Biomedical subjects

Mark L Parrish

Publications and source records attributed to Mark L Parrish.

5 recordsLinked to original sources

PubMed2004

A microarray platform comparison for neuroscience applications.

To address the need for high sensitivity in gene expression profiling of small neural tissue samples ( approximately 100 ng total RNA), we compared a novel RT-PCR-IVT protocol using fluor-reverse pairs on inkjet oligonucleotide microarrays and an RT-IVT protocol using 33P labeling on nylon cDNA arrays. The comparison protocol was designed to evaluate these systems for sensitivity, specificity, reproducibility, and linearity. We developed parameters, thresholds, and testing conditions that could be used to differentiate various systems that spanned detection chemistry and instrumentation; probe number and selection criteria; and sample processing protocols. We concluded that the inkjet system had better performance in sensitivity, specificity, and reproducibility than the nylon system, and similar performance in linearity. Between these two platforms, the data indicates that the inkjet system would perform better for the transcriptional profiling of 100 ng total RNA samples for neuroscience studies.

Animals↗

PubMed2003

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Alternative Splicing↗

PubMed2003

Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.

Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation.

DNA, Fungal↗

PubMed2002

Proliferation of the endoplasmic reticulum occurs normally in cells that lack a functional unfolded protein response.

Increased expression of certain ER membrane proteins leads to biogenesis of novel ER membrane arrays. These structures provide models in which to explore the mechanisms by which cells control the size and organization of organelles in response to changing physiological demands. In yeast, elevated levels of HMG-CoA reductase induce ER arrays known as karmellae. Cox and co-workers (1997) discovered that karmellae assembly is toxic to ire1 mutants. These mutants are unable to initiate the unfolded protein response, which enables cells to adjust levels of ER chaperones in response to stresses. We sought to determine whether the karmellae-dependent death of ire1 mutants was due to karmellae assembly or to increased levels of HMG-CoA reductase activity. Unexpectedly, we found that ire1 cells could assemble normal levels of karmellae that were structurally identical to those of wild-type cells. In addition, karmellae assembly did not itself induce the unfolded protein response. Certain ire1 strains produced significant numbers of transformants that were unable to utilize galactose as sole carbon source. These results suggest that the karmellae-dependent death of certain ire1 strains may simply reflect their inability to grow on galactose.

Endoplasmic Reticulum↗

PubMed2002

Mutations that affect vacuole biogenesis inhibit proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae.

In yeast, increased levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme, Hmg1p, induce assembly of nuclear-associated ER membranes called karmellae. To identify additional genes involved in karmellae assembly, we screened temperature-sensitive mutants for karmellae assembly defects. Two independently isolated, temperature-sensitive strains that were also defective for karmellae biogenesis carried mutations in VPS16, a gene involved in vacuolar protein sorting. Karmellae biogenesis was defective in all 13 other vacuole biogenesis mutants tested, although the severity of the karmellae assembly defect varied depending on the particular mutation. The hypersensitivity of 14 vacuole biogenesis mutants to tunicamycin was well correlated with pronounced defects in karmellae assembly, suggesting that the karmellae assembly defect reflected alteration of ER structure or function. Consistent with this hypothesis, seven of eight mutations causing defects in secretion also affected karmellae assembly. However, the vacuole biogenesis mutants were able to proliferate their ER in response to Hmg2p, indicating that the mutants did not have a global defect in the process of ER biogenesis.

Anti-Bacterial Agents↗