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Stephen J Kron

Publications and source records attributed to Stephen J Kron.

27 records · Page 2Linked to original sources
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↗
PubMedDate not supplied

Relevance and irrelevance of DNA damage response to radiotherapy.

Ionizing radiation (IR) has been used to treat human malignancies since the early part of the 20th century. To date, most of the advances in radiotherapy have focused on optimization of treatment delivery schedules and technologic improvements in the physical targeting of dose. By comparison, many of the discoveries regarding the molecular basis of DNA damage and repair have not yet been translated to clinical practice. This article offers some perspectives regarding modulators of radiation effects and the challenges faced as we approach newer molecular targets. Our goal is to frame the issues that contribute to the apparent disconnect between laboratory discoveries and improvements in clinically relevant therapeutics.

DNA↗
PubMedDate not supplied

Protein-acrylamide copolymer hydrogels for array-based detection of tyrosine kinase activity from cell lysates.

We describe the development of an array-based assay for the molecular level detection of tyrosine kinase activity directly from cellular extracts. Glutathione S-transferase-Crkl (GST-Crkl) fusion proteins are covalently immobilized into polyacrylamide gel pads via copolymerization of acrylic monomer and acrylic-functionalized GST-Crkl protein constructs on a polyacrylamide surface. The resulting hydrogels resist nonspecific protein adsorption, permitting quantitative and reproducible determination of Abl tyrosine kinase activity and inhibition, even in the presence of a complex cell lysate mixture. Half-maximal inhibition (IC50) values for imatinib mesylate inhibition of GST-Crkl (SH3) phosphorylation by v-Abl in a purified system and Bcr-Abl within a K562 cell lysate were determined to be 1.5 and 20 microM, respectively. Additionally, the protein-acrylamide copolymer arrays detected CML cell levels as low as 15% in a background of Bcr-Abl- leukemic cells and provided the framework for the parallel evaluation of six tyrosine kinase inhibitors. Such a system may have direct application to the detection and treatment of cancers resulting from upregulated tyrosine kinase activity, such as chronic myeloid leukemia (CML). These findings also establish a basis for screening tyrosine kinase inhibitors and provide a framework on which protein-protein interactions in other complex systems can be studied.

Acrylamides↗
PubMedDate not supplied

Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI-TOF-MS detection.

Thiophosphorylation provides a metabolically stable, chemically reactive phosphorylation analogue for analyzing the phosphoproteome in vitro and in vivo. We developed a MALDI-TOF-MS based assay for optimizing thiophosphopeptide production by a kinase even in the presence of Mg(2+) and ATP. We found that Abl kinase thiophosphorylation rates can be "rescued" using Mn(2+) in the presence of Mg(2+). Under our ideal conditions, titration of Mn(2+) and ATPgammaS in the presence of Mg(2+) allowed relatively rapid, highly specific thiophosphorylation by Abl tyrosine kinase, both as purified enzyme and in complex cell extracts.

Adenosine Triphosphate↗