Biomedical subjects

David S McNabb

Publications and source records attributed to David S McNabb.

5 recordsLinked to original sources

PubMed2005

Slowing DNA translocation in a solid-state nanopore.

Reducing a DNA molecule's translocation speed in a solid-state nanopore is a key step toward rapid single molecule identification. Here we demonstrate that DNA translocation speeds can be reduced by an order of magnitude over previous results. By controlling the electrolyte temperature, salt concentration, viscosity, and the electrical bias voltage across the nanopore, we obtain a 3 base/micros translocation speed for 3 kbp double-stranded DNA in a 4-8 nm diameter silicon nitride pore. Our results also indicate that the ionic conductivity inside such a nanopore is smaller than it is in bulk.

DNA↗

PubMed2005

Detecting single stranded DNA with a solid state nanopore.

Voltage biased solid-state nanopores are used to detect and characterize individual single stranded DNA molecules of fixed micrometer length by operating a nanopore detector at pH values greater than approximately 11.6. The distribution of observed molecular event durations and blockade currents shows that a significant fraction of the events obey a rule of constant event charge deficit (ecd) indicating that they correspond to molecules translocating through the nanopore in a distribution of folded and unfolded configurations. A surprisingly large component is unfolded. The result is an important milestone in developing solid-state nanopores for single molecule sequencing applications.

DNA, Single-Stranded↗

PubMed2005

Novel regulatory function for the CCAAT-binding factor in Candida albicans.

Candida albicans is an opportunistic human pathogen that can sense environmental changes and respond by altering its cell morphology and physiology. A number of environmental factors have been shown to influence this dimorphic transition, including pH, starvation, serum, and amino acids. In this report, we investigate the function of the C. albicans CCAAT-binding factor. In Saccharomyces cerevisiae, this heterooligomeric transcriptional activator stimulates the expression of genes that encode proteins involved in respiration. To examine the function of this transcription factor in C. albicans, we cloned CaHAP5 and generated a hap5delta/hap5delta mutant of C. albicans. Using mobility shift studies, we identified four separate complexes from C. albicans cell extracts whose DNA-binding activities were abolished in the hap5delta/hap5delta mutant, suggesting that they represented sequence-specific CCAAT-binding complexes. We found that the C. albicans hap5delta homozygote was defective in hyphal development under a variety of conditions, and the mutant displayed a carbon source-dependent "hyperfilamentation" phenotype under certain growth conditions. In addition, the mRNA levels for two enzymes involved in respiration, encoded by COX5 and CYC1, were overexpressed in the hap5delta/hap5delta mutant when grown in medium containing amino acids as the sole carbon and nitrogen source. Thus, the C. albicans CCAAT-binding factor appeared to function as a repressor of genes encoding mitochondrial electron transport components, in contrast to its activator function in S. cerevisiae. These data provide the first evidence that the CCAAT-binding factor can act as a transcriptional repressor and raise new and interesting questions about how carbon metabolism is regulated in this opportunistic human pathogen.

Amino Acid Sequence↗

PubMed2005

Assembly of the Hap2p/Hap3p/Hap4p/Hap5p-DNA complex in Saccharomyces cerevisiae.

The CCAAT-binding factor (CBF) is an evolutionarily conserved multimeric transcriptional activator in eukaryotes. In Saccharomyces cerevisiae, the CCAAT-binding factor is composed of four subunits, termed Hap2p, Hap3p, Hap4p, and Hap5p. The Hap2p/Hap3p/Hap5p heterotrimer is the DNA-binding component of the complex that binds to the consensus 5'-CCAAT-3' sequence in the promoter of target genes. The Hap4p subunit contains the transcriptional activation domain necessary for stimulating transcription after interacting with Hap2p/Hap3p/Hap5p. In this report, we demonstrate that Hap2p, Hap3p, and Hap5p assemble via a one-step pathway requiring all three subunits simultaneously, as opposed to the mammalian CCAAT-binding factor which has been shown to assemble via a two-step pathway with CBF-A (Hap3p homolog) and CBF-C (Hap5p homolog) forming a stable dimer before CBF-B (Hap2p homolog) can interact. We have also found that the interaction of Hap4p with Hap2p/Hap3p/Hap5p requires DNA binding as a prerequisite. To further understand the protein-protein and protein-DNA interactions of this transcription factor, we identified the minimal domain of Hap4p necessary for interaction with the Hap2p/Hap3p/Hap5p-DNA complex, and we demonstrate that this domain is sufficient to complement the respiratory deficiency of a hap4Delta mutant and activate transcription when fused with the VP16 activation domain. These studies provide a further understanding of the assembly of the yeast CCAAT-binding factor at target promoters and raise a number of questions concerning the protein-protein and protein-DNA interactions of this multisubunit transcription factor.

Amino Acid Sequence↗

PubMed2005

Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae.

A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multi-cloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements.

Base Sequence↗