Glyceraldehyde-3-phosphate Dehydrogenase Transcripts Research Articles

Electrochemical Genosensing of Overexpressed GAPDH Transcripts in Breast Cancer Exosomes.

Exosomes are receiving highlighted attention as new biomarkers for the detection of cancer since they are profusely released by tumor cells in different biological fluids. In this paper, the exosomes are preconcentrated from the serum by immunomagnetic separation (IMS) based on a CD326 receptor as a specific epithelial cancer-related biomarker and detected by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. Following the lysis of the captured exosomes, the released GAPDH transcripts are amplified by reverse transcription polymerase chain reaction (RT-PCR) with a double-tagging set of primers on poly(dT)-modified-MPs to increase the sensitivity. The double-tagged amplicon is then quantified by electrochemical genosensing. The IMS/double-tagging RT-PCR/electrochemical genosensing approach is first demonstrated for the sensitive detection of exosomes derived from MCF7 breast cancer cells and compared with CTCs in terms of the analytical performance, showing an LOD of 4 × 102 exosomes μL-1. The genosensor was applied to human samples by immunocapturing the exosomes directly from serum from breast cancer patients and showed a higher electrochemical signal (3.3-fold, p < 0.05), when compared with healthy controls, suggesting an overexpression of GAPDH on serum-derived exosomes from breast cancer patients. The detection of GAPDH transcripts is performed from only 1.0 mL of human serum using specific magnetic particles, improving the analytical simplification and avoiding ultracentrifugation steps, demonstrating to be a promising strategy for minimal invasive liquid biopsy.

Comparison of endogenous development, invasion ability and apoptotic features between diclazuril resistant and sensitive strains of Eimeria tenella

Diclazuril (DIC) is widely used in the poultry industry to control coccidiosis. However, drug resistance makes it less effective, and the underlying mechanism remains unclear. One DIC-resistant E. tenella (RE) isolate and one sensitive E. tenella (SE) isolate were used to compare the differences in their endogenous development, pathogenicity, invasion-related gene expression and apoptotic characteristics. Chickens were allocated into four groups to receive RE or SE strain and their corresponding DIC treatment or not. Caeca tissues were sampled at 96 h, 120 h and 144 h post-infection (PI) for pathological analysis. Meanwhile, second-generation merozoites (Mz2) were separated at 120 h PI to detect alterations in mitochondrial membrane potential (MMP), apoptotic rate and caspase-3 activity and mRNA expression of protein phosphatase 5 (PP5), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin depolymerizing factor (ADF) and microneme proteins (MICs). Haematoxylin and eosin staining revealed that DIC treatment strictly blocked the development of the SE strain but slightly affected the RE strain. Meanwhile, the number of SE Mz2 and their MMP decreased at the same time the apoptotic rate increased after DIC treatment. Real-time quantitative PCR and caspase-3 activity studies demonstrated that Mz2 from the RE strain had higher mRNA expression of ADF and MICs along with no significant changes in GAPDH and caspase-3 activity under DIC pressure compared to its control; in contrast, the mRNA expression of ADF, MICs and PP5 was markedly suppressed in Mz2 from SE with upregulated caspase-3 activity and GAPDH transcription. In addition, the mRNA expression of GAPDH and PP5 in Mz2 from RE was remarkably higher than that of SE. Taken together, the higher mRNA expression of invasion-related genes and almost unaffected endogenous development provide a better understanding of coccidian resistance to DIC.

Effect of MG‐132 on myofibrillogenesis and the ubiquitination of GAPDH in quail myotubes

