Theses (Biochemistry and Microbiology)
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Item Intron 5 of Ars2 contains internal ribosomal entry sites that control expression of cytoplasmic ARS2 protein isoforms(2024) Ngo, Quang Loc; Howard, Perry L.Ars2 is an essential gene in RNA metabolism that can undergo alternative splicing in which either all or a portion of its highly conserved intron 5 is retained. The retention of this intron 5, while resulting in transcripts with long 5’UTRs and uORFs that are sensitive to nonsense-mediated decay and are suboptimal to cap-dependent translation, generates cytoplasmic protein isoform of ARS2 (ARS2c) that are required for response to arsenic stress. Using a reporter plasmid that can generate circular RNAs, I demonstrated that mouse Ars2 intron 5 contains at least 3 internal ribosomal entry sites (IRESs). The presence of these IRESs within intron 5 may explain the translation of ARS2c, and its upregulation in arsenic stress, which normally result in global attenuation of cap-dependent translation. Structural prediction in combination with mutagenesis indicate that these IRESs are highly structured and form pseudoknots at their 3’ ends which are important for their activity. Additionally, my reporter system also indicated that intron 5 and its splice variants contain regulatory elements that modulate the activity of the IRESs. Taken together, my findings provide mechanistic insights into the translation controls of ARS2c. Given that ARS2c is involved in cellular stress response and is a potential cancer suppressor, this work may aid in the development of novel cancer therapeutic strategies.Item Characterization of epigenetic changes in Rana [Lithobates] catesbeiana tissues during natural and induced thyroid hormone-dependent metamorphosis using mass spectrometry(2024) Kuecks-Winger, Haley Noah; Helbing, Caren C.Thyroid hormone (TH) signaling is critical for proper development, growth and metabolism in all vertebrates. Amphibian metamorphosis is a TH-dependent developmental process that requires coordinated physical and biochemical changes to facilitate the transition from a tadpole to a frog. Metamorphosis involves extensive tissue-specific changes in the gene expression of differentiated tissues. Rana catesbeiana (American bullfrog) metamorphosis can be precociously induced by treatment with exogenous TH. However, metamorphosis is temperature-dependent and does not proceed at 5°C even in the presence of TH. Remarkably, a subsequent shift to permissive temperatures (24°C) results in an accelerated metamorphosis, implying that TH establishes a molecular memory at 5°C. Previous studies suggest that epigenetic processes, including histone variant incorporation and post-translational modifications, are involved in TH-signalling during natural metamorphosis and during temperature-modulated, TH-induced metamorphosis. Herein, we use mass spectrometry to characterize the histone composition of R. catesbeiana blood, liver, and tailfin during natural and temperature-modulated induced metamorphosis. The natural metamorphosis model identified tissue- and developmental stage-specific changes in histone abundance and PTMs. The temperature-modulated TH-induced metamorphic model demonstrated temperature- and tissue-specific changes in the abundance and PTMs of histones and other chromatin-binding proteins. To our knowledge, this represents the first unbiased analysis of the chromatin-associated proteins during amphibian metamorphosis. The findings presented herein expand our understanding of putative epigenetic factors involved in regulating TH-dependent development, which has broad relevance to all vertebrate species due to the conserved nature of TH action.Item Advancing T cell-based immunotherapies through targeted engineering with CRISPR-Cas9(2024) Carleton, Gillian; Lum, JulianT cell-based immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy have undoubtably revolutionized the treatment of cancer. However, the broad effectiveness of CAR-T cell therapy is hindered by several unresolved problems, most notably a lack of therapeutic efficacy in treating solid tumor cancers. A second challenge stems from the widespread use of viral vectors in CAR-T manufacturing, which poses safety risks to patients receiving treatment. Here, we showed that genome editing with CRISPR-Cas9 can be used to overcome both of these issues. As the solid tumor microenvironment (TME) is known to be metabolically suppressive, we devised a single-step editing method to enhance the metabolism and effector function of CAR-T cells. This approach combined CRISPR-mediated homology-directed repair with a gene-trap approach to link CAR