN-alpha in physiological conditions, an initial study was carried out in which the cellular distribution of immunoreactive IFN-alpha was studied in formalin fixed paraffin embedded normal adult human tissues from 38 different organs using various immunocytochemical techniques. These samples were drawn from over 300 individuals none of whom had evidence of viral infection. Tissue histiocytes from all organs in the body, with the exception of cerebral and cerebellar cortex in brain and renal cortex and medulla, stained positively for IFN-alpha. Kupffer cells, pulmonary alveolar macrophages and lymph node macrophages were also positive for IFN-alpha. Parenchymal cells in some other organs also contained immunoreactive IFN-alpha. These included syncytiotrophoblast in first, second and third trimester placentas, cuboidal lining cells of the choroid plexus in the brain, thyroid follicullar cells, pituitary endocrine cells, adrenocortical cells and parathyroid endocrine cells. These findings are compatible with previous suggestions that IFN-alpha may be synthesized and released in the absence of viral infection and may have a role in normal physiology. The presence of IFN-alpha in most cells of the mononuclear phagocyte system suggests that these cells play a major role in the defence against viral infection. This speculation, however does not preclude other possible roles for IFN-alpha, such as modulation of cell growth, major histocompitability antigen expression etc. The demonstration of immunoreactive IFN-alpha in formalin fixed paraffin embedded normal adult human tissues prompted other studies. In the first of these studies the cellular distribution of immunoreactive IFN-alpha was studied in formalin fixed paraffin embedded normal human autopsy tissues from 32 fetuses (7-42 weeks gestation) and 20 infants (aged from a few hours to 24 months). This study was performed to test the hypothesis that microbes have a role in switching on IFN-alpha synthesis. Fetal tissues are "germ free" while the infants had been exposed to a normal microbial flora. Immunoreactive IFN-alpha was first seen at 9 weeks gestation in macrophages in the fetal liver and thereafter was seen in macrophages in most other organs except in kidneys and cerebral and cerebellar cortex. When infant lungs were compared with fetal lungs a statistically significant increase in the number of macrophages (P< 0.0001, Mann-Whitney test) and the percentage of these cells expressing IFN-alpha (P <0.0005, Mann-Whitney test) was noted in infant lungs. No such changes were observed in spleen, liver and thymus following birth. These findings suggested that there is a basal level of IFN-alpha production by macrophages, which is not dependent on microbial products, but that such microbial products can enhance synthesis of this cytokine. Immunoreactive IFN-alpha was also demonstrated in parenchymal cells of thyroid gland, choroid plexus in brain, anterior pituitary gland and adrenal gland in the fetal and infant tissues. These findings were almost identical to those seen in adult tissues. In a separate study an attempt was made to extract, detect and quantify IFN-alpha in human tissues using protein extraction and an immunoradiometric assay kit for the detection of IFN-alpha. 20 placentas (14-42 weeks gestation) obtained fresh within 1-2 hours of vaginal delivery, 4 specimens of amniotic fluid obtained at the time of caesarean section from 37-39 weeks gestation pregnancies, 10 samples of choroid plexus and cerebral cortex, 11 thyroid glands and 9 fetal adrenal glands from adult and fetal autopsies performed within 10-24 hours of death were studied. IFN-alpha was detected in 9 placentas, 1 adult thyroid gland and all 4 amniotic fluids. However, this study failed to detect IFN-alpha in the remaining placentas and adult thyroid glands and in all choroid plexuses, cerebral cortex and fetal adrenal glands. Formalin fixed paraffin embedded tissues from these organs did show immunoreactive IFN-alpha in cells using the immunocytochemical techniques. Finally an attempt was made to detect IFN-alpha messenger RNA (mRNA) in normal human tissues using an in situ hybridization method

Genes: Multigene Families, Control of Gene Expression, Genetic contributions to Human Diseases, including Chromosomal Fragile Sites and ‘Dynamic’ and ‘Non-self’ Mutations

