Errors in segregation of genetic materials are detrimental to all organisms. The budding yeast ensures accurate chromosome segregation by employing a system called the spindle checkpoint. The spindle checkpoint, which consists of proteins such as Mad1, Mad2, Mad3, Bub1, and Bub3, monitors the attachment of microtubules to the chromosomes and prevents cell cycle progression until all chromosomes are properly attached. To understand how the spindle checkpoint arrests cells in response to attachment errors at the chromosomes, we recruited different checkpoint proteins to an ectopic site on the chromosome by taking advantage of the binding of the lactose repressor (LacI) to the lactose operator (LacO). We found that cells expressing Bub1-LacI arrest in metaphase. The phenotype is in fact caused by dimerization of Bub1 when it is fused to LacI rather than the recruitment of Bub1 to chromosome. The cell cycle arrest by the Bub1 dimer depends on the presence of other checkpoint proteins, suggesting that the dimerization of Bub1 represents an upstream event in the spindle checkpoint pathway. The results with the Bub1 dimer inspired us to fuse checkpoint proteins to each other to mimic protein interactions that may contribute to checkpoint activation. We showed that fusing Mad2 and Mad3 arrests cells in mitosis and that this arrest is independent of other checkpoint proteins. We believe that combining Mad2 and Mad3 arrests cells because both proteins can bind weakly to Cdc20, the main target of the spindle checkpoint, and the sum of these two weak bindings creates a hybrid protein that binds tightly to Cdc20. We reasoned that if Mad3's role is to make Mad2 bind tightly, artificially tethering Mad2 directly to Cdc20 should also arrest cells and this arrest should not depend on any other checkpoint components. Our experiments confirmed these predictions, suggesting that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, that this binding is sufficient to inhibit APC activity, and that this reaction is the most downstream event in spindle checkpoint activation. The interactions among spindle checkpoint proteins thus play an important role in cell cycle arrest and must be carefully regulated.

"Thesis submitted for the degree of B.S.A."

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 36-37). Also available via the Internet.

Yes / We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division. / Biotechnology and Biological Sciences Research Council (BB/K009346/1)

3 pp. / Citrus budding is a plant propagation technique that any homeowner can do. Once the technique is learned, homeowners can add citrus tree. This publication addresses the budding techniques of citrus trees. Topics include the preparation prior to budding, selecting budsticks, storing budsticks, selecting and preparing the budding location, cutting the bud, inserting the bud in the t cut, and forcing the bud.

The operating principles of complex regulatory networks are more easily understood with mathematical modelling than by intuitive reasoning. In this thesis, I study the dynamics of the mitotic exit control system in budding yeast. I present a comprehensive mathematical model, which provides a system’s-level understanding of the mitotic exit process. This model captures the dynamics of classic experimental situations reported in the literature, and overcomes a number of limitations present in previous models. Analysis of the model led to a number of breakthroughs in the understanding of mitotic exit control. Firstly, numerical analysis of the model quantified the dependence of mitotic exit on the proteolytic and non-proteolytic functions of separase. It was shown that the requirement for the non-proteolytic function of separase depends on cyclin-dependent kinase activity. Secondly, APC/Cdc20 is a critical node that controls the phosphatase (Cdc14) branch and both cyclin (Clb2 and Clb5) branches of the cell cycle regulatory network. Thirdly, the model proved to be a useful tool for the systematic analysis of the recently discovered phenomenon of Cdc14 endocycles. Most proteins belonging to the cell cycle control network are regulated at the level of synthesis, degradation and activity. Presumably, these multiple layers of regulation facilitate robust cell cycle behaviour in the face of genetic and environmental perturbations. To falsify this hypothesis, I subjected the model to global parameter perturbations and tested viability against pre-defined criteria. According to these analyses, the regulated transcription and degradation of proteins make different contributions to cell cycle control. Regulated degradation confers cell cycle oscillations with robustness against perturbations, while regulated transcription plays a major role in controlling the period of these oscillations. Both regulated transcription and degradation are part of important feedback loops, that combined promote robust behaviour in the face of parametric variations.