integration with simultaneous deletion of a metabolic gene of interest. For proof-of-concept, we targeted the folate receptor alpha (aFR) CAR to the locus of the essential autophagy gene ATG5, and showed that editing at ATG5 could be achieved with a high level of on-target specificity. Functionally, deletion of ATG5 led to alterations in glucose and glutamine metabolism and enhanced CAR-T cell efficacy under nutrient-restricted conditions in vitro and in vivo. To address the safety concerns associated with viral transduction, we developed a process for nonviral manufacturing of clinical-grade CAR-T cells for B-cell malignancies. This approach used electroporation of a Cas9 ribonucleoprotein complexed with a linear double-stranded DNA template to facilitate site-specific insertion of a CD22 CAR at the T cell receptor alpha chain (TRAC) locus. In vitro, nonviral CD22 CAR-T cells exhibited comparable antitumor activity to lentiviral CD22 CAR-T cells. thereby establishing feasibility of our nonviral manufacturing process. Taken together, the results of these studies highlight the broad applicability of CRISPR-Cas9 as a tool for engineering safer, more effective T cell-based immunotherapies for patients with cancer.Item The cloning, purification and preliminary functional studies on lytB in Escherichia coli(2000) Zhang, LimeiThe lytB gene is located at about 0.5 min on Escherichia coli genetic linkage map. It is part of an operon, designated ileS-lsp, which consists of 5 genes: ribF, ileS, lsp, slpA and lytB (5' to 3'). Previous studies on the lytB gene suggested that it may be involved in the regulation of the starvation stress response, known as the stringent response, and in mediating tolerance to penicillin. However, recent studies indicate that the original lytB mutant actually carried multiple mutations, and the major phenotypic characteristics were due to a temperature-sensitive mutation in the ileS gene. A second unusual and unidentified dominant "temperature-sensitive mutation lies downstream of the lytB. Importantly, no mutation was found in lytB, and the function of this gene therefore currently unknown. The 5 genes in the ileS-lsp operon share no obvious relationship to each other, and there are no clues here regarding a possible function for lytB. The main objective of this thesis was to clone and express the E. coli lytB gene as a first step toward determining the biological function of LytB. The lytB gene was amplified from a previously cloned copy of ileS-lsp operon by polymerase chain reaction. It was cloned expressed from three different expression vectors. It was most efficiently expressed as a glutathione S-tranferase fusion protein from the cloning vector, pGEX-5X-1, and was purified in this form in high yield. The purified LytB protein was >95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blot analysis. The lytB gene is predicted to encode a 35.5-kDa protein, and this was confirmed in this study. Although the protein has not been quantified, preliminary experiments involving a Western blot analysis of crude cell extracts from wild type bacteria with a polyclonal anti-LytB antiserum indicate that LytB is a low abundance protein. The protein appeared to be unstable, and this characteristic may be worth further study. Several attempts were made to disrupt the chromosomal copy of the lytB gene. These attempts involved the in vitro insertion of a genetic element known as the n interposon into the lytB gene cloned into a co1E1 vector. The Ω interposon encodes spectinomycin resistance which offers the advantage of a direct selection for constructs. It also contains efficient transcriptional and translational terminators on its 3'-end and is consequently an effective gene disruptor. Three independent attempts were made to transform the constructed plasmid into a po1A (DNA polymerase 1-deficient) mutant strain. Co1E1 plasmids are unstable in Po1A mutants, and this method is used to force the insertion of cloned genes into the chromosome of the host by homologous recombination. All 3 attempts were unsuccessful. About 97% of the spectinomycin-resistant transformants still carried the plasmid in a stable form and presumably had developed a way of stabilizing Co1E1 plasmids, possibly through spontaneous suppressor mutations. The remaining 3% of the spectinomycin-resistant transformants carried the Ω interposon in their chromosomes, but they still produced LytB as determined by Western blot analysis. The disrupted lytB gene apparently entered the chromosome of these strains by illegitimate recombination. Two other approaches to disrupt lytB were also unsuccessful. An attempt to identify prtoeins that interact with LytB by the yeast