Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe an improved highly sensitive method for generating cDNA libraries containing a high proportion of cDNAs enriched with 5'-coding sequences from single human preimplantation embryos and a 10 week old whole foetus. The embryonic mRNA was isolated using oligo-(dT) linked to magnetic beads. First-strand cDNA synthesis was carried out directly on the bound mRNA, followed by PCR designed to amplify the cDNA molecules synthesized in their entirety. The complexities of the libraries are between 10(5) and 10(6) independent clones. The average cDNA size is 1.0 kb, and the size range is 0.5-3.0 kb. PCR analysis of the embryonic libraries for specific genes has revealed transcripts for genes known to be transcribed in preimplantation stages, such as the imprinted gene SNRPN, developmental genes WNT11, HOX, OCT-1 and the embryonic OCT-4, cytoskeletal genes keratin-18 and beta-actin, the cell cycle gene C-MOS, and housekeeping genes GAPDH and HPRT. Sequencing of random clones showed the presence of a variety of sequences, such as human chorionic gonadotrophin, ubiquitin, TFIIA, guanine nucleotide-binding protein (beta-subunit), annexin I, a gene encoding a kinesin-like protein, and TWIST, which encodes a basic helix-loop-helix (bHLH) transcription factor implicated in Saethre-Chotzen syndrome (characterized by craniofacial and limb anomalies). Approximately 40% of these randomly analysed clones were full length. In addition to cDNAs matching known ESTs (Expressed Sequence Tags) in the GenBank and dbEST databases, novel sequences were detected at a frequency of 16% of randomly picked clones. The libraries are a valuable resource, providing longer cDNAs representing genes expressed during human preimplantation development.
...
PMID:Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos. 1052 61

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.
...
PMID:Differentiating embryonic stem cells: GAPDH, but neither HPRT nor beta-tubulin is suitable as an internal standard for measuring RNA levels. 1220 95

The circadian expression patterns of genes encoding for proteins that make up the core of the circadian clock were measured in rat retina using real-time quantitative PCR (qPCR). Transcript levels of several genes previously used for normalization of qPCR assays were determined and the effect of ischemia-reperfusion on the expression of clock genes was studied. Statistically significant circadian changes in transcript levels were found for: Per2, Per3, Cry2, Bmal1, Rora, Rorb, and Rorc with changes ranging between 1.6- and 2.6-fold. No changes were found for Per1, Cry1, Clock, Rev-erb alpha, and Rev-erb beta. Significant differences in transcript levels were observed for several candidate reference genes: HPRT, GAPDH, rhodopsin, and Thy1 and, consequently, the use of these genes for normalization purposes in qPCR or Northern blots may lead to erroneous conclusions. Ischemia-reperfusion leads to a persistent decrease of Per1 and Cry2, which may be related to the selective degeneration of amacrine and ganglion cells. We conclude that while all clock genes are expressed in the retina, only a few show a clear circadian pattern.
...
PMID:Circadian expression of clock genes and clock-controlled genes in the rat retina. 1578 Dec 26

Preeclampsia and diabetes are complications of pregnancy that contribute to maternal and perinatal mortality worldwide. Results emerging from molecular studies of placentae may elucidate etiologically important genomic alterations. Appropriate application of real time reverse transcription (RT) PCR in comparative gene expression studies requires endogenous housekeeping genes to normalize between sample variations. Ideal housekeeping genes must have stable tissue expression, but few have been specifically studied in the placenta. We sought to identify candidate control genes by analyzing seven functionally distinct housekeeping genes (B2M, GAPDH, HMBS, HPRT, SDHA, TBP, YWHAZ) for their expression stability and level in the placenta. mRNA isolated from 20 placentae was analyzed for gene expression using RT-PCR. Expression stability (M) was assessed using normalization strategies previously used for other tissues. TBP and SDHA were the most stable, with an average expression stability of M = 0.43, followed by YWHAZ (M = 0.44) > HPRT (M = 0.53) > HMBS (M = 0.57) > GAPDH (M = 0.61) > B2M (M = 0.69). The genes tested ranged in abundance, with an approximately 300-fold increase from the lowest (HMBS) to the highest (B2M). By using TBP, SDHA and YWHAZ, with greater expression stability than those housekeeping genes commonly used in placenta studies, gene expression profile comparisons will have more sensitivity and specificity.
...
PMID:Evaluation of housekeeping genes in placental comparative expression studies. 1608 39

