Steven B. Haase

Isolation of human sequences that replicate autonomously in human cells.

We have isolated a heterogeneous collection of human genomic sequences which replicate autonomously when introduced into human cells. The novel strategy for the isolation of these sequences involved cloning random human DNA fragments into a defective Epstein-Barr virus vector. This vector alone was unable to replicate in human cells, but appeared to provide for the nuclear retention of linked DNA. The human sequences persist in a long-term replication assay (greater than 2 months) in the presence of the viral nuclear retention sequences. Using a short-term (4-day) assay, we showed that the human sequences are able to replicate in the absence of all viral sequences. The plasmids bearing human sequences were shown to replicate based on the persistence of MboI-sensitive plasmid DNA in the long-term assay and the appearance of DpnI-resistant DNA in the short-term assay. The human sequences were shown to be responsible for the replication activity and may represent authentic human origins of replication.

Global control of cell-cycle transcription by coupled CDK and network oscillators

A significant fraction of the Saccharomyces cerevisiae genome is transcribed periodically during the cell division cycle, indicating that properly timed gene expression is important for regulating cell-cycle events. Genomic analyses of the localization and expression dynamics of transcription factors suggest that a network of sequentially expressed transcription factors could control the temporal programme of transcription during the cell cycle. However, directed studies interrogating small numbers of genes indicate that their periodic transcription is governed by the activity of cyclin-dependent kinases (CDKs). To determine the extent to which the global cell-cycle transcription programme is controlled by cyclin–CDK complexes, we examined genome-wide transcription dynamics in budding yeast mutant cells that do not express S-phase and mitotic cyclins. Here we show that a significant fraction of periodic genes are aberrantly expressed in the cyclin mutant. Although cells lacking cyclins are blocked at the G1/S border, nearly 70% of periodic genes continued to be expressed periodically and on schedule. Our findings reveal that although CDKs have a function in the regulation of cell-cycle transcription, they are not solely responsible for establishing the global periodic transcription programme. We propose that periodic transcription is an emergent property of a transcription factor network that can function as a cell-cycle oscillator independently of, and in tandem with, the CDK oscillator.

Improved flow cytometric analysis of the budding yeast cell cycle.

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size. Key Words: Flow cytometry, Cell cycle, Saccharomyces cerevisiae, SYTOX Green, Propidium iodide

Evidence that a free-running oscillator drives G1 events in the budding yeast cell cycle.

In yeast and somatic cells, mechanisms ensure cell-cycle events are initiated only when preceding events have been completed. In contrast, interruption of specific cell-cycle processes in early embryonic cells of many organisms does not affect the timing of subsequent events, indicating that cell-cycle events are triggered by a free-running cell-cycle oscillator. Here we present evidence for an independent cell-cycle oscillator in the budding yeast Saccharomyces cerevisiae. We observed periodic activation of events normally restricted to the G1 phase of the cell cycle, in cells lacking mitotic cyclin-dependent kinase activities that are essential for cell-cycle progression. As in embryonic cells, G1 events cycled on schedule, in the absence of S phase or mitosis, with a period similar to the cell-cycle time of wild-type cells. Oscillations of similar periodicity were observed in cells responding to mating pheromone in the absence of G1 cyclin (Cln)- and mitotic cyclin (Clb)-associated kinase activity, indicating that the oscillator may function independently of cyclin-dependent kinase dynamics. We also show that Clb-associated kinase activity is essential for ensuring dependencies by preventing the initiation of new G1 events when cell-cycle progression is delayed.

Multi-step control of spindle pole body duplication by cyclin-dependent kinase.

Organelles called centrosomes in metazoans or spindle pole bodies (SPBs) in yeast direct the assembly of a bipolar spindle that is essential for faithful segregation of chromosomes during mitosis. Abnormal accumulation of multiple centrosomes leads to genome instability, and has been observed in both tumour cells and cells with targeted mutations in tumour-suppressor genes. The defects that lead to centrosome amplification are not understood. We have recapitulated the multiple-centrosome phenotype in budding yeast by disrupting the activity of specific cyclin-dependent kinase (CDK) complexes. Our observations are reminiscent of mechanisms that govern DNA replication, and show that specific cyclin/CDK activities function both to promote SPB duplication and to prevent SPB reduplication.

Flow cytometric analysis of DNA content in budding yeast

Publisher Summary This chapter discusses the flow cytometric analysis of DNA content in budding yeast. The budding yeast, Saccharomyces cerevisiae, is a popular model organism for the study of eukaryotic cell biology. Saccharomyces cerevisiae was initially an attractive model system for cell cycle research because progression through the cell cycle was mirrored by changes in cell morphology. With advances in cell cycle research, alternative methods for assaying cell cycle progression have become increasingly important. This chapter outlines the methods for the preparation and staining of budding yeast for the analysis of DNA content. It also discusses the machine settings and collection parameters used for the acquisition of data on the flow cytometer. Further, this chapter discusses the interpretation of flow cytometer data, concentrating on points that are unique to the analysis of budding yeast cells.

