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Stephen J Kron

Publications and source records attributed to Stephen J Kron.

13 recordsLinked to original sources
PubMed2003

Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy.

Based on the role of phosphorylation of the histone H2A variant H2AX in recruitment of DNA repair and checkpoint proteins to the sites of DNA damage, we have investigated gammaH2AX as a reporter of tumor radiosensitivity and a potential target to enhance the effectiveness of radiation therapy. Clinically relevant ionizing radiation (IR) doses induced similar patterns of gammaH2AX focus formation or immunoreactivity in radiosensitive and radioresistant human tumor cell lines and xenografted tumors. However, radiosensitive tumor cells and xenografts retained gammaH2AX for a greater duration than radioresistant cells and tumors. These results suggest that persistence of gammaH2AX after IR may predict tumor response to radiotherapy. We synthesized peptide mimics of the H2AX carboxyl-terminal tail to test whether antagonizing H2AX function affects tumor cell survival following IR. The peptides did not alter the viability of unirradiated tumor cells, but both blocked induction of gammaH2AX foci by IR and enhanced cell death in irradiated radioresistant tumor cells. These results suggest that H2AX is a potential molecular target to enhance the effects of radiotherapy.

Animals↗
PubMed2003

Asynchronous cell cycle and asymmetric vacuolar inheritance in true hyphae of Candida albicans.

Candida albicans forms unconstricted hyphae in serum-containing medium that are divided into discrete compartments. Time-lapse photomicroscopy, flow cytometry, and a novel three-dimensional imaging system were used to demonstrate that the kinetics and cell cycle events accompanying hyphal development were correlated with dynamic changes in vacuole morphology and the pattern of vacuole inheritance. Apical cells of hyphae underwent continuous extension before and after the first cytokinesis event. However, the resulting mother cell and sub-apical compartments did not immediately reenter the cell cycle and instead underwent cell cycle arrest before reentering the cycle. Vacuole was inherited asymmetrically at cytokinesis so that the distal, arrested compartments inherited most vacuole and the growing apical cell inherited most cytoplasm. Hydroxyurea release experiments demonstrated that the arrested, vacuolated hyphal compartments were in the G(1) phase of the cycle. The period of cell cycle arrest was decreased by the provision of assimilatable forms of nitrogen, suggesting that the hyphal cell cycle is regulated by nitrogen limitation that results in sup-apical cell cycle arrest. This pattern of growth is distinct from that of the synchronous, symmetrical development of pseudohyphae of C. albicans and other yeast species. These observations suggest that the cellular vacuole space correlates with alterations in the cell cycles of different cell types and that the total organelle space may influence size-regulated functions and hence the timing of the eukaryotic cell cycle.

Animals↗
PubMed2002

Design and implementation of algorithms for focus automation in digital imaging time-lapse microscopy.

BACKGROUND: Digital time-lapse microscopy using Nomarski-DIC requires that an autofocusing system adapt to changes in cell shape, size, and position while contending with drift, noise, and hysteresis in the microscope and imager. We have designed and implemented an autofocusing system that tracks subjects under dynamic conditions and maintains focus within a threshold of discriminability. METHODS: With the use of proven and novel algorithms for autofocusing in Nomarski, we performed "virtual" experiments on recorded image stacks to simulate drift and sudden displacements and test the search algorithm response. RESULTS: We found that combining a simple [1, -1] contrast function with an adaptive "warmer-colder" focusing algorithm yields a reasonable compromise between focusing precision and noise tolerance. This method was implemented to record growth kinetics of yeast cells in single and multiple fields of view over several hours. CONCLUSIONS: We have implemented a robust digital autofocus that maintains focus on optically complex samples imaged at high resolution. The tolerance of this system of drift and vibration suggests that it is a practical system for time-lapse imaging in many biological applications.

Algorithms↗
PubMed2002

An essential function of yeast cyclin-dependent kinase Cdc28 maintains chromosome stability.