two-hybrid technology was also unsuccessful. A major conclusion of this study is that the lytB gene in E. coli appears to be indispensable.Item Characterization of the sperm chromatin structure in Mytilus californianus and Spisula solidissima(1996) Zhang, FanThe aim of this thesis was to establish the chromatin structure and organization in the sperm of two bivalve mollusks: Mytilus californianus and Spisula solidissima. Nuclease digestion and crosslinking studies showed that the sperm chromatin of Mytilus califomianus has a organization consisting of small groups of oligonucleosomes interspersed between long stretches of nucleoprotamine filaments. The nucleoprotamine complexes result from the association of PL-II, PL-III and PL-IV with DNA. The spatial arrangement of different PL proteins within these complexes is discussed. In the case of Spisula, it was found that its major protamine-like component (PL-I) contains at least two cysteines located between tryptophan and tyrosine residues of the globular region of the molecule. It was also found that the PL-I exhibits a head to tail alignment in the sperm.Item Isolation and characterization of certain ribosomal domains : the 5S RNA-protein domain from Escherichia coli and the 'A' protein domain from wheat germ(1982) Watt, Paul WilliamRibosomes are believed to be composed of many structurally and functionally important, protein-protein and RNA-protein domains. Two of these domains have been investigated. These are the 5S RNA-protein domain and the ribosomal 'A' protein domain. (i) An attempt has been made to isolate the 5S RNA-protein domain from the large ribosomal subunit of the eubacterium Escherichia coli, as the first step in a project to isolate this domain from an archaebacterial source. E. coli 50S subunits were subjected to a low concentration of Mg²⁺ (2 mM), EDTA•Na₂(10 mM) and NH₄Cl (1 M), (A. Liljas, unpublished), as a possible method to remove the 5S RNA protein complex from the ribosome. The suspension was centrifuged at 35,000 rpm for 15 hours in a Beckman Ti60 rotor and the supernatant obtained was passed through a 5-20% sucrose gradient. A fraction was obtained which contained several r-proteins and 5S RNA. In an attempt to purify the complex. further, the fraction was passed through an S200 column. Although evidence suggests that a complex was obtained, attempts to identify the composition were not successful. (ii) An attempt was also made to isolate the ribosomal 'A' protein domain, (equivalent to EL7/EL12-EL10 from E. coli), from the large subunit of wheat germ ribosomes, and to isolate and characterize its individual components. A putative complex has been found , of molecular weight 58, 000, containing three r-proteins with molecular weights 15, 000, 13, 700 and 32, 000 respectively. The N-terminal portions of each of these r-proteins has been sequenced. One protein (protein 8) was identified by its sequence as the ribosomal 'A' protein , while another (protein 7) has no homology with the N-terminal portion to protein 8. The third protein (protein 10) has been obtained and differs substantially from the other two proteins in molecular weight, but shows an identical N-terminal amino acid sequence to protein 7.Item The Identification and characterization of highly immunogenic internal antigens of African trypanosomes(1996) Tuckey, Corinna DawnSix monoclonal antibodies previously derived against internal Trypanosoma brucei rhodesiense ViTat 1.1 procyclic culture form antigens were characterized for species- and life cycle stage-specificity by immunoblotting. Five of the mAbs were specific for T. brucei spp. while the sixth (mAb #20) recognized an antigen in all trypanosome species and life cycle stages tested. Immunogold electron microscopy revealed that mAb #20 immunoreactivity localized in the mitochondrion. The antigen recognized by mAb #20 was partially purified and the N-terminal sequence showed significant identity with T. cruzi Hsp 60 and E. coli GroEL mitochondrial chaperones. Using a combination of expression library screening and polymerase chain reaction amplification, I isolated the cDNA encoding the trypanosome antigen. The translated sequence matched sequences of mitochondrial chaperones from several species. Taken together, the results indicate that the antigen recognized by mAb #20 is the T. brucei spp Hsp60 mitochondrial chaperone. The mAb #20 or the Hsp60 protein may be used in a simple immunodiagnostic test for African sleeping sickness.Item Cloning, expression, and nucleotide sequence of the glycerophospholipid : cholesterol acyltransferase gene from Aeromonas hydrophila(1988) Thornton, Julian C.The purpose of this study was to examine the extracellular glycerophospholipid:cholesterol acyltransferase (GCAT) from & hydrophila. The primary structure of GCAT was determined by sequencing the GCAT gene using the chain termination method of Sanger (Sanger et al. 1979). This gene, gcatA, was subsequently cloned into a wide-host-range expression vector (pMMB66; Furst et al. 1986), and the ability of both Escherichia coli and Aeromonas salmonicida to secrete the protein was studied. It was revealed that gcatA from & hydrophila encoded a 31 kD enzyme capable of carrying out acyltransfer between glycerophospholipids and cholesterol.