Transcription inhibits the replication of autonomously replicating plasmids in human cells.

This study addresses the effect of transcription on replication, using a system based on autonomously replicating plasmids in human cells. We added transcriptional elements from the human cytomegalovirus promoter/enhancer and the human beta-actin promoter to autonomously replicating plasmids based on human sequences and found that the transcriptional elements inhibited plasmid replication. Furthermore, conditional inhibition of plasmid replication was demonstrated by using a tetracycline-responsive promoter. We found that replication activity of plasmids carrying this promoter was inversely correlated with promoter activity. Replication activity was partially restored on plasmids when a transcriptional termination sequence was placed directly downstream of the promoter element. Transcriptional activity of the promoters and the efficacy of the terminator sequence were confirmed by using steady-state RNA analysis. These experiments suggest that transcription inhibits DNA replication on these plasmids and that the degree of inhibition is dependent on transcription strength. The possible significance of these results for chromosomal DNA replication are discussed.

Distinct mechanisms control the stability of the related S-phase cyclins Clb5 and Clb6.

ABSTRACT The yeast S-phase cyclins Clb5 and Clb6 are closely related proteins that are synthesized late in G1. Although often grouped together with respect to function, Clb5 and Clb6 exhibit differences in their ability to promote S-phase progression. DNA replication is significantly slowed in clb5Δ mutants but not in clb6Δ mutants. We have examined the basis for the differential functions of Clb5 and Clb6 and determined that unlike Clb5, which is stable until mitosis, Clb6 is degraded rapidly at the G1/S border. N-terminal deletions of CLB6 were hyperstabilized, suggesting that the sequences responsible for directing the destruction of Clb6 reside in the N terminus. Clb6 lacks the destruction box motif responsible for the anaphase promoting complex-mediated destruction of Clb5 but contains putative Cdc4 degron motifs in the N terminus. Clb6 was hyperstabilized in cdc34-3 and cdc4-3 mutants at restrictive temperatures and when S/T-P phosphorylation sites in the N terminus were mutated to nonphosphorylatable residues. Efficient degradation of Clb6 requires the activities of both Cdc28 and Pho85. Finally, hyperstabilized Clb6 expressed from the CLB6 promoter rescued the slow S-phase defect exhibited by clb5Δ cells. Taken together, these findings suggest that the SCFCdc4 ubiquitin ligase complex regulates Clb6 turnover and that the functional differences exhibited by Clb5 and Clb6 arise from the distinct mechanisms controlling their stability.

Design and analysis of large-scale biological rhythm studies: a comparison of algorithms for detecting periodic signals in biological data

MOTIVATION To discover and study periodic processes in biological systems, we sought to identify periodic patterns in their gene expression data. We surveyed a large number of available methods for identifying periodicity in time series data and chose representatives of different mathematical perspectives that performed well on both synthetic data and biological data. Synthetic data were used to evaluate how each algorithm responds to different curve shapes, periods, phase shifts, noise levels and sampling rates. The biological datasets we tested represent a variety of periodic processes from different organisms, including the cell cycle and metabolic cycle in Saccharomyces cerevisiae, circadian rhythms in Mus musculus and the root clock in Arabidopsis thaliana. RESULTS From these results, we discovered that each algorithm had different strengths. Based on our findings, we make recommendations for selecting and applying these methods depending on the nature of the data and the periodic patterns of interest. Additionally, these results can also be used to inform the design of large-scale biological rhythm experiments so that the resulting data can be used with these algorithms to detect periodic signals more effectively.

Cyclin-dependent kinases are regulators and effectors of oscillations driven by a transcription factor network

During embryonic cell cycles, B-cyclin-CDKs function as the core component of an autonomous oscillator. Current models for the cell-cycle oscillator in nonembryonic cells are slightly more complex, incorporating multiple G1, S phase, and mitotic cyclin-CDK complexes. However, periodic events persist in yeast cells lacking all S phase and mitotic B-cyclin genes, challenging the assertion that cyclin-CDK complexes are essential for oscillations. These and other results led to the proposal that a network of sequentially activated transcription factors functions as an underlying cell-cycle oscillator. Here we examine the individual contributions of a transcription factor network and cyclin-CDKs to the maintenance of cell-cycle oscillations. Our findings suggest that while cyclin-CDKs are not required for oscillations, they do contribute to oscillation robustness. A model emerges in which cyclin expression (thereby, CDK activity) is entrained to an autonomous transcriptional oscillator. CDKs then modulate oscillator function and serve as effectors of the oscillator.