Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.61 (porphobilinogen deaminase)
 637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The regional gene assignments for human porphobilinogen deaminase (PBGD; EC 4.3.1.8) and esterase A4 (ESA4; EC3.1.1.1) chromosome 11 have been determined with somatic cell hybridization and immunologic, electrophoretic, and cytogenetic techniques. Dimethyl sulfoxide-induced erythroid differentiation of hybrid clones derived from the fusion of tetraploid Friend murine erythroleukemia (2S MEL) cells deficient in thymidine kinase and human Lesch--Nyhan fibroblasts (HLN) deficient in hypoxanthine phosphoribosyltransferase (HPRT-; EC 2.4.2.8) were examined for expression of human PBGD, ESA4, and lactate dehydrogenase A (LDHA; EC 1.1.1.27). Human PBGD was detected by rocket immunoelectrophoresis with rabbit anti-human PBGD IgG and by isoelectric focusing. The human chromosome complement of each clone was determined by cytogenetic and enzyme marker analyses. Of the five primary 2S MEL--HLN clones examined, three were positive for human PBGD. These were subcloned to yield a total of 10 secondary, tertiary, or quaternary clones. Analyses of these subclones permitted the regional assignment of human PBGD and ESA4 to the long arm of chromosome 11. Finer regional assignment of the loci for human PBGD and ESA4 was facilitated when two 2S MEL (HPRT-)--human fibroblast (HX/11) hybrids, each containing the X chromosome--autosome translocation (der11), t(X;11)(q25-26;q23) as the only human chromosome, were examined for expression of human PBGD, ESA4, and LDHA. One clone, HX/11-2, contained the intact X/11 translocated chromosome; in the other, HX/11-3, 11p was deleted, and the human X/11 derivative was translocated onto a mouse chromosome. HX/11-2 expressed human LDHA, but HX/11-3 did not, verifying that the latter human 11/X derivative did not include 11pter leads to 11p12; PBGD and ESA4 were not detected in either hybrid. These results confirm the location of the gene for human PBGD on chromosome 11 and establish the assignment of the loci for PBGD and ESA4 in the region 11q23 leads to 11qter.
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PMID:Regional gene assignment of human porphobilinogen deaminase and esterase A4 to chromosome 11q23 leads to 11qter. 694 13

We developed a one-step real-time duplex reverse transcription PCR (RT-PCR) method using the LightCycler platform. This method allows simultaneous reverse transcription and real-time PCR amplification of two mRNAs of specific genes of interest (analyte genes) and mRNA of constantly transcribed genes (housekeeping genes) in a single-tube reaction. Specimen total nucleic acids were used because eukaryotic cDNA is discriminated from genomic DNA using exon-spanning primers and/or fluorescence resonance energy transfer (FRET) probes. Transcripts of murine arginase I and hypoxanthine-phosphoribosyl transferase (HPRT; housekeeping gene) or murine arginase II analyte and porphobilinogen deaminase (PBGD; housekeeping gene) were quantified in such duplex RT-PCRs. Twenty-minute reverse transcription reactions at 55 degrees C followed by 18 high-stringency step-down thermal cycles and 25 relaxed-stringency fluorescence acquisition cycles produced sensitive and accurate RT-PCR results. Fluorescent signal spillover between channels was fully compensated. A matrix of duplex PCRs at variable ratios of target standards yielded equations for factors that correct PCR-specific target ratio-dependent deviations in quantification. The one-step real-time duplex RT-PCRs reliably and accurately determined 10-10,000 copies of each target over a 100,000-fold range of target copy ratios (analyte to housekeeping mRNA = 10(-2.5)-10(2.5)) in a single assay.
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PMID:One-step real-time duplex reverse transcription PCRs simultaneously quantify analyte and housekeeping gene mRNAs. 1503 67

Quantitative polymerase chain reaction (Q-PCR) studies of urine sediments have demonstrated an increased expression of cytotoxin genes during episodes of acute rejection of renal allografts. To compensate for differences in initial sample size and cDNA preparation, standard Q-PCR experiments involve normalization to a reference gene. Although stable expression of reference genes is a prerequisite for any Q-PCR analysis, commonly used reference genes have demonstrated a varying expression across tissues and various stimuli. In this study, cellular expression of several reference genes was investigated in a mixed lymphocyte reaction as a model of gene expression during alloreactive T-lymphocyte activation and acute rejection. Gene expression was quantified using Q-PCR, normalized to cell counts obtained by DNA quantification and corrected for cell polyploidy using flow cytometry. Examined reference genes were 18S rRNA, beta-actin (ACTB), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1) and peptidylprolyle isomerase B (PPIB). This study also examined two novel T-lymphocyte-specific reference genes: CD3E and CD8B. HMBS and HPRT were 18.8- and 7.4-fold upregulated, respectively, ACTB was 5.3-fold upregulated, PPIB was 3.2-fold upregulated while 18S rRNA remained stably expressed. The T-lymphocyte-specific reference gene CD3E remained stable while CD8B was upregulated 2.3-fold. In conclusion, several commonly used reference genes were actively regulated during alloreactive T-lymphocyte activation. Additionally, we introduce two stable T-lymphocyte-specific reference genes that might be useful in a Q-PCR analysis of T-lymphocyte-specific cytotoxin genes in urine sediments, as they overcome the contribution of reference gene mRNA from cells irrelevant for diagnosis.
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PMID:Commonly used reference genes are actively regulated in in vitro stimulated lymphocytes. 1725 26

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.
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PMID:Selection of endogenous control genes for normalization of gene expression analysis after experimental brain trauma in mice. 1862 56

Comprehensive analyses of gene expression have been carried out by the development of microarrays and deep sequencers. However, it is difficult to obtain comprehensive information on gene expression from a small amount of ribonucleic acid (RNA). Therefore, we investigated the reproducibility and application of T7 RNA polymerase-mediated transcription, adaptor ligation and polymerase chain reaction (PCR) amplification, followed by T7 transcription (TALPAT), an efficient method for amplifying poly (A)-positive RNA, such as messenger RNA (mRNA). When amplified complementary RNA (cRNA) was electrophoresed, a large number of amplified cRNA was detected in the size of 0.2-0.5 kb. This indicates that the region up to 0.2-0.5 kb from the 3' end of the original mRNA was amplified by the TALPAT method. Seven housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1), ribosomal protein L13a (RPL13A), succinate dehydrogenase complex (SDHA), TATA box-binding protein (TBP) and ubiquitin C (UBC), showed high reproducibility (square of the correlation coefficient, R2=0.9954), according to scatter plots of Ct values obtained in the real-time PCR analysis of amplified cRNA. In addition, relative expression ratios of amplified cRNA of the seven housekeeping genes were approximately equal to the ratio of the original RNA solution. Furthermore, cRNA was amplified from 20 pg total RNA. In the present study, we confirmed the characteristics of mRNA amplification using the TALPAT method. This method may be applicable to mRNA and poly (A)-positive non-coding RNA amplification, using a small amount of RNA from single, laser-captured and sorted cells, as well as exosomes from serum, urine and body fluids.
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PMID:An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA. 2464 3