· This enzyme, which is normally extracellular in cultures of & hydrophila (MacIntyre and Buckley 1978), was secreted extracellularly by the related bacteria & salmonicida when the & hydrophila GCAT gene was present on a wide-host-range plasmid. In contrast, !L.. coli clones bearing the same recombinant plasmid only secreted GCAT to their periplasmic space, thus suggesting a lack of the necessary GCAT secretory machinery in the !L._ coli outer membrane. A typical !L._ coli consensus-like promoter was not seen in the nucleotide sequence upstream of the GCAT gene. Although a region sharing some similarities with the CAP binding-site consensus sequence of !L._ coli (de Crombrugghe et al. 1984) was identified approximately 130-150 nucleotides upstream of the start codon of the GCAT gene. The sequence of the structural gene, gcatA, contained two regions that share sequence simtlarities with other lipases. These regions are believed to be the active site and the interfacial lipid-binding site of the other lipases (Maragnore and Henrickson 1986}. Polyclonal antibodies directed against GCAT from both hydrophila and salmonicida cross reacted with both proteins in ELISA and in Western immunoblots. This suggests that although the molecular weights and amino acid compositions of the two related proteins are quite different, some of the antigenic determinants of GCAT are conserved in these two proteins.Item Nucleotide sequence of murE gene of escherichia coli and preliminary studies on extragenic suppressor mutations of murE(1989) Tao, Jing-SongTwo approaches were used to study the murE gene encoding the diaminopimelic acid adding enzyme of Escherichia coli. In the first approach, the cloning and nucleotide sequencing of the murE gene has been performed. The coding region of murE consisted of 1,413 base pairs. The open reading frame of murE gene was separated from the ftsl ( penicillin-binding protein 3 ) gene by 61 base pairs, and it overlapped the initiation codon of the murF ( D-Ala-D-Ala adding enzyme) gene by one base pair. The deduced primary structure of MurE comprised 41 7 amino acid residues with a molecular mass of 50.6 kilodaltons. Amino acid sequences corresponding to the domains A and B of proposed ATP binding sites were identified in the deduced amino acid sequence of MurE. The close linkage of the murE gene to its upstream and downstream neighbouring genes was similar to the close linkage of the other genes in 2-min region of E. coli genetic linkage map. The interesting physical arrangement of the genes in this region and the fact that they are all involved in cell envelope metabolism or cell division has prompted the speculation of some novel form of regulation. As a second approach, extragenic suppressor mutations, which suppress the temperature- sensitive lysis phenotype of the murE16 allele, have been isolated and preliminarily characterized. Growth studies indicated that the murE16 mutation was at least partially osmoremedial, Further more, the two suppressor mutations characterized in this study functioned as suppressors of murE16 only in high osmolarity medium. The attempt to map the suppressor mutations were unsuccessful, and the products of the suppressor genes have not been identified.Item Item Structural and functional characterization of the secreted acid phosphatase produced by Leishmania donovani(1992) Sigurdson, Andrea GailSecreted acid phosphatase (sACPase) was purified from the supernatants of in vitro Leishmania donovani 1S2D cultures. The sACPase was found to be a high molecular weight polydisperse glycoprotein species having a strong net negative charge. The N-terminal sequence of the sACPase protein was determined and compared to previously reported sequences of Leishmania sACPases. L. donovani lipophosphoglycan (LPG)-specific monoclonal antibodies (mAbs) were tested for binding to the sACPase. MAbs L98 and L157, specific for the LPG-associated protein did not bind the sACPase, nor did the sACPase-specific mAb 16Al cross-react with LPG; however, the anti-LPG phosphodisaccharide mAb CA7AE bound the sACPase, indicating the presence of an LPG-like [-PO₄-6Galactose (βl-4)Manoseαl-] epitope on the sACPase. Enzyme activity trapping assays and immunoblots using mAb CA7AE demonstrated that the phosphodisaccharide epitope of the sACPase was very strongly associated with the enzyme. Experiments were performed to characterize the nature of the linkage between this unusual phosphodisaccharide structure and the sACPase protein. Growth of L. donovani promastigotes in the presence of asparagine-linked glycosylation inhibitors did not eliminate the phosphodisaccharide epitope. Digestion of native sACPase with endo-β-N-acetylglucosaminidases also failed to remove the phosphorylated structure. These data suggested that the phosphodisaccharide was not attached to the protein through an N-linked oligosaccharide structure, although the actual mode of attachment was not determined. SACPase produced by promastigotes cultured in the presence of N-glycosylation inhibitors showed no enzyme activity, indicating that glycosylation may be required for enzyme activity. Endoglycosidase-mediated removal of the N-linked oligosaccharides and exoglycosidase-mediated cleavage of mannose residues from native sACPase caused a gradual loss of enzymatic activity. This suggested that the N-glycosylations were essential for the maintenance of the enzyme activity even after synthesis and secretion. This role of the oligosaccharides was not altered when Leishmania promastigotes were grown in the presence of glycosylation processing inhibitors, nor was it affected by the presence or absence of the phosphodisaccharide structure. The far ultraviolet circular dichroism spectra of native and endoglycosidase-digested sACPase indicated possible changes in the secondary or tertiary structure of the protein upon deglycosylation, providing preliminary evidence for a role of the sACPase N-linked oligosaccharides in the maintenance of the active protein conformation. Potential physiological roles for the sACPase were studied through characterization of the substrate specificity of the enzyme. The sACPase was found to dephosphorylate various inositol phosphates at a rate that may be physiologically significant. In addition, mannose-6-phosphate was identified as an optimal monophosphorylated monosaccharide substrate for the sACPase. The enzyme showed some activity toward phosphorylated amino acids, but did not hydrolyze a phosphopeptide substrate. Together, these data suggest that the L. donovani sACPase may be an important factor in the host-parasite relationship that is established during Leishmania infection.Item The effects of ultraviolet-B radiation on gene expression in Douglas fir(2001) Schmidt, Anna-maryDepletion of the stratospheric ozone layer is leading to an increase in ultraviolet-8 (UV-B) radiation reaching the earth's surface. The effects of UV-Bon gene expression in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) were examined during seed germination and early seedling development. Douglas-fir seeds were stratified, then germinated in a controlled environment chamber and exposed to UV-B doses of 0 or 7 kJ m-2 d-1. Transcript levels for hsp90, BiP (luminal binding protein), cpr (NADPH:cytochrome P450 reductase) and ubiquitin increased in response to UYV-B exposure. Correspondingly, UV-B treatment led to increases in HSP90, BiP and CPR protein levels. In contrast, cab (chlorophyll a/b-binding protein) transcript amounts were marginally reduced in response to UV-B. These results suggest a role for heal shock proteins, CPR and ubiquitin in the protection of Douglas-fir from the effects of UV-B radiation.Item The characterization of lytF, a new member of the murH gene family of Escherichia coli(1993) Noble, Michael-AnneThe characterization of a new gene, lytF, is described. The lytF mutation was mapped to 62.4 minutes on the Escherichia coli genetic linkage map, and was found to confer a temperature-sensitive colony forming and lysis phenotype. This mutant was of interest because its lytic phenotype resembled other previously described mutants that were defective in peptidoglycan metabolism. To further characterize the role of lytF, two extragenic suppressors of murH, a mutant whose lysis phenotype is similar to that of lytF and which appears to be defective in peptidoglycan metabolism, were transduced into the lytF strain. Both of these suppressors, designated smhA1 and smhB1, were found to suppress both the temperaturesensitive colony formation and lysis associated with the lytF mutation at high temperature. The smhA1- or smhB1-mediated suppression of the lytF temperature-sensitive colony formation was salt dependent, whereas the suppression of lysis was independent of the medium osmolarity. Because the ability