Thesis (M.S.)--University of Michigan, 2002. / Includes bibliographical references.

In Cellulomonas fimi the cenB gene encodes a secreted endoglucanase (EngB) involved in the degradation of cellulose. The cenB gene carried on a 5.6 kb C fimi DNA fragment encodes a polypeptide of Mr 110,000 in Escherichia coli. The level of expression of the gene was significantly increased by replacing its normal transcriptional and translational regulatory signals with those of the E. coli lac operon. The intact EngB polypeptide is not required for enzymatic activity: active polypeptides of Mr 95,000 and 82,000 also appear in E. coli and a deletion mutant of cenB encodes an active polypeptide of Mr 72,000. The intact and truncated EngB both bind to microcrystalline cellulose. A simple, rapid affinity chromatography procedure on Avicel was developed for the purification of intact EngB and of the 72,000 deletion derivative. Alignment of the amino-terminal amino acid sequence of the purified intact EngB from E. coli with the partial nucleotide sequence of the cloned C. fimi DNA showed that the mature EngB is preceded by a sequence encoding a putative signal polypeptide of 32 amino acids, a translational initiation codon and a sequence resembling an E. coli ribosome binding site 4 nucleotides before the initiation codon. The signal peptide functions and is correctly processed in E. coli, even when its first 15 amino acids are replaced by the first 7 amino acids of β-galactosidase. The truncation of EngB does not affect its export to the periplasm of E.coli. In the intact EngB, 25% of the residues are hydroxyamino acids. It displays features common to endo-β-1 ,4-glucanases, since it has a high activity on carboxymethylcellulose. The kinetic parameters for carboxymethylcellulose hydrolysis of both intact and truncated EngB are not significantly different. C. fimi protease cleaves intact EngB, in a specific manner, to generate two polypeptides of Mr 65,000 and 43,000; the former has the capacity to bind Avicel. A polyclonal antibody raised against the purified intact EngB recognizes a C. fimi extracellular protein of M 110,000 as well as 5 polypeptides of lower molecular weight. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Leishmania major (L. major) is an intra-macrophage protozoan parasite which differentiates from a promastigote to an amastigote upon transmission from its insect vector at 25°C to its mammalian host at 37°C. This temperature shift occurs in the same range as that used to elicit the heat shock response in prokaryotes and higher eukaryotes in which the induction of genes encoding heat shock proteins is seen. Ubiquitin is a heat inducible protein and one of the most conserved eukaryotic proteins known. Genomic libraries made from major DNA were initially screened with the ubiquitin gene from yeast. DNA sequence analyses of positive clones revealed at least 5 ubiquitin coding elements arranged head to tail without intervening sequences. The predicted protein sequence showed that ubiquitin in Leishmania differs from that of yeast and barley at 5 out of 76 amino acid positions and from that of human at only 2 positions. Further characterization revealed another ubiquitin encoding locus believed to carry only one ubiquitin encoding element. Comparisons of ubiquitin mRNA levels from L. major grown at 26°C, 37°C, and 42°C suggest that ubiquitin gene expression in these particular parasites is constitutive and that prolonged exposure at a non-lethal temperature results in a reduction of ubiquitin-specific mRNA. However, a direct correlation between parasite differentiation and ubiquitin gene expression was not defined as it could not be determined whether the described experimental conditions actually established differentiated states of L. major. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Previous studies have suggested that manganese (Mn) deficiency is associated with increased pancreatic amylase activity in rats. The present study investigated whether this increase in amylase activity is a result of increased pancreatic amylase messenger RNA (mRNA) levels. Weanling rats were fed a high carbohydrate diet containing either 39.6 ppm (control) or 0.5 ppm (deficient) manganese for 4 to 8 weeks. Manganese deficiency was confirmed by determining hepatic manganese content which was significantly lower in Mn-deficient rats than in the respective controls. Pancreatic RNA was size-fractionated on formaldehyde gels, and hybridized with 32P-labeled complementary DNAs (cDNA) for amylase and trypsinogen. Amylase mRNA levels were increased significantly in both 4 week (200%) and 8 week (250%) Mn-deficient rats when compared with their respective controls. In contrast, manganese deficiency was not associated with alternations in trypsinogen mRNA levels. Moreover, in vitro translation of the pancreatic mRNA indicated that manganese deficiency increased amylase mRNA levels supporting the Northern Blot analysis. Insulin and corticosterone, hormones known to increase amylase mRNA levels, were not affected by Mn-deficiency. These observations suggest that manganese may participate in the regulation of amylase gene expression.

Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Mannan polysaccharides occur in the hemicellulose fraction of plant cell walls, either as structural polymers or as reserve carbohydrates. They are found predominantly in the seeds of leguminous plants in the form of galactomannan, and in softwoods as galactoglucomannan. Endo-I,4-I3-mannanases hydrolyze mannan polysaccharides to oligosaccharides of various lengths. These enzymes are secreted as single catalytic modules or as part of multi-modular proteins by fungi, bacteria, plants and animals. For example, the l3-mannanase of Aspergillus aculeatus, designated Aa-Man5A, is secreted as a single catalytic module, whereas that of Trichoderma reesei, designated Tr-Man5A, contains a l3-mannanase catalytic module linked to a cellulose-binding module by a Pro- Ser-Thr-rich linker. Heterologous gene expression in yeast provides the opportunity to produce individual hydrolytic enzymes in a host expression system devoid of related activities. Saccharomyces cerevisiae has a well-developed expression system and has frequently been used as a model organism for heterologous gene expression. A number of autoselection systems have been devised so that recombinant S. cerevisiae strains can be cultivated in any medium of choice without exerting selective pressure. An autoselection system based on defective chromosomal ura3 andfurl genes involved in the pyrimidine biosynthesis pathway of S. cerevisiae, and complementation of the ura3 gene with a multicopy plasmid-borne URA3 gene, were used in this study. The man1 of A. aculeatus gene encoding Aa-Man5A was cloned and expressed in autoselective S. cerevisiae under the regulation of the alcohol dehydrogenase (ADH2PT) and the phosphoglycerate kinase (PGK1PT) promoter and terminator sequences. Expression of man1 under both promoters resulted in high production levels of Aa- Man5A. The production levels were significantly higher than the levels of endo-l,4-13- mannanases produced by heterologous expression in Escherichia coli, and were comparable to the production levels of enzymes produced in Pichia pastoris, which presumably has a higher secretion capacity than S. cerevisiae. The recombinant yeast strain expressing man1 under the regulation of the PGK1p promoter displayed stunted biomass formation during the logarithmic phase, which was relieved when the native f3- mannanase secretion signal was replaced with the yeast MFuis secretion signal. The recombinant Aa-Man5A displayed biochemical properties similar to those of the native Aa-Man5A. The recombinant enzyme hydrolyzed unsubstituted mannan to predominantly mannose, mannobiose, and mannotriose. The expression of the man1 and man1 ácbd gene constructs of T reesei in S. cerevisiae fur 1::LEU2 strains under the regulation of the PGK1 PT promoter and terminator resulted in the production and secretion of Tr-Man5A and Tr-Man5A~CBD (lacking the cellulose binding module), respectively. However, the production levels of both proteins were approximately I5-fold lower than the production levels of Aa-Man5A. These levels did not improve after replacement of the native secretion signal with the MFuis secretion signal. Interestingly, reducing the cultivation temperature from 30°C to 20°C led to a five-fold increase in the secreted levels of Tr-Man5A, but a three-fold decrease in the production of Aa-Man5A. A preliminary investigation was performed to evaluate the possibility of using the recombinant Aa-Man5A in the processing of instant coffee. Arabica coffee extracts treated with Aa-Man5A displayed low viscosity in comparison to the untreated extract and showed better retention of volatile/aromatic compounds than the autoclaved extract. The results indicated that Aa-Man5A is capable of hydrolyzing coffee galactomannan and can be used for