Multiple surveillance pathways maintain genomic integrity in yeast during mitosis. Although the cyclin-dependent kinase Cdc28 is a well established regulator of mitotic progression, evidence for a direct role in mitotic surveillance has been lacking. We have now implicated a conserved sequence in the Cdc28 carboxyl terminus in maintaining chromosome stability through mitosis. Six temperature-sensitive mutants were isolated via random mutagenesis of 13 carboxyl-terminal residues. These mutants identify a Cdc28 domain necessary for proper mitotic arrest in the face of kinetochore defects or microtubule inhibitors. These chromosome stability-defective cdc28(CST) mutants inappropriately continue mitosis when the mitotic spindle is disrupted at 23 degrees C, display high rates of spontaneous chromosome loss at 30 degrees C, and suffer catastrophic aneuploidy at 35 degrees C. A dosage suppression screen identified Cak1, a kinase known to phosphorylate and activate Cdc28, as a specific high copy suppressor of cdc28(CST) temperature sensitivity and chromosome instability. Suppression is independent of the kinase activity of Cak1, suggesting that Cak1 may bind to the carboxyl terminus to serve a non-catalytic role in assembly and/or stabilization of active Cdc28 complexes. Significantly, these studies implicate Cdc28 and Cak1 in an essential surveillance function required to maintain genetic stability through mitosis.

Amino Acid Sequence↗
PubMed2002

Bcl-x(L) complements Saccharomyces cerevisiae genes that facilitate the switch from glycolytic to oxidative metabolism.

All eukaryotic organisms have mechanisms to adapt to changing metabolic conditions. The mammalian cell survival gene Bcl-x(L) enables cells to adapt to changes in cellular metabolism. To identify genes whose function can be substituted by Bcl-x(L) in a unicellular eukaryote, a genetic screen was performed using the yeast Saccharomyces cerevisiae. S. cerevisiae grows by anaerobic glycolysis when glucose is available, switching to oxidative phosphorylation when carbohydrate in the media becomes limiting (diauxic shift). Given that Bcl-x(L) appears to facilitate the switch from glycolytic to oxidative metabolism in mammalian cells, a library of yeast mutants was tested for the ability to efficiently undergo diauxic shift in the presence and absence of Bcl-x(L). Several mutants were identified that have a defect in growth when switched from a fermentable to a nonfermentable carbon source that is corrected by the expression of Bcl-x(L). These genes include the mitochondrial chaperonin TCM62, as well as previously uncharacterized genes. One of these uncharacterized genes, SVF1, promotes cell survival in mammalian cells in response to multiple apoptotic stimuli. The finding that TCM62 and the analogous human prohibitin gene also inhibit mammalian cell death following growth factor withdrawal implicates mitochondrial chaperones as regulators of apoptosis. Further characterization of the genes identified in this screen may enhance our understanding of Bcl-x(L) function in mammalian cells, and of cell survival pathways in general.

Animals↗
PubMed2002

Robust G1 checkpoint arrest in budding yeast: dependence on DNA damage signaling and repair.

Although most eukaryotes can arrest in G1 after ionizing radiation, the existence or significance of a G1 checkpoint in S. cerevisiae has been challenged. Previous studies of G1 response to chemical mutagens, X-ray or UV irradiation indicate that the delay before replication is transient and may reflect a strong intra-S-phase checkpoint. We examined the yeast response to double-stranded breaks in G1 using gamma irradiation. G1 irradiation induces repair foci on chromosome spreads and a Rad53 band shift characteristic of activation, which suggest an active DNA damage response. Consistent with a G1 arrest, bud emergence, spindle pole duplication and DNA replication are each delayed in a dose-dependent manner. Sensitivity to mating pheromone is prolonged to over 18 hours when G1 cells are lethally gamma or UV irradiated. Strikingly, G1 delay is the predominant response to continuous gamma irradiation at a dose that confers no loss of viability but delays cell division. Like the G2/M checkpoint, G1 delay is completely dependent on both RAD9 and RAD24 epistasis groups but independent of POL(epsilon). Lethally irradiated rad9 mutants rapidly exit G1 but perform a slow S phase, whereas rad17 and rad24 mutants are completely arrest deficient. Distinct from gamma irradiation, G1 arrest after UV is RAD14 dependent, suggesting that DNA damage processing is required for checkpoint activation. Therefore, as in the yeast G2/M checkpoint response, free DNA ends and/or single-stranded DNA are necessary and sufficient to induce a bona fide G1 checkpoint arrest.

Cell Cycle Proteins↗
PubMed2002

Marker-fusion PCR for one-step mutagenesis of essential genes in yeast.