of the smhA1 and smhB1 mutations to suppress lysis does not extend to mutational or antibiotic induced blockages in peptidoglycan biosynthesis, those mutations whose lytic phenotypes are suppressed by smhA1 or smhB1 have been proposed to be functionally related, and they have been designated as the murH family. Three observations indicate that the lytF gene may belong to the murH family. (i) The temperature-sensitive colony forming and lysis phenotype associated with lytF is similar to that expressed by other mutants in the murH family (murH, lytD, lytE). (ii) The lytF phenotypes are suppressed by the presence of smhAJ or smhBJ, which are specific to this family in terms of their suppression phenotypes. (iii) It is possible to select spontaneous smhA or smhB-like mutations in a lytF genetic background, indicating that the ability of smhA and smhB to suppress the lytF phenotype is more than just coincidence. It was also observed that the phenotype associated with the smhB locus may be variable depending on the allele. All smhB alleles characterized in this study share a common genetic linkage to the zbc-103 :: TnJO insertion, and are phenotypically dominant over the wild type smhB+ gene in complementation assays. However, the physical and suppression characteristics can differ substantially among alleles. It is currently unknown whether these differences in phenotype are due to different mutations in a single multifunctional gene, or different mutations in discrete, but closely linked genes.Item Isolation, characterization, and developmental regulation of 2S seed storage protein gene in Pseudotsuga menziesii (douglas fir)(1996) Machander, DavidStudies on the developmental regulation and characterization of genes from Douglas fir were undertaken. A clone (p900) was isolated from a cDNA library prepared from poly A+ mRNA isolated from the mid-stage of Douglas fir embryogenesis. Northern hybridization studies suggested the cDNA to be a full length clone. The gene is clearly developmentally regulated at the transcriptional level rn both the megagametophyte and slightly differently (temporally) in the zygotic embryo. Sequencing data confirmed the clone to be a 2S albumin, yet a unique isoform. Nucleotide sequence alignments showed an overall similarity to Picea glauca albumin and Pinus strobus albumins 1, 2, and 3 of 48.8%. An open reading frame of 140 amino acids encoding a 15.8 kDa preprotein precursor was seen from the predicted amino acid sequence data. A signal peptide of 21 aa was present as predicted by signal peptide cleavage site rules and confirmed by hydropathy plotting. The protein is predicted to have a lack of N-linked glycosylation and is a basic protein. A 30.5% overall amino acid similarity is seen between p900 and other gymnosperm albumins. The protein is high in arginine and glutamine/glutamate at 15.7% and 12.2%, respectively, indicative of the role of albumin as a nitrogen source during seed germination. Additionally, the p900 albumin has a high cysteine content (2.1 % ) and shows a strict conservation in the positioning of these cysteines. Unique features of this isoform include the deletion of two regions where cysteines are typically conserved.Item Secretion of proaerolysin via the general secretion pathway(1998) Yu, LiProaerolysin is secreted by Aeromonas salmonicida and Aeromonas hydrophila via the General Secretion Pathway (GSP). Research presented in this thesis focused on the second step of the GSP, the Main Terminal Branch (MTB). Several approaches were taken to learn more about this process. Although a large amount was secreted into the extracellular medium, some proaerolysin accumulated inside the cell. The intracellular proaerolysin was localized both in the periplasm and in association with the inner membrane. The proaerolysin was calculated. A study of the effect of proaerolysin secretion on protease secretion showed that the appearance of protease in the culture supernatant was greatly reduced when proaerolysin secretion approached the maximum, suggesting that there was an upper limit to the GSP-MTB. A study of secretion of domain II-IV of proaerolysin showed that domain II-IV could be secreted by itself. The secretion was poor compared to secretion of wild-type proaerolysin. When coexpressed with domain I of proaerolysin, domain II-IV did not show a great increase in secretion. However, parallel studies in our laboratory showed that secretion of both domain I and domain II-IV were increased when they were coexpressed in another way (Diep et al., in press). The Clostridium septicum α-toxin could not be secreted by A. salmonicida, despite the similarity in DNA sequence between α-toxin and aerolysin.Item Isolation