Undergraduate honors paper--Mount Holyoke College, 2008. Dept of Biologically Sciences. / Includes bibliographical references (leaves 100-105).

In this thesis, the main objective is to perform statistical analysis of parameters yeast cell cycle regulated genes. We have known that there are 800 cell cycle regulated genes from Spellman et al. [9] (Spellman¡¦s 800) and 687 cell cycle regulated genes from MIPS database (MIPS¡¦s 687). We analyze yeast cell cycle regulated genes with statistical methods and models. The four main index statistics considered are as follow: 1. numbers of triscription factors bind to promoters, 2. coherence of genes with 104 known regulated genes (KRG) in alpha-factor experiment, 3. coherence of genes with 104 KRG in cdc15 experiment, 4. coherence of genes with 104 KRG in cdc28 experiment. Although, binding numbers can be fitted to geometric distribution in two subgroups of genes, we found that it is infeasible to classify the cell cycle regulated genes by only using HMM, and the coherence method also improve Spellman classification results if the standard is MIPS database or the 104 KRG. Finally, the cell cycle classification criterion in MIPS¡¦s 687 genes are found to include the information given by Spellman¡¦s 800.

Thesis (PhD)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

No description available.

Herpes simplex virus protein VP16 activates immediate-early (IE) viral gene expression upon infection. VP16-mediated transactivation depends on formation of a multi protein complex with cellular factors on a cis-acting TAATGARA T sequence present in the IE promoters. The potent acidic activation domain, contained within the carboxyl terminus of VP16, is dispensable for the complex formation. The amino terminal part of VP16, which is inert in transactivation in mammalian cells, is sufficient for selective interactions with cellular factors, one of which has been identified as the ubiquitous transcription factor Oct-1. The yeast two-hybrid system was utilized to isolate the cellular factor(s) necessary in addition to Oct-1 for VP16 induced complex formation. This system, designed to directly clone proteins interacting with a given protein of interest, employs the yeast transcriptional activator GAL4. An interaction between VP16 and the cellular factor(s), fused to GAL4 DNA binding and activation domain, respectively, reconstitutes a hybrid transactivator that stimulates expression of a reporter lacZ gene in yeast. Thus, (beta)-galactosidase activity serves as a positive signal for protein-protein interaction. As a prelude of using this method for isolation of VP16-interacting cellular proteins, the system was tested with HSV-1 protein vhs, known to bind to VP16 in vitro. The obtained data demonstrated an interaction between VP16 and vhs in the two-hybrid system and deletion analysis revealed that VP16 sequence contained within the first 369 amino acids is required for binding to vhs. Thus, VP16 residues necessary for interaction with vhs in vivo coincide with these identified previously for VP16-vhs complex formation in vitro. VP16 fused to the GAL4 DNA binding domain activated expression of the reporter lacZ gene in yeast, despite the absence of its acidic activation domain. Deletion analysis showed that the amino terminal 369 residues of VP16 were sufficient for transactivation in yeast. Similar GAL4-VP16 derivatives were inactive in mammalian cells as measured by transient transfection assays. Thus, unlike in yeast, VP16 lacking the acidic activation domain is deficient in transactivation in mammalian cells even if it is directly bound to a promoter. VP16 sequences required for complex formation with vhs overlaps with those implicated in interaction with the mammalian factors, indicating that this region is involved in protein-protein interactions with both cellular and viral factors. Consistent with this, VP16 interaction with a yeast factor supplying an activation domain in trans would explain VP16-dependent transactivation in the absence of its acidic activation domain. Alternatively, a yeast specific activation domain might be present in the amino terminal part of VP16. / Thesis / Master of Science (MS)

Being at the center of most metabolic pathways and also one of the best known pathways, the glycolytic pathway has been of interest to modeling studies. This study is composed of our attempts to model ethanolic fermentation by yeast through kinetic equations of glycolytic steps and its branches. Model was based totally on experimentally measured kinetics of enzymes and transport steps, either obtained in this study or from the literature. Effect of ethanol on enzyme activities was tested in the range of ethanol 0 to 20% (v/v) in assay mixture. All enzymes were inhibited by ethanol to some degree and these inhibitions started at different ethanol concentrations, the least affected being the pyruvate kinase and the most inhibited ones being glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphogluco kinase, and alcohol dehydrogenase (forward). Effect of temperature on the activities of enzymes was tested within 10-30 &deg / C with five degrees of increments. Activation energies of enzymes were calculated using the Arrhenius equation. Activation energies of upper part of the glycolysis and the glycerol branch (glycerol-3-phosphate dehydrogenase) were relatively higher than that of lower part enzymes as well as the ethanol branch (alcohol dehydrogenase). Results obtained from these in vitro studies were incorporated into the model as mathematical relations. Model output thus obtained was compared with results of experiments conducted at several temperatures and initial ethanol concentrations. Model could estimate general trend in ethanolic fermentation that fermentation is inhibited by increasing concentrations of ethanol. Decrease in glycerol yields at lower temperatures was also estimated by the model. However, model did not fit exactly to experimental results, especially at low temperature and high ethanol concentrations. This could be attributed to stress responses of cells under these conditions, which are not considered in the model.