In the three-step myofibrillogenesis model, mature myofibrils are formed through two intermediate structures: premyofibrils and nascent myofibrils. We have recently reported that several inhibitors of the Ubiquitin Proteosome System, for example, MG-132, and DBeQ, reversibly block progression of nascent myofibrils to mature myofibrils. In this investigation, we studied the effects of MG132 and DBeQ on the expression of various myofibrillar proteins including actin, myosin light and heavy chains, tropomyosin, myomesin, and myosin binding protein-C in cultured embryonic quail myotubes by western blotting using two loading controls-α-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Surprisingly, we found that MG-132 affected the level of expression of GAPDH but DBeQ did not. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription-PCR (qRT-PCR) showed no significant effect of MG-132 on GAPDH transcription. Two-dimensional (2D) western blot analyses with extracts of control and MG-132-treated cells using anti-ubiquitin antibody indicated that MG132-treated myotubes show a stronger emitter-coupled logic signal. However, Spot% and Spot volume calculations for all spots from both western blot film signals and matched Coomassie-stained 2D polyacrylamide gel electrophoresis showed that the intensity of staining in a spot of ~39 kDa protein is 3.5-fold lower in the gel of MG-132-treated extracts. Mass spectrometry analyses identified the ~39 kDa protein as quail GAPDH. Immunohistochemical analysis of fixed MG-132-treated myotubes with anti-GAPDH antibody showed extensive clump formation, which may be analogous to granule formation by stress response factors in MG132-treated cells. This is the first report on in vivo ubiquitination of GAPDH. This may be essential for the moonlighting (Jeffery, 1999) activity of GAPDH for tailoring stress in myotubes.

Suitable primers for GAPDH reference gene amplification in quantitative RT-PCR analysis of human gene expression

In molecular biology, real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) has been commonly used for the evaluation of gene expression in human cells and tissues. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is commonly used as a reference gene for the analysis of RT-qPCR experiments. However, recent studies have recognized several variants of GAPDH gene transcripts in human tissues. Thus, the application of the GAPDH gene in RT-qPCR amplification was also established with false interpretations of differential gene expression analysis. Thereof, a consistent amplification of reference gene transcript is essentially required from various tissues or cells treated under differential conditions. In this study, we designed a set of oligo primers for consistent amplification of the human GAPDH transcripts as a reference for human gene amplification. The designed primers for the GAPDH gene showed equalized amplification of GAPDH transcripts from human fibroblasts cells for differential gene expression analysis under various experimental conditions.

Expression of p53 isoforms in response to two levels of hypoxia and effects of p53 knock‐down in glyceraldehyde‐3‐phosphate dehydrogenase in the white shrimp Litopenaeus vannamei

In response to hypoxia, aquatic organisms rely on anaerobic glycolysis. In the white shrimp Litopenaeus vannamei, hypoxia induces glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, a key enzyme in glycolysis. In mammals, GAPDH has additional functions in cellular activities besides glucose metabolism, such as interaction with the transcription factor p53 to form a complex that participates in apoptosis. We investigated the effect of two levels of hypoxia (1.5 and 1.0 mg dissolved oxygen (DO) L−1) on the expression of the two shrimp p53 isoforms known, FLp53 and ∆Np53, and the effect of p53 silencing during hypoxia on GAPDH expression and activity in hepatopancreas. The FLp53 transcripts were down-regulated after 24 and 48 h exposure to both hypoxic conditions, but not the ∆Np53. p53 silencing using RNAi, induced expression of GAPDH at 48 h in the 1.5 mg DO L−1 hypoxic condition compared with the unsilenced shrimp, contrary to the 1.0 mg DO L−1 where expression was induced in the silenced animals in normoxia after 48 h, but no significant changes were detected in enzyme activity in any case. These results indicate differential expression of p53 isoforms under two hypoxic conditions and maintenance of GAPDH transcripts and activity in p53-silenced shrimp.

Regulation of glyceraldehyde-3-phosphate dehydrogenase by hypoxia inducible factor 1 in the white shrimp Litopenaeus vannamei during hypoxia and reoxygenation

Hypoxia is a frequent source of stress in the estuarine habitat of the white shrimp Litopenaeus vannamei. During hypoxia, L. vannamei gill cells rely more heavily on anaerobic glycolysis to obtain ATP. This is mediated by transcriptional up-regulation of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The hypoxia inducible factor 1 (HIF-1) is an important transcriptional activator of several glycolytic enzymes during hypoxia in diverse animals, including crustaceans. In this work, we cloned and sequenced a fragment corresponding to the 5′ flank of the GAPDH gene and identified a putative HIF-1 binding site, as well as sites for other transcription factors involved in the hypoxia signaling pathway. To investigate the role of HIF-1 in GAPDH regulation, we simultaneously injected double-stranded RNA (dsRNA) into shrimp to silence HIF-1α and HIF-1β under normoxia, hypoxia, and hypoxia followed by reoxygenation, and then measured gill HIF-1α, HIF-1β expression, GAPDH expression and activity, and glucose and lactate concentrations at 0, 3, 24 and 48 h. During normoxia, HIF-1 silencing induced up-regulation of GAPDH transcripts and activity, suggesting that expression is down-regulated via HIF-1 under these conditions. In contrast, HIF-1 silencing during hypoxia abolished the increases in GAPDH expression and activity, glucose and lactate concentrations. Finally, HIF-1 silencing during hypoxia-reoxygenation prevented the increase in GAPDH expression, however, those changes were not reflected in GAPDH activity and lactate accumulation. Altogether, these results indicate that GAPDH and glycolysis are transcriptionally regulated by HIF-1 in gills of white shrimp.

