Biomedical subjects

P Scott Eastman

Publications and source records attributed to P Scott Eastman.

4 recordsLinked to original sources

PubMed2006

Global analysis of X-chromosome dosage compensation.

BACKGROUND: Drosophila melanogaster females have two X chromosomes and two autosome sets (XX;AA), while males have a single X chromosome and two autosome sets (X;AA). Drosophila male somatic cells compensate for a single copy of the X chromosome by deploying male-specific-lethal (MSL) complexes that increase transcription from the X chromosome. Male germ cells lack MSL complexes, indicating that either germline X-chromosome dosage compensation is MSL-independent, or that germ cells do not carry out dosage compensation. RESULTS: To investigate whether dosage compensation occurs in germ cells, we directly assayed X-chromosome transcripts using DNA microarrays and show equivalent expression in XX;AA and X;AA germline tissues. In X;AA germ cells, expression from the single X chromosome is about twice that of a single autosome. This mechanism ensures balanced X-chromosome expression between the sexes and, more importantly, it ensures balanced expression between the single X chromosome and the autosome set. Oddly, the inactivation of an X chromosome in mammalian females reduces the effective X-chromosome dose and means that females face the same X-chromosome transcript deficiency as males. Contrary to most current dosage-compensation models, we also show increased X-chromosome expression in X;AA and XX;AA somatic cells of Caenorhabditis elegans and mice. CONCLUSION: Drosophila germ cells compensate for X-chromosome dose. This occurs by equilibrating X-chromosome and autosome expression in X;AA cells. Increased expression of the X chromosome in X;AA individuals appears to be phylogenetically conserved.

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PubMed2006

Qdot nanobarcodes for multiplexed gene expression analysis.

We report a quantum dot (Qdot) nanobarcode-based microbead random array platform for accurate and reproducible gene expression profiling in a high-throughput and multiplexed format. Four different sizes of Qdots, with emissions at 525, 545, 565, and 585 nm are mixed with a polymer and coated onto the 8-mum-diameter magnetic microbeads to generate a nanobarcoded bead termed as QBeads. Twelve intensity levels for each of the four colors were used. Gene-specific oligonucleotide probes are conjugated to the surface of each spectrally nanobarcoded bead to create a multiplexed panel, and biotinylated cRNAs are generated from sample total RNA and hybridized to the gene probes on the microbeads. A fifth streptavidin Qdot (655 nm or infrared Qdot) binds to biotin on the cRNA, acting as a quantification reporter. Target identity was decoded based on spectral profile and intensity ratios of the four coding Qdots (525, 545, 565, and 585 nm). The intensity of the 655 nm Qdot reflects the level of biotinylated cRNA captured on the beads and provides the quantification for the corresponding target gene. The system shows a sensitivity of < or =10(4) target molecules detectable with T7 amplification, a level that is better than the 10(5) number achievable with a high-density microarray system, and approaching the 10(3)-10(4) level usually observed for quantitative PCR (qPCR). The QBead nanobarcode system has a dynamic range of 3.5 logs, better than the 2-3 logs observed on various microarray platforms. The hybridization reaction is performed in liquid phase and completed in 1-2 hours, at least 1 order of magnitude faster than microarray-based hybridizations. Detectable fold change is lower than 1.4-fold, showing high precision even at close to single copy per cell level. Reproducibility for this proof-of-concept study approaches that of Affymetrix GeneChip microarray, with an R(2) value between two repeats at 0.984, and interwell CV around 5%. In addition, it provides increased flexibility, convenience, and cost-effectiveness in comparison to conventional gene expression profiling methods.

Electronic Data Processing↗

PubMed2005

Defective expression of Tamm-Horsfall protein/uromodulin in COX-2-deficient mice increases their susceptibility to urinary tract infections.

Mice lacking a functional cyclooxygenase-2 (COX-2) gene develop abnormal kidneys that contain hypoplastic glomeruli and reduced proximal tubular mass, and they often die of renal failure. A comparison of kidney-specific gene expression between wild-type and COX-2-deficient mice by cDNA microarrays revealed that although more than 500 mRNAs were differentially expressed between the two strains of mice depending on their ages, the genes encoding pre-pro-epidermal growth factor (pre-pro-EGF) and Tamm-Horsfall protein (THP)/uromodulin were aberrantly expressed in the kidneys of COX-2 -/- mice at all stages of their development. Downregulation of EGF could potentially affect renal development, and THP/uromodulin gene has been implicated in abnormal kidney development and end-stage renal failure in humans. We assessed in detail mechanism of defective THP/uromodulin gene expression and its potential consequences in COX-2-deficient mice. Consistent with the microarray data, the steady-state levels of THP/uromodulin mRNA were severely reduced in the COX-2 -/- kidney. Furthermore, reduced expression of renal THP/uromodulin, as assessed by Western blot and immunohistological methods, was closely corroborated by a corresponding decline in the urinary secretion of THP/uromodulin in COX-2 -/- mice. Finally, we demonstrate that the bladders of COX-2 -/- mice, in contrast to those of the wild-type mice, are highly susceptible to colonization by uropathogenic Escherichia coli.

Animals↗

PubMed2003

Selectively reduced expression of synaptic plasticity-related genes in amyloid precursor protein + presenilin-1 transgenic mice.

A critical question in Alzheimer's disease (AD) research is the cause of memory loss that leads to dementia. The amyloid precursor protein + presenilin-1 (APP+PS1) transgenic mouse is a model for amyloid deposition, and like AD, the mice develop memory deficits as amyloid deposits accumulate. We profiled gene expression in these transgenic mice by microarray and quantitative RT-PCR (qRT-PCR). At the age when these animals developed cognitive dysfunction, they had reduced mRNA expression of several genes essential for long-term potentiation and memory formation (Arc, Zif268, NR2B, GluR1, Homer-1a, Nur77/TR3). These changes appeared to be related to amyloid deposition, because mRNA expression was unchanged in the regions that did not accumulate amyloid. Transgene expression was similar in both amyloid-containing and amyloid-free regions of the brain. Interestingly, these changes occurred without apparent changes in synaptic structure, because a number of presynaptic marker mRNAs (growth-associated protein-43, synapsin, synaptophysin, synaptopodin, synaptotagmin, syntaxin) remained stable. Additionally, a number of genes related to inflammation were elevated in transgenic mice, primarily in the regions containing amyloid. In AD cortical tissue, the same memory-associated genes were downregulated. However, all synaptic and neuronal transcripts were reduced, implying that the loss of neurons and synapses contributed to these changes. We conclude that reduced expression of selected genes associated with memory consolidation are linked to memory loss in both circumstances. This suggests that the memory loss in APP+PS1 transgenic mice may model the early memory dysfunction in AD before the degeneration of synapses and neurons.

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