Organisms must constantly face and adapt to environmental change. Although unpredictable events may inevitably impose threats, temporally correlated changes may also provide opportunities from which an organism can profit. An evolutionarily successful microbe must collect enough information to distinguish threats from opportunities. Indeed, for nutrient transport, it is not clear how organisms distinguish one from the other. Fluctuations in nutrient levels can quickly render any transporter's capabilities obsolete. Identifying the environment's dynamic identity is therefore a highly valuable asset for a cell to elicit an accurate physiological response. Recent evidence suggests that the baker's yeast Saccharomyces cerevisiae can exert anticipatory responses to environmental shifts. Nevertheless, the mechanisms by which cells are able to incorporate information from the environment's dynamic features is not understood. A potential source of complex information processing is a highly intricate biochemical network that controls glucose transport. The understanding of this network, however, has revolved around its ability to adjust expression of 17 hexose transporter genes (HXT) to glucose levels. In this thesis, I postulate that instead the glucose sensing network is dynamically controlling the 7 major hexose transporters. By studying transporter dynamics in several scenarios, I provide substantial evidence for this hypothesis. I find that hexose transporters with similar reported affinities (Hxt2 and Hxt4) are robustly allocated to separate stages of growth for multiple initial glucose concentrations. Using single-cell studies, I show that Hxt4 expresses exclusively during glucose downshifts, in contrast with Hxt2. From multiple approaches, I demonstrate that Mig1 is mostly responsible for reporting on the time derivative of glucose, and harnessing it to differentially regulate both transporters. I also provide evidence for the roles of Rgt2 and Std1 in modulating long-term glucose repression of Hxt4. This work extends our ideas on the functionality of transport and gene regulation beyond the established steady-state models. The ability to decode environmental dynamics is likely to be present in other signaling systems and may impact a cell's decision to use fermentation - a decision which is of fundamental interest both for cancer research and for biotechnology.

Synthetic biology is an emergent field incorporating aspects of computer science molecular biology-based methodologies in a systems biology context, taking naturally occurring cellular systems, pathways, and molecules, and selectively engineering them for the generation of novel or beneficial synthetic behaviour. This study described the construction of a novel synthetic gene circuit, which utilises the inducible downstream transcriptional activation properties of the pheromone-response pathway in the budding yeast Saccharomyces cerevisiae as the basis for initiation. The circuit was composed of three novel yeast expression plasmids; (1) a reporter plasmid in which the luciferase reporter gene was fused to the iron response element (IRE), and expressed under the control of the pheromone-inducible FUS1 promoter, (2) a repressor plasmid which constitutively expressed the mammalian iron response protein (IRP), which can bind to the IRE in the luciferase mRNA transcript, blocking translation, and (3) a de-repressor plasmid which also utilised the pheromone-inducible FUS1 promoter to express the bacterial LexA protein that represses transcription of the IRP gene, and thereby de-represses luciferase translation. Yeast cultures were propagated in media that selected for cells containing all three plasmid components of the gene circuit. In these cells, during vegetative growth conditions, reporter gene translation is constitutively repressed by IRP until addition of pheromone. Upon pheromone-induction, the pheromone response pathway up-regulated the expression of the LexA protein which represses transcription of IRP, enabling the translation of luciferase, which is itself up-regulated by the pheromone response pathway. The combination of the repressors functioned to increase the ratio of induction of the reporter gene between pheromone-induced and un-induced states. Proteins and mRNA species expressed by each plasmid were semi-quantified using SDS-PAGE, Western blot, and RT-qPCR. Luciferase expression was measured using an in vitro whole cell luminescence assay, and the data used to define the circuit 'output'. Metabolic control analysis was used prior to building the circuit in silico, and identified the transcription of IRP, as well as the IRP protein half-life as significant control points for increasing the expression of luciferase in vivo. Modelling resulted in the development of multiple variations of the circuit, incorporating strong and weak constitutive promoters for the IRP. For the degradation rate, the IRP was fused with a degradation tag from the PEST rich C-terminal residue of the Cln2 protein, forming IRPPEST , with approximately a 10-fold reduced half-life compared to wild type. By varying the promoter strength and half-life of the IRP, the circuit could be tuned in terms of the amplitude and period of luciferase expression during pheromone induction. Simulated annealing and Hooke-Jeeves algorithms were used to estimate model parameter values from the experimental luminescence data, refining the modelling such that it produced accurate time course simulation of the circuit output. While further characterisation of the individual components would be advantageous, the construction of the system represents a completed cycle of extensive modelling, experimentation, and further model refinement.

