Sequence of human glucose-6-phosphate dehydrogenase cloned in plasmids and a yeast artificial chromosome

Chen, Ellson Y.; Cheng, Andre; Lee, Angela; Kuang, Wun Jing; Hillier, LaDeana W.; Green, Philip; Schlessinger, David; Ciccodicola, Alfredo; D’Urso, Michele

doi:10.1016/0888-7543(91)90465-q

Cited by 105 publications

(71 citation statements)

References 28 publications

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“…The estimated size of the amplified fragments matched the calculated size 18,19 for b3a2 BCR-ABL (671 bp), b2a2 BCR-ABL (596 bp), b3a3 BCR-ABL (497 bp), b2a3 BCR-ABL (422 bp), e1a2 BCR-ABL (540 bp), ABL (386 bp), and G6PD (343 bp) in all cases.…”

Section: Real-time Quantitative Pcrmentioning

confidence: 77%

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

et al. 1999

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We sought to establish a rapid and reliable RT-PCR approach for detection and quantification of BCR-ABL fusion transcripts using the LightCycler technology. This device combines rapid thermocycling with online detection of PCR product formation and is based on the fluorescence resonance energy transfer (FRET) between two adjacent hybridization probes carrying donor and acceptor fluorophores. A pair of probes was designed that was complementary to ABL exon 3, thus enabling detection of all known BCR-ABL variants and also normal ABL as an internal control. Conditions were established to amplify less than 10 target molecules/reaction and to detect one CML cell in 10 5 cells from healthy donors. To determine the utility of the assay, we quantified BCR-ABL and ABL transcripts in 254 samples (222 peripheral blood, 32 bone marrow) from 120 patients with CML after therapy with IFN-␣ (n ‫؍‬ 219), allogeneic BMT (n ‫؍‬ 17), chemotherapy (n ‫؍‬ 11), or at diagnosis (n ‫؍‬ 7). The level of residual disease in the 245 BCR-ABL positive specimens was expressed as the ratio of BCR-ABL/ABL. This ratio was compared to results obtained by three established methods from contemporaneous specimens. A highly significant correlation was seen between the BCR-ABL/ABL ratios determined by the LightCycler and (1) the BCR-ABL/ABL ratios obtained by nested competitive RT-PCR (n ‫؍‬ 201, r ‫؍‬ 0.90, P Ͻ 0.0001); (2) the proportion of Philadelphia chromosome positive metaphases determined by cytogenetics (n ‫؍‬ 81, P Ͻ 0.0001); and (3) the BCR ratio determined by Southern blot analysis (n ‫؍‬ 122, P Ͻ 0.0001). We conclude that real-time PCR with hybridization probes is a reliable and sensitive method to monitor CML patients after therapy. The major advantages of the methodology are (1) amplification and product analysis are performed in the same reaction vessel, avoiding the risk of contamination; (2) the results are standardized by the quantification of housekeeping genes; and (3) the complete PCR analysis takes less than 60 min.

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Section: Real-time Quantitative Pcrmentioning

confidence: 77%

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

et al. 1999

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“…The cells were cultured and stably transfected as described elsewhere (17). The plasmid pGD15neo has been described (18); it also contains a Neo gene driven by the SV40 early promoter. Control HeLa cells were transfected with the same plasmid devoid of all the human G6PD coding sequences.…”

Section: Methodsmentioning

confidence: 99%

Enhanced Glutathione Levels and Oxidoresistance Mediated by Increased Glucose-6-phosphate Dehydrogenase Expression

Salvemini¹,

Franzè²,

Iervolino³

et al. 1999

Journal of Biological Chemistry

299

186

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Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway that is responsible for the generation of NADPH, which is required in many detoxifying reactions. We have recently demonstrated that G6PD expression is induced by a variety of chemical agents acting at different steps in the biochemical pathway controlling the intracellular redox status. Although we obtained evidence that the oxidative stress-mediated enhancement of G6PD expression is a general phenomenon, the functional significance of such G6PD induction after oxidant insult is still poorly understood. In this report, we used a GSHdepleting drug that determines a marked decrease in the intracellular pool of reduced glutathione and a gradual but notable increase in G6PD expression. Both effects are seen soon after drug addition. Once G6PD activity has reached the maximum, the GSH pool is restored. We suggest and also provide the first direct evidence that G6PD induction serves to maintain and regenerate the intracellular GSH pool. We used HeLa cell clones stably transfected with the human G6PD gene that display higher G6PD activity than the parent HeLa cells. Although the activities of glutathione peroxidase, glutathione reductase, and catalase were comparable in all strains, the concentrations of GSH were significantly higher in G6PD-overexpressing clones. A direct consequence of GSH increase in these cells is a decreased reactive oxygen species production, which makes these cells less sensitive to the oxidative burst produced by external stimuli. Indeed, all clones that constitutively overexpress G6PD exhibited strong protection against oxidants-mediated cell killing. We also observe that NF-B activation, in response to tumor necrosis factor-␣ treatment, is strongly reduced in human HeLa cells overexpressing G6PD.

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“…The G6PD gene exists on the X chromosome distributed in 13 exons (Chen et al 1991). Almost all G6PD deficiencies are caused by one amino acid change caused by a point mutation of the genomic DNA, and more than 100 molecular variants of the G6PD genotype have been identified .…”

Section: Introductionmentioning

confidence: 99%

Glucose-6-phosphate dehydrogenase (G6PD) mutations in Myanmar: G6PD Mahidol (487G>A) is the most common variant in the Myanmar population

et al. 2004

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We conducted a survey of malaria diagnoses and treatments in remote areas of Myanmar. Blood specimens from more than 1,000 people were collected by the finger-prick method, and 121 (11%) of these people were found to be glucose-6-phosphate dehydrogenase (G6PD) deficient. Of these 121, 50 consented to analysis of the G6PD genome. We read the G6PD sequences of these subjects and found 45 cases of G6PD Mahidol (487G>A), two of G6PD Coimbra (592C>T), two of G6PD Union (1360C>T), and one of G6PD Canton (1376G>T). Taken together with data from our previous report, 91.3% (73/80) of G6PD variants were G6PD Mahidol. This finding suggests that the Myanmar population is derived from homogeneous ancestries and are different from Thai, Malaysian, and Indonesian populations.

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Sequence of human glucose-6-phosphate dehydrogenase cloned in plasmids and a yeast artificial chromosome

Cited by 105 publications

References 28 publications

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

Enhanced Glutathione Levels and Oxidoresistance Mediated by Increased Glucose-6-phosphate Dehydrogenase Expression

Glucose-6-phosphate dehydrogenase (G6PD) mutations in Myanmar: G6PD Mahidol (487G>A) is the most common variant in the Myanmar population

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