processing instant coffee. / AFRIKAANSE OPSOMMING: Mannaanpolisakkariede kom in die hemisellulose fraksie van plantselwande as strukturele polimere of reserwe koolstofbron voor. Mannaan word hoofsaaklik in die sade van peulplante, III die vorm van galaktomannaan, en III sagtehout as galaktoglucomannaan aangetref. Endo-I,4-j3-mannanase kan mannaanpolisakkariede na oligosakkariede van verskillende lengtes afbreek. Hierdie ensieme word deur fungi, bakterieë, plante en diere as enkele katalitiese modules of as deel van multi-modulêre proteïene uitgeskei. Die j3-mannanase (Aa-Man5A) van Aspergillus aculeatus is byvoorbeeld 'n enkele katalitiese module, maar die j3-mannanase (Tr-Man5A) van Trichoderma reesei bestaan uit 'n j3-mannanase katalitiese module gekoppel aan 'n sellulose-bindingsmodule deur middel van 'n Pro-Ser- Thr-ryke koppelstuk. Heteroloë geenuitdrukking in gIS bied die geleentheid om individuele hydrolitiese ensieme in 'n gasheer uitdrukkingsisteem sonder verwante aktiwiteite te produseer. Saccharomyces cerevisiae het 'n goed ontwikkelde uitdrukkingsisteem en word as model organisme vir heteroloë geenuitdrukking gebruik. 'n Aantal outoseleksiesisteme is ontwikkel, waardeur rekombinante S. cerevisiae-tese in enige medium sonder selektiewe druk gekweek kan word. 'n Outoseleksiesisteem, gebaseer op defektiewe chromosomale ura3 en furl gene wat vir ensieme in die pirimidien biosinteseweg kodeer, en komplementasie van die ura3-geen met die wilde-tipe URA3-geen wat op In multikopie plasmied teenwoordig is, is vir hierdie studie gebruik. Die manl-geen, wat vir die Aa-Man5A j3-mannanase van A. aculeatus kodeer, is gekloneer en in outoselektiewe S. cerevisiae onder die regulering van die alkoholdehidrogenase 2 (ADH2PT) en fosfogliseraatkinase 1 (POKl PT) promotor- en termineerderopeenvolgings uitgedruk. Uitdrukking van die manl-geen onder albei promotors het hoë produksievlakke van Aa-Man5A gelewer. Die produksievlakke was aansienlik hoër as die endo-I,4-j3-mannanase-vlakke wat deur heteroloë geenuitdrukking in Escherichia coli geproduseer was, en kon vergelyk word met die produksievlakke van ensieme in Pichia pastoris. P. pastoris is veronderstel om In hoër sekresiekapasiteit as S. cerevisiae te hê. Die rekombinante gisras wat die manl-geen onder beheer van die PGKl p promotor uitgedruk het, se biomassavorming was belemmer gedurende die laat logaritmiese fase. Die belemmering is opgehef nadat die natuurlike sekresiesein van 13-mannanasemet die MFais sekresiesein vervang is. Die rekombinante Aa-ManSA het soortgelyke biochemiese eienskappe as die natuurlike Aa-ManSA getoon. Die rekombinante ensiem het onvertakte mannaan tot hoofsaaklik mannose, mannobiose en mannotriose gehidroliseer. Die uitdrukking van die manl- en manl Licbd-geenkonstrukte van T. reesei in S. cerevisiae furl::LEU2-rasse onder regulering van die PGKlPT promotor en termineerder het tot die produksie en sekresie van onderskeidelik die Tr-ManSA en Tr-ManSAilCBD (sonder die sellulose-bindingsdomein) ensieme gelei. Die produksievlakke van beide proteïene was egter ongeveer IS-voudig laer as die vlakke van Aa-ManSA. Hierdie vlakke het egter nie verbeter nadat die natuurlike sekresiesein met die MFais sekresiesein vervang is nie. Interessant is die feit dat 'n afname in opkwekingstemperatuur vanaf 30°C tot 20°C tot 'n vyf-voudige toename m sekresievlakke van die Tr-ManSA gelei het, maar tot 'n drie-voudige afname in die produksie van Aa-ManSA. 'n Voorlopige ondersoek na die moontlike gebruik van rekombinante Aa-ManSA in kitskoffieprosessering is ondersoek.. Arabica koffie-ekstrak wat met Aa-ManSA behandel is, het 'n laer viskositeit in vergelyking met onbehandelde ekstrak getoon, asook beter behoud van vlugtige/aromatiese verbindings in vergelyking met geoutoklaveerde ekstrak. Hierdie resultate toon dat Aa-ManSA in staat is om koffee galaktomannaan te hidroliseer en dat dit vir die prosessering van kitskoffie gebruik kan word.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Master / Master of Medical Sciences