Baigiamojo darbo tikslas – ištirti Paramyxoviridae šeimos virusų nukleokapsidės baltymų sintezės mielėse Saccharomyces cerevisiae galimybes ir nustatyti ar mielėse susintetintus nukleokapsidės baltymus galima taikyti Paramyxoviridae šeimos virusų infekcijų serologinei diagnostikai. Tikslas pasiektas ištyrus Paramyxoviridae šeimos Sendai, žmogaus parainfluenza, žmogaus respiracinio sincitinio, Nipah, Hendra ir Menangle virusų nukleokapsidės baltymų sintezę mielių Saccharomyces cerevisiae kamiene 214∆pep4. Nustatyta, kad mielėse susintetinti nukleokapsidės baltymai pasižymi natyviems analogiškų virusų nukleokapsidės baltymams būdingomis savybėmis: yra tirpūs, formuoja virusų nukleokpsidę primenančias daleles, kurios nespecifiškai įjungia ribonukleorūgštis, pasižymi antigeninėmis savybėmis, būdingomis natyviems analogiškų virusų nukleokapsidės baltymams. Taip pat mielėse susintetinti nukleokapsidės baltymai išgryninti juos centrifuguojant per tankų sacharozės tirpalo sluoksnį ir CsCl tankio gradiente. Įsitikinta, kad mielėse Saccharomyces cerevisiae susintetinti Paramyxoviridae šeimos virusų nukleokapsidės baltymai reaguoja su atitinkamais šios šeimos virusais užsikrėtusių žmonių ir gyvūnų kraujo serumo antikūnais, todėl tinka šių virusų infekcijų serologinės diagnostikos sistemų kūrimui. Naudojant mielėse susintetintus nukleokapsidės baltymus buvo gauti specifiniai monokloniniai antikūnai reaguojantys su natyviais virusų nukleokapsidės baltymais. Įvertinos gautų monokloninių... [toliau žr. visą tekstą] / The aims of this study was to investigate synthesis of SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV nucleocapside (N) proteins in yeast Saccharomyces cerevisiae, to determine properties of recombinant N proteins and evaluate the feasibility to use them in diagnostics. In this study it was demonstrated, that yeast S.cerevisiae is an excellent host for a high-level production of proteins SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV N proteins as virus nukleokapsid-like particles (vNLP). The yeast-expressed hPIV1, hPIV3, hRSV, NiV, HeV and MenV vNLPs represent useful tools for the development of new virus detection systems and demonstrate the effectiveness of yeast as a host for generation of recombinant proteins organized in complex structures like human virus NLPs.

The aims of this study was to investigate synthesis of SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV nucleocapside (N) proteins in yeast Saccharomyces cerevisiae, to determine properties of recombinant N proteins and evaluate the feasibility to use them in diagnostics. In this study it was demonstrated, that yeast S.cerevisiae is an excellent host for a high-level production of proteins SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV N proteins as virus nukleokapsid-like particles (vNLP). The yeast-expressed hPIV1, hPIV3, hRSV, NiV, HeV and MenV vNLPs represent useful tools for the development of new virus detection systems and demonstrate the effectiveness of yeast as a host for generation of recombinant proteins organized in complex structures like human virus NLPs. / Baigiamojo darbo tikslas – ištirti Paramyxoviridae šeimos virusų nukleokapsidės baltymų sintezės mielėse Saccharomyces cerevisiae galimybes ir nustatyti ar mielėse susintetintus nukleokapsidės baltymus galima taikyti Paramyxoviridae šeimos virusų infekcijų serologinei diagnostikai. Tikslas pasiektas ištyrus Paramyxoviridae šeimos Sendai, žmogaus parainfluenza, žmogaus respiracinio sincitinio, Nipah, Hendra ir Menangle virusų nukleokapsidės baltymų sintezę mielių Saccharomyces cerevisiae kamiene 214∆pep4. Nustatyta, kad mielėse susintetinti nukleokapsidės baltymai pasižymi natyviems analogiškų virusų nukleokapsidės baltymams būdingomis savybėmis: yra tirpūs, formuoja virusų nukleokpsidę primenančias daleles, kurios nespecifiškai įjungia ribonukleorūgštis, pasižymi antigeninėmis savybėmis, būdingomis natyviems analogiškų virusų nukleokapsidės baltymams. Taip pat mielėse susintetinti nukleokapsidės baltymai išgryninti juos centrifuguojant per tankų sacharozės tirpalo sluoksnį ir CsCl tankio gradiente. Įsitikinta, kad mielėse Saccharomyces cerevisiae susintetinti Paramyxoviridae šeimos virusų nukleokapsidės baltymai reaguoja su atitinkamais šios šeimos virusais užsikrėtusių žmonių ir gyvūnų kraujo serumo antikūnais, todėl tinka šių virusų infekcijų serologinės diagnostikos sistemų kūrimui. Naudojant mielėse susintetintus nukleokapsidės baltymus buvo gauti specifiniai monokloniniai antikūnai reaguojantys su natyviais virusų nukleokapsidės baltymais. Įvertinos gautų monokloninių... [toliau žr. visą tekstą]