Verifying the Stability of Selected Genes for Normalization in Q PCR Experiments of Spodoptera frugiperda Cells during AcMNPV Infection

It is challenging to find genes with stable transcripts for use as reference genes for quantitative realtime polymerase chain reaction (qRT-PCR) during viral infection. Autographa californica nucleopolyhedrovirus (AcMNPV) is known to globally shut off host gene transcription in Sf21 cells and to modify their cytoskeletons. In this study, seven host genes were selected for validation as references for gene expression experiments using qRT-PCR. Two of them, ecdysoneless (ECD) and myosin showed stable RNA levels in our previous microarray study at 6, 12, and 24 hpi for both genes and 48 hpi for ECD. The others, actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 28S ribosome (28S), are commonly employed as reference genes for qRT-PCR. Ribosomal protein L35 (L35) gene was selected to test if ribosomal protein genes show stable RNA transcript levels similar to 28S and 18S rRNA and to validate the microarray data. In addition to 28S, previously known to have stable transcript levels, qRT-PCR showed that ECD transcript levels remained constant throughout the time course of AcMNPV infection. Transcripts of cytoskeleton genes such as actin, tubulin, and myosin declined dramatically as the infection progressed. GAPDH and L35 transcripts also declined over time. These results indicate that ECD is a reliable reference gene for qRT-PCR experiments during AcMNPV infection of Spodoptera frugiperda cells. Although 28S could be used as a reference gene for these experiments, it is less useful than ECD because of its abundance, which might make it difficult to establish an accurate baseline value for data analysis.

Aromatic hydrocarbons upregulate glyceraldehyde-3-phosphate dehydrogenase and induce changes in actin cytoskeleton. Role of the aryl hydrocarbon receptor (AhR)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme involved in several cellular functions including glycolysis, membrane transport, microtubule assembly, DNA replication and repair, nuclear RNA export, apoptosis, and the detection of nitric oxide stress. Therefore, modifications in the regulatory ability and function of GAPDH may alter cellular homeostasis. We report here that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and β-naphthoflavone, which are well-known ligands for the aryl hydrocarbon receptor (AhR), increase GAPDH mRNA levels in vivo and in vitro, respectively. These compounds fail to induce GAPDH transcription in an AhR-null mouse model, suggesting that the increase in GAPDH level is dependent upon AhR activation. To analyse the consequences of AhR ligands on GAPDH function, mice were treated with TCDD and the level of liver activity of GAPDH was determined. The results showed that TCDD treatment increased GAPDH activity. On the other hand, treatment of Hepa-1 cells with β-naphthoflavone leads to an increase in microfilament density when compared to untreated cultures. Collectively, these results suggest that AhR ligands, such as polycyclic hydrocarbons, can modify GAPDH expression and, therefore, have the potential to alter the multiple functions of this enzyme.

Regulation of Glyceraldehyde 3-Phosphate Dehydrogenase Expression by Metformin in HepG2 Cells

Biguanides are known to have a serious side effect, lactic acidosis. We previously reported that buformin suppressed the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPD) and suggested that this decrease was one of the causes of lactic acidosis. In this study, we examined the signaling pathway and regulatory factors for the expression of the GAPD gene triggered by metformin in HepG2 cells. The mRNA and protein expression of GAPD, detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively, decreased upon treatment of the cells with 10 mM metformin for 24 h. Under the conditions, metformin induced phosphorylation of AMP-activated protein kinase (AMPK). The expression of GAPD mRNA decreased on treatment with an activator for AMPK, 5-amino-imidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). Inhibitors for signal transducers, Compound C, H-89, and MDL-12,330A, restored the level of GAPD mRNA. A luciferase reporter plasmid containing bp -1795 to +57 of the 5'-flanking region of the GAPD gene was constructed for a reporter gene assay. The luciferase activity in transfectants decreased on incubation with metformin. A mutant reporter plasmid with an altered cAMP-response element (CRE) counteracted the metformin-mediated repression of GAPD transcription. These results suggest that signal transducers, adenylate cyclase (AC), protein kinase A (PKA), and AMPK, are involved in the signaling pathway triggered by metformin and CRE-binding protein is one of the transcription factors for the GAPD gene down-regulated by metformin.