We have established an easy real-time PCR assay, which allows the precise quantification of changes in the expression level of 6 relevant porcine cytokines, and 3 housekeeping genes. This assay simultaneously detects 9 sequences by measuring 3 x 3 targets in a triplex-format. The mRNA of the lymphokines IL-2, IL-4, IL-10, and IFN-gamma, of the proinflammatory cytokines IL-1alpha and IL-6, and of the housekeeping genes are quantified using TaqMan-probes by means of standard dilution series on the iCycler iQ. The standard consists of equal aliquots of the experimental cDNAs under investigation. Simultaneously the most suitable combination of 3 out of the four housekeeping genes beta-actin, HPRT, GAPDH, and cyclophilin can be selected, and their averaged expression values constitute a normalisation factor. The raw data of all targets of interest is then calculated relative to this normalisation factor, making eventual changes of the relative expression level of the single housekeeping genes controllable and quantifiable. We have applied this assay to quantify changes in the cytokine mRNA levels of porcine stimulated with various concentrations of LPS and ConA, known to induce different cytokine expression patterns. We have shown, that even small differences in the expression level (less than 2-fold) can be precisely quantified, and reveal statistically significant changes, when using the normalisation factor. This assay will be useful for studying changes in the expression of relevant porcine cytokines and will help to further improve the investigation of immune responses in the pig.
...
PMID:Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines. 1622 7

Valid housekeeping genes (HKG) are a prerequisite for accurate gene quantification. We performed real-time reverse transcription-polymerase chain reaction to investigate the gene expression of five commonly used HKGs (beta-actin, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ubiquitin C [UBC], hypoxanthine phosphoribosyl-transferase [HPRT], and cyclophilin A [CYPa]) and antioxidant enzymes in the liver of young and old male Fischer rats. A wide variation in HKG expression existed during the aging process, and HPRT was identified as the most stable HKG in rat liver aging. When Cu/Zn-superoxide dismutase gene expression was normalized to HPRT, there was no detectable difference between young and old rats; however, a significant difference was seen when it was normalized to UBC. The variation of UBC caused the misinterpretation of Cu/Zn-superoxide dismutase expression. Catalase expression was significantly decreased, whereas glutathione peroxidase expression was not altered with age. We demonstrated that HPRT was an appropriate HKG, validation of HKGs was vital for accurate quantification, and decreased catalase expression might be involved in the decline of antioxidant defenses during rat liver aging.
...
PMID:Identification of valid housekeeping genes and antioxidant enzyme gene expression change in the aging rat liver. 1645 91

The non-invasive parasitic cestode Hymenolepis diminuta induces hypertrophy, hyperplasia and other changes in cell activity in the intestine of rats which are indicated in the expression of mRNA. We have investigated various house-keeping genes (GAPDH, beta-actin, 18S and HPRT) and other internal controls (total RNA/unit biomass, total RNA/unit length of intestine) to validate gene expression in the rat intestine after cestode infection and drug-induced neuromodulation. Variation in GAPDH, beta-actin, 18S and HPRT expression was observed in rat jejunal tissue according to treatment. Total RNA/unit length of intestine was found to be the most suitable internal control for normalizing target gene mRNA expression in both infected and/or drug-induced rat intestine. This normalization method may be applied to studies of gene expression levels in intestinal tissue where hypertrophy, hyperplasia, rapid growth and cell differentiation generally occur.
...
PMID:Validation of internal controls for gene expression analysis in the intestine of rats infected with Hymenolepis diminuta. 1766 83