The early work in this thesis utilizes the general approach of comparative analysis. In order to find out the relationship between entities (either functional or genetic) my colleagues and I have attempted to identify the important elements by detecting similarity between those entities that act in a similar manner. The philosophy behind this approach is simply that when two distinct objects perform a similar process then the requirements essential for that process will be revealed as similarities between those objects above a noise of difference between them. The use of comparative analysis in biological systems is an attempt to identify natural order from apparent chaos. This work includes but is not limited to :- 1. discovery of the family of kallikrein genes and exploration of their roles in biology, 2. identification of the DNA sequence elements required for hormonal and heavy metal control of metallothionein gene expression 3. discovery of at least some of the necessary and sufficient conditions for the appearance of fragile sites on chromosomes, and their consequent contributions to disease, 4. the molecular properties of repeat DNA sequence expansion that lead to dynamic mutation and consequent fragile site expression and / or disease pathogenesis. In a sense the use of genetic animal models in order to study gene function and pathogenesis follows similar logic of comparative analysis – the mutation of a single endogenous gene or the expression of a single introduced mutated gene in a (presumed) constant genetic background to enable the biological consequences of the genetic mutation or aberrant gene expression by comparing animals from the ‘wild-type’ or parent line with those that now carry the mutation or altered gene. This approach has been utilized in the most recent work contained herein as a means to determine gene function and / or to model human genetic disease pathogenesis, specifically pathogenic mechanisms of the protein WWOX in cancer and expanded repeat RNAs in neurodegenerative diseases. The culmination of this recent work is the development of an hypothesis – 4. that expanded repeat double-stranded RNA leads to neurodegeneration through its recognition by the RNA-binding pattern recognition receptors as a ‘non-self’ or foreign nucleic acid due to a paucity of RNA modification. The resultant pathogenic mechanism is therefore autoinflammatory disease. Given the wide range and variety of evidence of inflammatory activation in neurodegenerative diseases in general, this mechanism is therefore hypothesized to be the general causal mechanism for most (or all) of these diseases. A specific Introduction - highlighting the nature and significance of the work, and a Conclusion – of how this work has contributed to knowledge, are given at the start of each chapter, while the impact of the various components of this work is indicated by the number of citations for each of the included publications. Authorship contributions to each of the included publications in this work are also indicated with each specific reference.Thesis (DSc) -- University of Adelaide, School of Biological Sciences, 202

The 37kDa/67kDa laminin receptor as a therapeutic target in prion diseases: potency of antisense LRP RNA, siRNAs specific for LRP mRNA and a LRP decoy mutant