Mandibular condylar cartilage (MCC) consists of fibrous (F), proliferative (P), mature (M) and hypertrophic (H) zones, and exhibits distinctive biological features in physiology and function. Accordingly, the genetic regulation of MCC is expected to be different from other articular cartilages. Combined lasercapture microdissection (LCM) and microarray analysis (MAA) approach allows large-scale screening of zone-specific gene expressions. A few investigators have attempted to apply this approach on different cartilages, but not on MCC yet. Therefore, this study aimed to: 1) optimize an LCM protocol for isolating homogenous cell populations from MCC zones; 2) perform a zone-specific comprehensive gene expression analysis for MCC using LCM & MAA; and 3) find a set of genes, following the validation of MAA data using in-vivo and invitro quantitative reverse transcription-polymerase chain reaction (qRT-PCR), which could potentially distinguish MCC zones from each other and from articular chondrocytes. MCC and femoral condylar cartilage (FCC) specimens were harvested from normal 5-week-old SD rats, and formalin-fixed sections and cryosections were compared histologically. LCM samples for five groups (FCC and four MCC zones) were prepared, and then RNAs were extracted and evaluated for integrity. For MAA experiment, LCM samples were amplified before microarray hybridization. MAA data were analyzed using GeneSpring software. cDNA from unamplified LCM-RNA samples were also prepared for the five groups for in-vivo qRT-PCR validation of 48 genes selected from MAA data, 10 of which were additionally validated by cultivating ATDC5 cells and extracting RNA at different time points for in-vitro qRT-PCR validation. Factors enhancing tissue visualization, LCM efficiency, LCM specificity, and RNA yield and integrity were optimized in the suggested LCM protocol. At a 2-fold change, 8353 (26.86%) transcripts were differentially expressed among the MCC zones and FCC. Subsequent data mining allowed the creation of seven subsets of 127 genes. Forty-eight genes were selected for validation based on their signal intensities, clustering classification, and gene ontology. In-vivo and in-vitro qRT-PCR showed high consistency with the MAA data. Results revealed robust gene expression differences among MCC zones, and between articular chondrocytes and MCC cells. The F & P zones could be characterized by upregulation of Crabp1, Dpt, Fndc1, Aspn, Tnmd, Bcl11b, Angptl1, Col14a1, and downregulation of Mug1, Foxa2, Lect1, and Matn3. Opposite modulation of the same genes may characterize M & H zones. In addition, unizonal distinct profiles were also identified; upregulated Igfbp6, Igha, Hils1, and Ptgds genes might be considered as potential markers for F, P, M, and H zones, respectively. In conclusion, this study sets up an LCM protocol that enables isolating homogenous zone-specific cell populations from the MCC, and obtaining highquality RNAs for subsequent gene expression analysis. Comprehensive gene profiling has been successfully achieved with high fidelity; using minute RNA amounts via the LCM & MAA combined approach. The MCC cells clearly exhibit distinguishable phenotypes from the articular chondrocytes, and a set of genes has been determined as potential unizonal/bizonal markers to identify MCC zones. Generating accurate regional data enhances our understanding of MCC biology and provides invaluable insights for tissue-engineering and cellbased therapeutic strategies. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

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