Biomedical subjects

Michael J Holland

Publications and source records attributed to Michael J Holland.

6 recordsLinked to original sources

PubMed2005

PrimerSelect: a transcriptome-wide oligonucleotide primer pair design program for kinetic RT-PCR-based transcript profiling.

We describe PrimerSelect, a program capable of transcriptome-wide design of primer pairs for optimal performance in kinetic reverse-transcriptase polymerase chain reaction (RT-PCR). For the yeast Saccharomyces cerevisiae, PrimerSelect designs primer pairs for 86% of genomic open reading frames (ORFs) using design criteria we previously established to be optimal for kinetic RT-PCR (kRT-PCR)-based transcript quantitation. Primer pairs designed by PrimerSelect for 230 yeast ORFs were evaluated for primer dimer potential, PCR cyclewise yield, and cross-priming. Performance of 95% of these primer pairs is optimal with respect to primer dimer potential and PCR cyclewise yield for quantitating even the rarest yeast transcript. All of the primer pairs produced a single amplicon of the expected size from yeast genomic DNA template. The utility of PrimerSelect for designing primer pairs complementary to ORF sequences defined for multiple isolates of the human bacterial pathogens Helicobacter pylori and Staphylococcus aureus is also demonstrated.

Computer-Aided Design↗

PubMed2005

Mutations in the nucleosome core enhance transcriptional silencing.

Transcriptional silencing in Saccharomyces requires specific nucleosome modifications promoted in part by a complex of Sir proteins that binds to the modified nucleosomes. Recent evidence suggests that modifications of both the histone amino termini and the core domain of nucleosomes contribute to silencing. We previously identified histone H4 mutations affecting residues in the core of the nucleosome that yield enhanced silencing at telomeres. Here we show that enhanced silencing induced by these mutations increases the proportion of cells in which telomeres and silent mating-type loci are in the silent state. One H4 mutation affects the expression of a subset of genes whose expression is altered by deletion of HTZ1, which encodes the histone variant H2A.Z, suggesting that the mutation may antagonize H2A.Z incorporation into nucleosomes. A second mutation causes the spread of silencing into subtelomeric regions that are not normally silenced in wild-type cells. Mechanistically, this mutation does not significantly accelerate the formation of silent chromatin but, rather, reduces the rate of decay of the silenced state. We propose that these mutations use distinct mechanisms to affect the dynamic interplay between activation and repression at the boundary between active and silent chromatin.

Alleles↗

PubMed2004

Interaction between transcription elongation factors and mRNA 3'-end formation at the Saccharomyces cerevisiae GAL10-GAL7 locus.

Spt6 is a conserved transcription factor that associates with RNA polymerase II (pol II) during elongation. Spt6 is essential for viability in Saccharomyces cerevisiae and regulates chromatin structure during pol II transcription. Here we present evidence that mutations that impair Spt6, a second elongation factor, Spt4, and pol II can affect 3'-end formation at GAL10. Additional analysis suggests that Spt6 is required for cotranscriptional association of the factor Ctr9, a member of the Paf1 complex, with GAL10 and GAL7, and that Ctr9 association with chromatin 3' of GAL10 is regulated by the GAL10 polyadenylation signal. Overall, these results provide new evidence for a connection between the transcription elongation factor Spt6 and 3'-end formation in vivo.

3' Untranslated Regions↗

PubMed2004

Increased sample capacity for genotyping and expression profiling by kinetic polymerase chain reaction.

We fabricated and evaluated high-throughput kinetic thermal cyclers with 768-reaction capacity for kinetic polymerase chain reaction (kPCR)-based genotyping and kinetic reverse transcription (kRT)-PCR-based transcript quantitation. The system uses dye-based detection with ethidium bromide and a single DNA polymerase-based PCR or RT-PCR assay. Allele-specific detection of the two most common hereditary hemochromotosis mutant alleles, C282Y and H63D, was reliably measured by kPCR using human DNA templates as low as 10 genome equivalents per assay. Transcript profiling was performed for 16 yeast transcripts ranging in intracellular abundance over four orders of magnitude. Standard deviations of the PCR cycle threshold values determined from multiple kRT-PCR assays in three different instruments ranged from 0.11 to 0.97 PCR cycles and were reproducible, transcript specific, and instrument independent. The effects of the sin3, gal11, and snf2 knockout mutations on expression of 385 yeast genes were evaluated by kRT-PCR and compared to published values determined by high-density oligonucleotide array and/or microarray analysis for snf2 and sin3. The 768-reaction kinetic thermalcyclers, each with a capacity for more than a half million assays per year, are well suited to genomics applications such as single nucleotide polymorphism/disease association studies and genomewide transcription profiling where high sensitivity and accuracy are required.

Base Sequence↗

PubMed2003

Enzymatic function of loop movement in enolase: preparation and some properties of H159N, H159A, H159F, and N207A enolases.

The hypothesis that His159 in yeast enolase moves on a polypeptide loop to protonate the phosphoryl of 2-phosphoglycerate to initiate its conversion to phosphoenolpyruvate was tested by preparing H159N, H159A, and H159F enolases. These have 0.07%-0.25% of the native activity under standard assay conditions and the pH dependence of maximum velocities of H159A and H159N mutants is markedly altered. Activation by Mg2+ is biphasic, with the smaller Mg2+ activation constant closer to that of the "catalytic" Mg2+ binding site of native enolase and the larger in the mM range in which native enolase is inhibited. A third Mg2+ may bind to the phosphoryl, functionally replacing proton donation by His159. N207A enolase lacks an intersubunit interaction that stabilizes the closed loop(s) conformation when 2-phosphoglycerate binds. It has 21% of the native activity, also exhibits biphasic Mg2+ activation, and its reaction with the aldehyde analogue of the substrate is more strongly inhibited than is its normal enzymatic reaction. Polypeptide loop(s) closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilize a carbanion intermediate.

Animals↗

PubMed2002

Transcript abundance in yeast varies over six orders of magnitude.

In the current era of functional genomics, it is remarkable that the intracellular range of transcript abundance is largely unknown. For the yeast Saccharomyces cerevisiae, hybridization-based complexity analysis and SAGE analysis showed that the majority of yeast mRNAs are present at one or fewer copies per cell; however, neither method provides an accurate estimate of the full range of low abundance transcripts. Here we examine the range of intracellular transcript abundance in yeast using kinetically monitored, reverse transcriptase-initiated PCR (kRT-PCR). Steady-state transcript levels encoded by all 65 genes on the left arm of chromosome III and 185 transcription factor genes are quantitated. Abundant transcripts encoded by glycolytic genes, previously quantitated by kRT-PCR, are present at a few hundred copies per cell whereas genes encoding physiologically important transcription factors are expressed at levels as low as one-thousandth transcript per cell. Of the genes assessed, only the silent mating type loci, HML and HMR, are transcriptionally silent. The results show that transcript abundance in yeast varies over six orders of magnitude. Finally, kRT-PCR, cDNA microarray, and high density oligonucleotide array assays are compared for their ability to detect and quantitate the complete yeast transcriptome.

Genes, Fungal↗