We describe a one-step gene replacement method based on fusion PCR that can be used to mutagenize essential genes at their endogenous locus. Marker-fusion PCR can facilitate transfer of alleles between strains as well as PCR-based techniques, such as site-directed and error-prone PCR mutagenesis, all without cloning or strain constructions. With this method, PCR is used to fuse a mutagenized fragment to an overlapping fragment containing a selectable marker flanked by regions of homology to the target. By transforming yeast with these PCR products, specific mutations are introduced at the endogenous locus through homologous recombination. We tested the 'marker-fusion PCR' method using the budding yeast CDC28 gene and were able to efficiently introduce site-directed mutations and integrate genomic or plasmid-borne mutant alleles. As a further application for this method, we used a spiked oligonucleotide to randomize the coding sequence for a single domain of CDC28 and were able to construct highly mutagenized libraries for this region.

Genes, Fungal↗
PubMed2002

Peptide chips for the quantitative evaluation of protein kinase activity.

Peptide chips are an emerging technology that could replace many of the bioanalytical methods currently used in drug discovery, diagnostics, and cell biology. Despite the promise of these chips, their development for quantitative assays has been limited by several factors, including a lack of well-defined surface chemistries to immobilize peptides, the heterogeneous presentation of immobilized ligands, and nonspecific adsorption of protein to the substrate. This paper describes a peptide chip that overcomes these limitations, and demonstrates its utility in activity assays of the nonreceptor tyrosine kinase c-Src. The chip was prepared by the Diels-Alder-mediated immobilization of the kinase substrate AcIYGEFKKKC-NH(2) on a self-assembled monolayer of alkanethiolates on gold. Phosphorylation of the immobilized peptides was characterized by surface plasmon resonance, fluorescence, and phosphorimaging. Three inhibitors of the enzyme were quantitatively evaluated in an array format on a single, homogeneous substrate.

Amino Acid Sequence↗
PubMed2002

Depression of Saccharomyces cerevisiae invasive growth on non-glucose carbon sources requires the Snf1 kinase.

Haploid Saccharomyces cerevisiae cells growing on media lacking glucose but containing high concentrations of carbon sources such as fructose, galactose, raffinose, and ethanol exhibit enhanced agar invasion. These carbon sources also promote diploid filamentous growth in response to nitrogen starvation. The enhanced invasive and filamentous growth phenotypes are suppressed by the addition of glucose to the media and require the Snf1 kinase. Mutations in the PGI1 and GND1 genes encoding carbon source utilization enzymes confer enhanced invasive growth that is unaffected by glucose but requires active Snf1. Carbon source does not modulate FLO11 flocculin expression, but enhanced polarized bud site selection is necessary for invasion on certain carbon sources. Interestingly, deletion of SNF1 blocks invasion without affecting bud site selection. Snf1 is also required for formation of spokes and hubs in multicellular mats. To examine glucose repression of invasive growth more broadly, we performed genome-wide microarray expression analysis in wild-type cells growing on glucose and galactose, and snf1 Delta cells on galactose. SNF1 probably mediates glucose repression of multiple genes potentially involved in invasive and filamentous growth. FLO11-independent cell-cell attachment, cell wall integrity, and/or polarized growth are affected by carbon source metabolism. In addition, derepression of cell cycle genes and signalling via the cAMP-PKA pathway appears to depend upon SNF1 activity during growth on galactose.

Carbon↗
PubMed2002

NuA4 subunit Yng2 function in intra-S-phase DNA damage response.

While regulated transcription requires acetylation of histone N-terminal tails to promote an open chromatin conformation, a similar role for histone acetylation in DNA replication and/or repair remains to be established. Cells lacking the NuA4 subunit Yng2 are viable but critically deficient for genome-wide nucleosomal histone H4 acetylation. We found that yng2 mutants are specifically sensitized to DNA damage in S phase induced by cdc8 or cdc9 mutations, hydroxyurea, camptothecin, or methylmethane sulfonate (MMS). In yng2, MMS treatment causes a persistent Mec1-dependent intra-S-phase checkpoint delay characterized by slow DNA repair. Restoring H4 acetylation with the histone deacetylase inhibitor trichostatin A promotes checkpoint recovery. In turn, mutants lacking the histone H3-specific acetyltransferase GCN5 are similarly sensitive to intra-S-phase DNA damage. The inviability of gcn5 yng2 double mutants suggests overlapping roles for H3 and H4 acetylation in DNA replication and repair. Paradoxically, haploid yng2 mutants do not tolerate mutations in genes important for nonhomologous end joining repair yet remain proficient for homologous recombination. Our results implicate nucleosomal histone acetylation in maintaining genomic integrity during chromosomal replication.

Acetylation↗