Prion diseases are a group of rare, fatal neurodegenerative diseases, also known as transmissible spongiform encephalopathies (TSEs), that affect both animals and humans and include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, chronic wasting disease in deer and elk and Creutzfeldt-Jakob disease (CJD) in humans. TSEs are usually rapidly progressive and clinical symptoms comprise dementia and loss of movement coordination. A common hallmark of TSEs is the accumulation of an abnormal isoform (PrPSc) of the host-encoded prion protein (PrPc) in the brains of affected animals and humans. PrPc is a highly conserved cell surface sialoglycoprotein that is expressed in several cell types, mainly neuronal cells, but its normal physiological function is still not known. However, PrPc is elementary for the acquisition and the replication of prion diseases. Several inhibitors of the PrPSc formation have been reported, but none of them showed great potency in an in vivo application. Thus, the identification of the 37kDa/67kDa laminin receptor (LRP/LR) as the cell surface receptor for prions opened a new direction for the development of a TSE therapy. Currently, no treatment to slow down or stop the disease process in humans with any form of CJD is established. However, several strategies have been investigated to find an anti-prion treatment including development of a vaccination therapy and screening for potent chemical compounds. In scrapie-infected neuronal cells, which represent a widely used and well characterized in vitro model for transmissible spongiform encephalopathies, the accumulation of PrPSc has been prevented by transfection of (i) antisense LRP RNA, (ii) small interfering RNAs targeting the LRP mRNA and (iii) incubation with the polyclonal anti-LRP antibody W3. Furthermore, the knock down of surface LRP/LR resulted in a reduction of the cellular PrP levels, suggesting an interference with the PrP internalization process. Thus, LRP/LR is required for the PrPSc propagation in vitro and involved in the PrPc metabolism.Due to the existence of several LR genes, a major step to investigate the role of the 37kDa/67kDa laminin receptor in scrapie pathogenesis in vivo is the generation of transgenic mice exhibiting a lower level of LRP/LR. Hemizygous transgenic mice that express LRP/LR antisense RNA under the control of the neuron-specific enolase (NSE) promoter were generated and showed a reduced LRP/LR protein level in the cerebellum and the hippocampus. Intracerebral inoculation of these transgenic mice with the scrapie agent will show, whether the accumulation of pathogenic PrPSc in the brain is delayed or prevented due to a reduced LRP/LR level. A further therapeutic anti-prion approach is given by LRP/LR deletion mutants that can be secreted to the cell culture medium and might act as decoys. Previously, it has been demonstrated that a transmembrane deletion mutant is able to prevent PrPc binding and internalization. In vitro studies using an N-terminally truncated LRP mutant, representing the extracellular domain of LRP/LR (LRP102-295::FLAG), revealed a reduced binding of (i) recombinant cellular PrP to mouse neuroblastoma cells, (ii) infectious moPrP 27-30 to BHK21 cells and (iii) interfered with the PrPSc propagation in chronically scrapie-infected mouse neuroblastoma cells. Furthermore, a cell free binding assay demonstrated the direct binding of the LRP102-295::FLAG mutant to both PrPc and PrPSc. These results together with the finding that that endogenous LRP levels remain unaffected by the expression of the mutant indicate that the secreted LRP102-295::FLAG mutant may act in a trans-dominant negative manner as a decoy by trapping PrP molecules. To investigate the therapeutic potential of the LRP102-295::FLAG decoy mutant in vivo transgenic mice were generated ectopically expressing LRP102-295::FLAG in the brain. Animals showed no phenotype and transgene expression was detected in cortical and cerebellar brain regions. An intracerebral prion inoculation of these mice will prove whether the expression of the LRP102-295::FLAG mutant can impair the PrPSc accumulation in the brain and can thus, act as a alternative therapeutic tool in prion diseases. The recent finding that experimental introduction of RNA can be used to interfere with the function of an endogenous gene (RNA interference) provided another tool for the development of gene-specific therapeutics. In order to evaluate a gene transfer therapeutic TSE strategy, human immunodeficiency virus (HIV)-derived vectors that express short hairpin RNA (shRNA) directed against the LRP mRNA were used. Following integration of LRP-shRNA-expressing lentiviral vectors into the genome of neuronal cells efficient LRP/LR downregulation was observed. In scrapie infected neuronal cells, downregulation of the LRP gene expression resulted in a diminishment of PrPSc propagation, providing a further therapeutic strategy in the development of a TSE treatment

Hair follicle germinative epidermal cells: a molecular study

At the base of the hair follicle epidermal matrix is a population of germinative epidermal (GE) cells that is in close communication with the dermal papilla. These GE cells are at the core of activities that comprise the fundamental processes of cell signalling and differentiation in the hair follicle. Since it is in the germinative region that the signals that produce hair are being received and transcribed, identification of genes expressed in the GE cells will be important for our understanding of hair growth control and the molecular mechanisms operating at the site of epidermal proliferation and differentiation. This study describes the production of a series of cDNA libraries, both by conventional means from rat vibrissa follicles and follicle end bulbs, and by PGR from the GE cells and the tissues of the upper end bulb. These libraries were then used for a variety of screening approaches to isolate cDNA clones, firstly for molecules which are known to be involved in the control of hair growth, and secondly for molecules which are differentially expressed in the follicular germinative epidermis. In order to identify such preferentially expressed genes, a dual labelling differential screen of the vibrissa follicle end bulb cDNA library was performed, using probes derived from the germinative epidermal and upper end bulb PGR generated libraries. Nine putative differentially expressed clones were isolated and sequenced. RNase protection analysis and non radioactive in situ hybridisation was then performed to confirm that these clones were expressed in the germinative epidermis of rat vibrissa follicles. Further characterisation by northern blotting revealed that several of the clones were expressed in multiple tissues. Nucleotide sequence analysis revealed that six of the clones had a concensus BG1 repeat sequence at the end of their 3'UTR. This has been implicated in post-transcriptional control of intracellular mRNA localisation. Three of these clones were related to genes implicated in induction and vesicle trafficking. These clones may therefore be involved in the signal transduction pathways operating in the germinative epidermis in response to primary signalling molecules received from the dermal papilla