and characterization of the extracellular acid phosphatase of Leishmania Donovani promastigotes(1992) Li, FengchunAn extracellular acid phosphatase was purified from the culture supernatants of L. donovani promastigotes by using ultrafiltration, Con-A and lentil lectin affinity chromatography, Mono-Q ion-exchange chromatography and DEAE-5PW ion-exchange chromatography. The overall yield of the enzyme was about 50%, representing approximately a 1000-fold purification. This exoenzyme displayed optimum activity at pH 5.0 and 42°C. A Kₘ value of 1.92 mM was obtained when the enzyme was assayed with p-nitrophenyl phosphate as the substrate. The enzyme was completely inactivated by 2.5 x 10⁻³ mM ammonium molybdate, 0.1 mM sodium fluoride and 2 .0 mM L-tartrate . Kinetic analysis of the inhibition showed that the ammonium molybdate and L-tartrate were competitive inhibitors of the enzyme, exhibiting a K; value of 6.0 nM and 2.55 uM, respectively. In contrast, sodium fluoride inhibited the acid phosphatase in a complex fashion. The extracellular acid phosphatase appeared to be heterogeneous upon electrophoresis in both nondissociating and dissociating polyacrylamide gels, suggesting a high degree of post-translational modification. The enzyme was subsequently found to be a phosphorylated glycoprotein by metabolic labelling, binding to Con-A and carbohydrate staining of the SDS-PAGE gels. A molecular weight of 145 K for the major component of this exoenzyme was estimated by SDS-P AGE. The amino acid composition was determined. An earlier determined N-terminal sequence was corroborated and two internal peptide sequences were also established. Carbohydrate compositional analysis provided evidence that the extracellular acid phosphatase contained N-linked oligosaccharides as well as O-linked oligosaccharides which were partially characterized by exoglycosidase digestions, HF-dephosphorylation and Bio-Gel P-4 chromatography after release by hydrazinolysis and reduction with NaB³H₄. The major fragment, which accounted for more than 90% of the total radioactive materials, was found to be a phosphorylated galactosyl-β-mannitol disaccharide. In addition, the charged nature of the oligosaccharides was demonstrated to be exclusively due to phosphate groups. These cumulative chemical results are in agreement with earlier immunological data which inferred that the extracellular acid phosphatase contained a lipophosphoglycan-like phosphoglycan structure.Item Item A structural analysis of Xenopus laevis oocyte 5S rRNA(1989) Leal Carretero, María Luisa IsabelThe purpose of this project was to investigate the secondary and tertiary structures of Xenopus laevis oocyte 5S rRNA using chemical and enzymatic probes. Mutations were introduced into the X. laevis 5S rRNA gene in order to study the effect that these mutations would have on the structure of 5S rRNA transcribed from the gene. Four different mutants were constructed by introducing base substitutions in loops D and E of the 5S rRNA structure. Three of the mutants have sequence substitutions in loop E (Xlo73-76, Xlo99-101 and Xlo96-101), and mutant Xlo87-90 has sequence substitutions in loop D. The accessibility of all 5S rRNAs to single-strand specific nucleases (T1,T2,A, and S1), and to a nuclease specific for double-stranded or structured regions (RNase V1), was determined. The reactivity of nucleobases at N7, N3 and Nl positions to chemical probes (DMS, DEPC, and CMCT), was investigated under native (SmM MgCl2, l00mM KCL, 20°C), and semi-denaturing (lmM EDTA, 20°C) conditions. N-ethylnitrosourea was used to identify phosphates not reactive to alkylation under native conditions. The enzymatic accessibility and chemical reactivity results obtained from all four mutants, as well as from the wild-type 5S rRNA, confirm the presence of the five helical stems predicted by the consensus secondary structure model of 5S rRNA. A comparison of the data obtained from the four mutants with that obtained from wild-type 5S rRNA yielded the following results: (i) The conformation of loops A, B, and C is virtually identical in wild-type 5S rRNA and in all four mutants. (ii) In mutants Xlo73-76 and Xlo99-101, region Eis more reactive to chemical probes and more susceptible to enzymatic clevage, and helices IV and V are less stable than the corresponding regions in wild-type 5S rRNA. In Xlo96-101, region E is less reactive, and helices IV and V more stable than their corresponding wild-type regions. (iii) In mutant Xlo87-90, loop D is more susceptible to enzymatic cleavage and more reactive to chemical probes, and helix IV is less stable than the same regions in wild-type 5S rRNA. These results support the conformation