Os frequentes aumentos de custos de produção de cana-de-açúcar aliado à ampliação da mecanização do plantio e colheita da cultura exigem a busca por canaviais mais produtivos. Nesse sentido é essencial o conhecimento da interferência dos propágulos e seus tratamentos no desenvolvimento e produção da cana-de-açúcar. O objetivo dessa pesquisa foi verificar a influência de três tipos de propágulos quanto ao seu número de gemas, bem como sua posição de origem no colmo, e o efeito da utilização de biorreguladores no desenvolvimento, produção e qualidade de cana planta. O experimento foi desenvolvido no município de Tabapuã (SP) e a variedade utilizada foi a RB83-5054. Classificaram-se os propágulos em função de sua posição de origem (apical, central e basal) e tipo, de acordo com o numero de gemas (duas, três ou quatro). Para verificar o efeito dos biorreguladores na cana-de-açúcar foi utilizado o produto comercial Stimulate® na dose de 0,5 litro por hectare. O delineamento experimental utilizado foi o de blocos inteiramente casualizados com três repetições e as variáveis analisadas foram: número de brotos, perfilhos, altura e diâmetro do colmo, produtividade, açúcar total recuperável (ATR) e toneladas de ATR por hectare. Os resultados obtidos foram submetidos a analise de variância e quando necessário os dados foram transformados. Posteriormente realizou-se o teste comparativo de médias pelo teste de Tukey ao nível de 5% de significância. Propágulos de origem apical e central dos colmos de cana-de-açúcar resultaram em canaviais com maior brotação, perfilhamento, altura de plantas, diâmetro de colmos, produtividade e toneladas de açúcar total recuperável por hectare do que os provenientes das posições basais. Canaviais originados de propágulos da posição basal dos colmos foram influenciados por seu tipo. Plantas mais altas, com maior produtividade agrícola e toneladas de açúcar total recuperável por hectare foram obtidas pelo plantio de propágulos contendo três e quatro gemas. A utilização do Stimulate® nos diferentes propágulos não interferiu, nas condições deste experimento, na brotação, desenvolvimento, produção e qualidade tecnológica da cultura da cana-de-açúcar. / Frequent increases in sugarcane production costs combined with the expansion of planting and harvest mechanization of crop requires the search for more productive sugarcane fields. In this regard, it is essential to know the interference of the propagules and their treatments in development and production of sugarcane. The purpose of this research was to investigate the influence of three types of propagules as to its numbers of buds, and its original position in the stalks, and the effect of use of bioregulators in development, production and quality of plant cane. The experiment was conducted in Tabapuã (SP) and the variety used was the RB83- 5054. The propagules were classified according to their origin position (apical, middle and basal) and its type, according to the number of gems (two, three or four). To verify the effect of the bioregulators on the sugarcane the commercial product Stimulate® at a dose of 0.5 liter per hectare was used. The experimental design used was the totally randomized blocks with three replications and the variables analyzed were: number of shoots, tillers, height and diameter of the stalks, productivity, total recoverable sugar (TRS) and tons of TRS per hectare. The results were submitted to analysis of variance and whenever necessary the data were transformed. Afterwards, the comparative test of means by Tukey test at 5% significance level was done. Propagules from an apical and central source of the sugarcane stalks resulted in sugarcane fields with more budding, tillering, plant height, stalk diameter, productivity and tons total recoverable sugar per hectare than those from basal positions. Sugarcane fields of propagules from the basal position of the stalks were influenced by their type. Taller plants, increased agricultural productivity and tons total reducing sugar per hectare were obtained by planting propagules containing three and four gems. The use of Stimulate® in different propagules, as to type and source position did not interfere, for the conditions of this experiment, with sprouting, development, technological quality production of the sugarcane culture.