Laser microdissection combined with real-time RT-PCR represents a powerful method to analyse the transcription efficiency of defined cell types. Therefore, a RNA-preserving immunolabelling method was established to identify neurons and astrocytes in persistently BDV-infected rat brain sections for subsequent laser microdissection and quantitation of viral gene products by real-time RT-PCR. Firstly, to ensure an accurate measurement of viral RNA after immunolabelling, different reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], succinate-ubiquinone reductase [SDHA], hypoxanthine phosphoribosyl-transferase-1 [HPRT]) were tested. Only normalisation with GAPDH yielded a stable relative expression of viral RNA encoding the nucleoprotein (BDV-N), the matrixprotein and the glycoprotein (intron I and intron II). The two remaining reference genes biased the ratios of BDV-transcripts in the immunolabelled brain sections significantly. Secondly, 100 immunolabelled neurons and astrocytes were harvested using laser microdissection and amplification of all viral transcripts revealed 681 and 168 (BDV-N), 573 and 254 (intron I), 324 and 133 (intron II) and 161 and 36 (GAPDH) absolute copy numbers in neurons and astrocytes, respectively. Thus, laser microdissection combined with real-time RT-PCR provides an effective tool for the analysis of cell-specific viral transcription efficiency and allows elucidating virus-host-interactions and virus persistence mechanisms in the CNS.
...
PMID:A rapid method for gene expression analysis of Borna disease virus in neurons and astrocytes using laser microdissection and real-time RT-PCR. 1805 93

Quantitative measurements of gene expression require correction for tissue sample size, RNA quantity, and reverse transcription efficiency. This can be achieved by normalization with control genes. The study was designed to identify candidates not altered after brain trauma. Male C57Bl/6 mice were anesthetized with isoflurane, and a pneumatic brain trauma was induced by controlled cortical impact (CCI) on the right parietal cortex. Brains were removed at 15 min, and 3, 6, 12 and 24 h after CCI and from naive animals (n = 6 each). Absolute copies of six control genes (beta-2-microglobin [B2M], cyclophilin A, beta-actin, hypoxanthine ribosyltransferase [HPRT], porphobilinogen deaminase [PBGD], and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and one example target gene (iNOS) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR; Lightcycler) in the traumatic focus and contralateral tissue. Control gene expression was stable until 12 h after CCI. At 24 h after CCI expression of B2M, cyclophilin A and HPRT remained stable in the contusion, while expression of beta-actin, GAPDH, and PBGD increased. Due to variations between animals (+/-85%), increases in beta-actin (+64%) and GAPDH (+59%) did not reach the level of significance. In non-contused tissue, expression of all genes dropped 24 h after CCI (range, -17% to -61%). Due to low variations between animals and stable expression after CCI, B2M and cyclophilin A seem to be suitable to serve as single normalizer. Normalization of the example target gene iNOS resulted in varying relative expression extending from onefold (PBDG) to 10-fold (HPRT). The results suggest that the knowledge of the temporal profile of control genes is essential to properly interpret results of mRNA quantification.
...
PMID:Selection of endogenous control genes for normalization of gene expression analysis after experimental brain trauma in mice. 1862 56

Careful validation of reference genes used for the normalization of real-time RT-PCR data is required to obtain accurate results regarding gene expression. We evaluated the stability of seven commonly used reference genes in the cerebral cortex and hippocampus of rats 3 days following traumatic brain injury (TBI). HPRT, SDHA, and GUSB were found to be the most stable reference genes in the cerebral cortex, whereas B2MG, TBP, and GAPDH were the most stable in the hippocampus. The use of three reference genes was determined to be the optimal number for accurate normalization of data. To illustrate this point, when our gene of interest, substance P (SP), was normalized against the three most stable reference genes in both brain areas, we found no significant difference between injured and uninjured rats at the 3-day time point. However, when our SP data were normalized to each reference gene individually, SP mRNA level was highly variable depending on the reference gene chosen. The results of the present study highlight the importance of validating reference genes to be used for real-time RT-PCR analysis. The use of the most stable reference genes presented here will allow more accurate normalization of gene expression data in TBI.
...
PMID:Validation of reference genes for normalization of real-time quantitative RT-PCR data in traumatic brain injury. 1871 51


1 2 3 4 Next >>

---