Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
 2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We constructed a subgenomic cosmid library of DNA replicated early in the S phase of normal human diploid fibroblasts. Cells were synchronized by release from confluence arrest and incubation in the presence of aphidicolin. Bromodeoxyuridine (BrdUrd) was added to aphidicolin-containing medium to label DNA replicated as cells entered S phase. Nuclear DNA was partially digested with Sau 3AI, and hybrid density DNA was separated in CsCl gradients. The purified early-replicating DNA was cloned into sCos1 cosmid vector. Clones were transferred individually into the wells of 96 microtiter plates (9,216 potential clones). Vigorous bacterial growth was detected in 8,742 of those wells. High-density colony hybridization filters (1, 536 clones/filter) were prepared from a set of replicas of the original plates. Bacteria remaining in the wells of replica plates were combined, mixed with freezing medium, and stored at -80 degrees C. These pooled stocks were analyzed by polymerase chain reaction to determine the presence of specific sequences in the library. Hybridization of high-density filters was used to identify the clones of interest, which were retrieved from the frozen cultures in the 96-well plates. In testing the library for the presence of 14 known early-replicating genes, we found sequences at or near 5 of them: APRT, beta-actin, beta-tubulin, c-myc, and HPRT. This library is a valuable resource for the isolation and analysis of certain DNA sequences replicated at the beginning of S phase, including potential origins of bidirectional replication.
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PMID:Construction of a cosmid library of DNA replicated early in the S phase of normal human fibroblasts. 1086 48

Embryonic stem (ES) cells are pluripotent cell lines that possess virtually unlimited self-renewal and differentiation capacity. Such characteristics make them potentially an invaluable cell source for diverse tissue-engineering applications. In vitro ES cell differentiation occurs spontaneously in three-dimensional structures termed "embryoid bodies" that mimic postimplantation embryonic tissue. HPRT, beta-tubulin, and GAPDH are commonly used as internal RNA standards in ES cell-derived gene transcription studies so that corrected sample mRNA levels can be obtained for (semi) quantitative gene expression data. However, if reliable data is to be obtained, it is essential that such housekeeping gene expression remains constant, and this has not been demonstrated for differentiating ES cell cultures, which represent a mixed and changing population of cells with time in culture. Therefore, in the present study, we tested the suitability of these housekeeping genes to act as true internal standards for differentiating murine ES cells cultured as embryoid bodies. PCR-amplified gene-specific products were quantified from digital images of ethidium bromide-stained gels using a computer software package. Both HPRT and beta-tubulin mRNA levels varied markedly in spontaneously differentiating and growth factor-supplemented (TGF-beta) ES cell cultures (p < 0.001, ANOVA), while GAPDH expression remained relatively constant (p > 0.2). Our results demonstrate the importance of fully validating housekeeping gene expression in in vitro ES cell gene transcription studies and suggest that GAPDH may be a suitable candidate to act as an internal RNA standard, while both HPRT and beta-tubulin appear to be inappropriate. Finally, we demonstrate enhanced mesodermal differentiation of ES cell-derived cultures by treatment with TGF-beta through significant upregulation of Brachyury T expression, with a concomitant decrease in expression of the undifferentiated ES cell marker Oct-4.
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PMID:Differentiating embryonic stem cells: GAPDH, but neither HPRT nor beta-tubulin is suitable as an internal standard for measuring RNA levels. 1220 95