Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 103-113).

DNA replication is initiated at replication origins, which are temporally regulated within the S phase of the cell cycle so that some origins initiate early, while others initiate late. S. cerevisiae telomeres of normal length replicate late during the S phase. However, shortened telomeres replicate early – coupling the regulation of length to the replication timing. The mechanism through which telomere length regulates the activation time of nearby replication origins is unknown. In this thesis, I find that Tel1, a homolog of human ATM kinase, is required for early replication of short telomeres. In the absence of Tel1, short telomeres of the yku70Δ mutant no longer replicate early. I tested the role of Histone H2A(X) phosphorylation at Serine-129, as the target of Tel1 kinase, in regulation of telomeric replication times. However, preventing this phosphorylation had only a minor effect, so H2A(X) is unlikely to be the sole Tel1 target in telomeric replication regulation. On the other hand, deletion of Rif1 – a telomeric length regulator – showed complete epistasis to tel1Δ in telomeric replication regulation, implying that Rif1 acts downstream of Tel1 in the regulation of telomeric replication. Proteomic analysis revealed Tel1-dependent phosphorylation of Rif1 upon telomere shortening, implicating Rif1 as a direct downstream target of Tel1. However, mutation of the Tel1-phosphorylation sites on Rif1 had only a small effect on telomere replication times, indicating that phosphorylation of Rif1 by Tel1 is not the sole mechanism governing replication timing of short telomeres.

I present the results of my analysis of the final stage of gene expression, the translation of messenger RNA. Translation is carried out by large molecular motors called ribosomes, which move along the mRNA molecule in a stepwise fashion, producing a polypeptide chain. Translation can be modelled as a driven diffusion process, and I extend the Totally Asymmetric Simple Exclusion Process (TASEP) to consider the possibility that ribosomes that terminate from the lattice can rejoin the lattice to reinitiate translation, known as ribosome recycling. Inclusion of recycling leads to marked changes in the phase transition boundaries when compared with a non-recycling lattice, with extension and expansion of the so-called maximal current regime, where protein production is at optimal levels. Recycling makes this phase accessible even at low values of de novo initiation. Furthermore, recycling leads to a sharp peak in particle current on the low density-high density phase boundary, with a decrease in current within the high density regime. Simulations of real biological sequences from the budding yeast Saccharomyces cerevisiae suggest that increasing ribosomal availability can, in fact, reduce the efficiency of protein production in a sequence-dependent manner, particularly for mRNAs encoding proteins with a stressresponse function. Consistent with this, separating the yeast transcriptome into phase transition classes brings to light class-dependent features including parameter values and protein functionality. The role ribosome recycling plays in determining the lifetime of a messenger RNA is also studied. Ribosome initiation has a stabilising effect; counteracting this is ribosome termination, which enhances removal of proteins that protect the mRNA 'tail', exposing it to degradation enzymes. Mathematical modelling of this process shows that recycling can affect mRNA lifetimes in a phase-dependent manner that is not straightforward. This indicates that there are additional, important factors involved in the regulation of decay beyond the interplay between termination and initiation. The research reported in this thesis shows that systems biology can offer insight into a complex stage of gene regulation, and has a significant role to play in future research.

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One of the leading aims of systems biology is the complete delineation of the organization and architecture of signaling networks. Within this aim, characterizing integrated circuits is a particular challenge. Integrated circuits are the sites of information multiplexing, where input from multiple sources are combined into a single output or channel. A number of quantitative methods for analyzing epistasis within integrated pathways have been developed, with limited success. Here I present Expression Component Analysis, a novel approach for determining quantitative epistasis within an integrated signaling circuit, and describe the application of Expression Component Analysis in analyzing an interesting and important integrated signaling circuit in the model eukaryote, S.cerevisiae.

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[URE3] priono indukcija priklauso nuo Ras/PKA signalinio kelio aktyvumo. Galima šio kelio įtaka [PSI+] priono formavimuisi gali būti numanoma iš natyvaus Sup35 baltymo struktūros. Šio baltymo struktūroje yra nustatytos kelios menamos PKA fosforilinimo vietos. Siekiant patikrinti šią galimybę buvo atliktas trijų, Ras/PKA signalinio kelio aktyvumu besiskiriančių, izogeninių kamienų – 6-α‘1-NB13-, 12-α‘1-NB13-, 7-α‘1-NB13 - [PSI+] priono indukcijos dažnio įvertinimas. Rezultatai parodė, kad terpėje esant turtingam azoto šaltiniui 6-α‘1-NB13- kamieno ląstelėse padidinta CYR1, BCY1 ir Ras2Val19 genų raiška sumažina [PSI+] priono indukcijos dažnį. Tuo tarpu, terpėje esant neturtingam azoto šaltiniui 7-α‘1-NB13 kamieno ląstelėse padidinta BCY1 geno raiška padidina [PSI+] priono indukcijos dažnį. Todėl galima daryti išvadą, kad [PSI+] priono indukcijos dažnis gali priklausyti nuo Ras/PKA signalinio kelio aktyvumo. / [URE3] prion induction depends on the activity of the Ras/PKA signal transduction pathway. Possible influence of this pathway on the formation of [PSI+] prion could be predicted from the structure of the native Sup35 protein. Several possible phosphorylation sites are known in the structure of this protein. In order to check this possibility analysis of prion induction frequency of three isogenic mutant strains – 6-α‘1-NB13-, 12-α‘1-NB13-, 7-α‘1-NB13 – different in the activity of the Ras/PKA signal transduction pathway, was performed. Results showed that in the presence of rich nitrogen source in cells of the strain 6-α‘1-NB13 the increased expression of CYR1, BCY1 and Ras2Val19 genes decreases the frequency of [PSI+] prion induction. Instead, in the presence of poor nitrogen source in cells of the strain 7-α‘1-NB13 the increased expression of BCY1 gene increases the frequency of [PSI+] prion induction. So, it is possible to make a conclusion that the frequency of induction of the [PSI+] prion depends on the activity of the Ras/PKA signal transduction pathway.