of loops€ and Das they appear in the computer graphic model proposed by Westhof et al. (in press) for the tertiary structure of X. laevis oocyte 5S rRNA. In particular, two important features of this model are supported by the structure probing of the four mutants studied here: (i) region E and loop D seem to contain unusual base pairing, consisting of non-canonical base-pairs; and (ii) no tertiary interactions appear to take place between loop C and either region E or loop D.Item Characterization of genes of stress-related proteins in conifers : low molecular weight heat shock proteins in douglas-fir (Pseudotsuga menziesii [Mirb.] Franco)(1996) Kaukinen, Karia HannahTwo cDNA clones (PM 18.2A; PM 18.2B) from Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco), which encode low molecular weight heat shock proteins (LMW HSPs) of 18.2 kDa. were characterized by Northern and Western blot analyses. A notable induction of LMW HSP transcripts occurred during post-gennination and early seedling growth. Unlike late embryogenesis abundant (LEA) protein genes, the expression of the PM HSPs appears to be primarily restricted to seedlings because mRNA transcripts were only detected at very low amounts during seed development and desiccation. The expression of LMW HSP genes in seedlings was shown to be regulated environmentally and hormonally. The LMW HSP proteins assemble into aggregates. The lack of coordinated gene expression of the PM LMW HSPs and HSP 90 suggests specific roles for the LMW HSPs in conifer seedling development. Studies of desiccation proteins in several conifers revealed a diverse array of LENdehydrins during somatic and zygotic embryogenesis and in mature dry seeds. Therefore, these studies suggest a role of stress-related proteins during seed desiccation, germination and growth.Item Flagella phase variation in the thermophilic campylobacters(1986) Harris, Lori-AnneThe purpose of this study was to examine the contribution of flagella to the widely used Lior heat-labile serotyping scheme for thermophilic Campylobacters. The serotype chosen for examination, serotype 8, contains strains of both Campylobacter jejuni and Campylobacter coli and is commonly isolated from man. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of cultures of strains belonging to the Lio 8 serogroup, showed that many of the strains produced flagellin molecules of two different molecular weights (MW). Radio-immunoprecipitation experiments with the Lio 8 typing antiserum revealed that while most Lio 8 strains produced flagellin which reacted with Lio 8 antiserum, some strains produced flagellin which did not react with this antiserum. This indicated that while the Lio 8 antiserum contained a high titer of serospecific antibodies to flagellin the serotype determinants for the Lio 8 serogroup were not carried on the flagella. Immune-electron microscopy with Lio 8 antiserum showed that some cultures were producing two antigenically distinct flagella. This observation together with the ability of strains to produce flagellins of different MW suggested that inf. jejuni and f. coli flagella were subject to antigenic phase variation. To show that a strain could switch from the production of one flagellin antigenic type to the production of a different flagellin antigenic type, cells originating from a single clone were selected which exhibited motility in the presence of Lio 8 antiserum. By SDS-PAGE analysis, flagellin produced before the switch (phase 1) was found to have a MW of 61,000, while flagellin produced after the switch (phase 2) had a MW of 57,000. This flagellin switching was shown by three different strains. In the case of the Lio 8 serogroup type strain VC 167, the rate of the phase 1 to phase 2 (1.9 x 10-5) switch was over ten times higher than the rate of the phase 2 to phase 1 switch (1.1 x 10-6). Although the two flagellins reacted with polyclonal antiserum to non surface-exposed conserved Campylobacter flagellin epitopes and to a monoclonal antibody which recognized a cross reactive internal Campylobacter flagellin epitope, only phase 1 flagellin reacted with Lio 8 antiserum by radioimmune-precipitation, enzyme-linked immunosorbent assay, or by . 0 1 . immune-e ectron microscopy. Absorption experiments confirmed that the X serospecific epitopes were exposed on the surface of the native phase 1 flagella filament. Biochemical analysis of the two flagellin molecules revealed significant structural conservation. The N-terminal amino acid sequences were identical for the first 22 residues, and peptide mapping showed many conserved peptides. However, the flagellins were different in MW and amino acid composition, and also contained unique pepides.