Os frequentes aumentos de custos de produção de cana-de-açúcar aliado à ampliação da mecanização do plantio e colheita da cultura exigem a busca por canaviais mais produtivos. Nesse sentido é essencial o conhecimento da interferência dos propágulos e seus tratamentos no desenvolvimento e produção da cana-de-açúcar. O objetivo dessa pesquisa foi verificar a influência de três tipos de propágulos quanto ao seu número de gemas, bem como sua posição de origem no colmo, e o efeito da utilização de biorreguladores no desenvolvimento, produção e qualidade de cana planta. O experimento foi desenvolvido no município de Tabapuã (SP) e a variedade utilizada foi a RB83-5054. Classificaram-se os propágulos em função de sua posição de origem (apical, central e basal) e tipo, de acordo com o numero de gemas (duas, três ou quatro). Para verificar o efeito dos biorreguladores na cana-de-açúcar foi utilizado o produto comercial Stimulate® na dose de 0,5 litro por hectare. O delineamento experimental utilizado foi o de blocos inteiramente casualizados com três repetições e as variáveis analisadas foram: número de brotos, perfilhos, altura e diâmetro do colmo, produtividade, açúcar total recuperável (ATR) e toneladas de ATR por hectare. Os resultados obtidos foram submetidos a analise de variância e quando necessário os dados foram transformados. Posteriormente realizou-se o teste comparativo de médias pelo teste de Tukey ao nível de 5% de significância. Propágulos de origem apical e central dos colmos de cana-de-açúcar resultaram em canaviais com maior brotação, perfilhamento, altura de plantas, diâmetro de colmos, produtividade e toneladas de açúcar total recuperável por hectare do que os provenientes das posições basais. Canaviais originados de propágulos da posição basal dos colmos foram influenciados por seu tipo. Plantas mais altas, com maior produtividade agrícola e toneladas de açúcar total recuperável por hectare foram obtidas pelo plantio de propágulos contendo três e quatro gemas. A utilização do Stimulate® nos diferentes propágulos não interferiu, nas condições deste experimento, na brotação, desenvolvimento, produção e qualidade tecnológica da cultura da cana-de-açúcar. / Frequent increases in sugarcane production costs combined with the expansion of planting and harvest mechanization of crop requires the search for more productive sugarcane fields. In this regard, it is essential to know the interference of the propagules and their treatments in development and production of sugarcane. The purpose of this research was to investigate the influence of three types of propagules as to its numbers of buds, and its original position in the stalks, and the effect of use of bioregulators in development, production and quality of plant cane. The experiment was conducted in Tabapuã (SP) and the variety used was the RB83- 5054. The propagules were classified according to their origin position (apical, middle and basal) and its type, according to the number of gems (two, three or four). To verify the effect of the bioregulators on the sugarcane the commercial product Stimulate® at a dose of 0.5 liter per hectare was used. The experimental design used was the totally randomized blocks with three replications and the variables analyzed were: number of shoots, tillers, height and diameter of the stalks, productivity, total recoverable sugar (TRS) and tons of TRS per hectare. The results were submitted to analysis of variance and whenever necessary the data were transformed. Afterwards, the comparative test of means by Tukey test at 5% significance level was done. Propagules from an apical and central source of the sugarcane stalks resulted in sugarcane fields with more budding, tillering, plant height, stalk diameter, productivity and tons total recoverable sugar per hectare than those from basal positions. Sugarcane fields of propagules from the basal position of the stalks were influenced by their type. Taller plants, increased agricultural productivity and tons total reducing sugar per hectare were obtained by planting propagules containing three and four gems. The use of Stimulate® in different propagules, as to type and source position did not interfere, for the conditions of this experiment, with sprouting, development, technological quality production of the sugarcane culture.

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G1 phase assembly of pre-replicative complexes. Only the Orc1-5 subunits are required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. In comparison to other eukaryotic Orc6 proteins, budding yeast Orc6 appears to be quite divergent. Two-hybrid analysis revealed that Orc6 only weakly interacts with other ORC subunits. In this assay Orc6 showed a strong ability to self-associate, although the significance of this dimerization or multimerization remains unclear. Imaging of Orc6-eYFP revealed a punctate sub-nuclear localization pattern throughout the cell cycle, representing the first visualization of replication foci in live budding yeast cells. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations from metazoans. An essential role for Orc6 in DNA replication was identified by depleting the protein before and during G1 phase. Surprisingly, Orc6 was required for entry into S phase after pre-replicative complex formation, in contrast to what has been observed for other ORC subunits. When Orc6 was depleted in late G1, Mcm2 and Mcm10 were displaced from chromatin, the efficiency of replication origin firing was severely compromised, and cells failed to progress through S phase. Depletion of Orc6 late in the cell cycle indicated that it was not required for mitosis or cytokinesis. However, Orc6 was shown to be associated with proteins involved in regulating these processes, suggesting that it may act as a signal to mark the completion of DNA replication and allow mitosis to commence.

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G1 phase assembly of pre-replicative complexes. Only the Orc1-5 subunits are required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. In comparison to other eukaryotic Orc6 proteins, budding yeast Orc6 appears to be quite divergent. Two-hybrid analysis revealed that Orc6 only weakly interacts with other ORC subunits. In this assay Orc6 showed a strong ability to self-associate, although the significance of this dimerization or multimerization remains unclear. Imaging of Orc6-eYFP revealed a punctate sub-nuclear localization pattern throughout the cell cycle, representing the first visualization of replication foci in live budding yeast cells. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations from metazoans. An essential role for Orc6 in DNA replication was identified by depleting the protein before and during G1 phase. Surprisingly, Orc6 was required for entry into S phase after pre-replicative complex formation, in contrast to what has been observed for other ORC subunits. When Orc6 was depleted in late G1, Mcm2 and Mcm10 were displaced from chromatin, the efficiency of replication origin firing was severely compromised, and cells failed to progress through S phase. Depletion of Orc6 late in the cell cycle indicated that it was not required for mitosis or cytokinesis. However, Orc6 was shown to be associated with proteins involved in regulating these processes, suggesting that it may act as a signal to mark the completion of DNA replication and allow mitosis to commence.