Clinical utility of quantitative RT‐PCR targeted to α1,4‐N‐acetylglucosaminyltransferase mRNA for detection of pancreatic cancer

alpha1,4-N-Acetylglucosaminyltransferase (alpha4GnT) is a glycosyltransferase responsible for the biosynthesis of alpha1,4-GlcNAc-capped O-glycans, and is frequently expressed in pancreatic cancer cells but not peripheral blood cells. In the present study, we tested the clinical utility of alpha4GnT mRNA expressed in the mononuclear cell fraction of peripheral blood as a biomarker of pancreatic cancer. Total RNA isolated from the peripheral blood mononuclear cells from 55 pancreatic cancer patients, 10 chronic pancreatitis patients, and 70 cancer-free volunteers was analyzed quantitatively by reverse transcription-polymerase chain reaction with primers specific for alpha4GnT, and the expression level of alpha4GnT mRNA relative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured. When the ratio of alpha4GnT to GAPDH transcripts exceeded a defined cut-off value, patients were considered to have pancreatic cancer. By these standards, 76.4% of the pancreatic cancer patients were detected by this assay. A strong correlation was obtained between positivity in this assay and the expression of alpha4GnT protein detected immunohistochemically in pancreatic cancer tissues resected subsequently, suggesting that alpha4GnT mRNA detected in the peripheral blood is derived from circulating pancreatic cancer cells. Although increased levels of alpha4GnT mRNA was detected in 40.0% of chronic pancreatitis patients and 17.1% of cancer-free volunteers, the expression levels were significantly lower than those seen in pancreatic cancer patients. These results suggest that quantitative analysis of alpha4GnT mRNA expressed in the mononuclear cell fraction of peripheral blood will contribute to the detection of pancreatic cancer.

Up-regulation of Surfactant Protein A in Chronic Sialadenitis

Salivary secretions play a critical role in maintaining the health of the oral cavity, which is the first gate of entry to the airways and thus is exposed to a variety of environmental insults. Surfactant protein A (SP-A) is a member of the collectin family and plays an important role in first-line airway defense. The objectives of this study were to examine the expression of SP-A messenger RNA and protein in human salivary glands and to investigate its up-regulation during inflammatory conditions. Reverse transcription-polymerase chain reaction was performed on salivary gland tissues from patients and a control group. The expression levels of SP-A to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcripts were semiquantified by densitometry. We also characterized the cellular localizations of SP-A protein immunohistochemically. Tertiary academic institution. Ten patients with chronic sialadenitis and 10 patients with healthy salivary glands. Surfactant protein A messenger RNA and protein were detected in glands of patients who were healthy and in those with chronic sialadenitis. The expression levels of SP-A messenger RNA in the salivary glands of patients with chronic sialadenitis was significantly increased compared with those in healthy salivary glands. Immunohistochemical staining revealed SP-A immunoreactivity in the ductal epithelia of healthy salivary glands and in the salivary glands of those with chronic sialadenitis, and stronger immunoreactivity was observed in those with chronic sialadenitis tissues. Surfactant protein A is present in the salivary gland epithelium and is up-regulated in individuals with chronic sialadenitis. These results suggest that salivary gland SP-A may play an important role in the innate host defense of human salivary glands.

Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane

A protocol for reverse transcription followed by real-time quantitative PCR (RT-qPCR) analysis of tissue-specific and genotype-variable gene expression in sugarcane (Saccharum sp.) was developed. A key requirement for this analysis was the identification of a housekeeping gene with transcript levels that were relatively stable across tissues and genotypes, suitable for use as a reference. Primers for β-actin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes and 25S ribosomal RNA were designed and tested by RT-qPCR, and formation of product in the reactions was measured with the SYBR green I dye system. Ribosomal RNA was the most sensitive and consistent as a reference gene. Determination of the expression levels of β-actin, β-tubulin, and GAPDH transcripts relative to that of 25S rRNA showed that GAPDH had the most consistent mRNA expression of protein-coding genes across different tissues. GAPDH also showed low variation in expression in maturing stem internodes when compared across 2 cultivars and 3 otherSaccharum species. GAPDH therefore appears to be a suitable “housekeeping gene” in addition to 25S rRNA as a reference for measuring the relative expression of other genes in sugarcane. With use of GAPDH as a reference, the relative expression of the sugarcane sugar transporter genePst2a was assessed in a range of tissues. The result obtained was similar to our previously published Northern blot analysis. The protocol described here, using GAPDH as a reference gene, is recommended for studying the expression of other genes of interest in diverse tissues and genotypes of sugarcane.

Glyceraldehyde-3-Phosphate Dehydrogenase Expression Varies With Age and Nutrition Status

Objective: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway, and it is a popular internal standard for northern blot analysis. We examined GAPDH expression early in life when feed is either provided or not provided to animals. Methods: Male broiler chickens were provided a standard starter diet plus Oasis nutritional supplement (fed group; Novus International, St. Louis, MO, USA) or no feed (starved group) for the first 3 d posthatch. Subsequently, the standard starter diet was provided to all chickens between 3 and 7 d posthatch. RNA was extracted from the pectoralis thoracicus, and GAPDH expression was evaluated with quantitative northern analysis. Results: GAPDH expression was significantly ( P < 0.05) higher in the fed than in the starved group at 3 d posthatch, suggesting that nutritional manipulations can alter GAPDH transcription. Similarly, GAPDH mRNA levels were significantly ( P < 0.05) higher at 7 d posthatch compared with all younger animals, suggesting that GAPDH is developmentally upregulated with advancing age. Conclusion: GAPDH expression changes with age and nutrition status in the early posthatch chick, suggesting that GAPDH is not a proper internal standard for muscle studies using quantitative northern analysis.

Simple quantitative assay of alpha-fetoprotein mRNA in liver tissue using the real-time detection polymerase chain reaction assay — its application for clinical use

Alpha-feto protein (AFP) mRNA levels increase in hepatocellular carcinoma (HCC) cells as compared with non-neoplastic tissue. Therefore, detection of AFP mRNA in blood nuclear cells is useful for the evaluation of treatment efficacy and prognosis of HCC. In this study, simple and reproducible methods were developed to quantify AFP mRNA using the real-time RT-PCR assay (Taq Man assay). By using in vitro synthesized AFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA, the sensitivity and dynamic range of the RT-PCR assay were established. AFP mRNA in both HCC and non-neoplastic tissue, as well as in cell lines, were measured using this assay system. The expression of the AFP mRNA level was normalized using the GAPDH house keeping gene product as an endogenous reference. AFP and GAPDH mRNA can be quantified in the range of 10–10 8 copies when using this quantitative assay. Among HCC cell lines, Huh 7 and HepG2 cells, respectively, represented 1.5×10 6 and 6.0×10 5 AFP mRNA/10 6 GAPDH mRNA, in contrast to 6, 23 and 230 AFP mRNA/10 6 GAPDH mRNA for HLE, HLF and PLC/PRF/5 cells, respectively. Other cell lines derived from stomach, pancreas, and colon cancers have 10 AFP mRNA copies/10 6 GAPDH mRNA. In liver tissue from patients with chronic hepatitis, and the non-neoplastic portion of the liver from HCC patients, AFP mRNA distributes from 2.5×10 3 to 5.8×10 4/10 6 GAPDH transcripts. In contrast, AFP mRNA in tumor cells were more than 100-fold higher than that found in corresponding non-neoplastic portions in two patients who had a high level of AFP in serum. The establishment of the TaqMan quantifying system for AFP mRNA may have important clinical implications.

The lymphocytic choriomeningitis virus RING protein Z associates with eukaryotic initiation factor 4E and selectively represses translation in a RING-dependent manner.

Only a few host cell proteins that associate with arenaviruses have been identified. To date, the arenavirus Z protein associates with the promyelocytic leukemia protein PML and the ribosomal P proteins. The majority of PML is present in nuclear bodies which are translocated to the cytoplasm by infection with the arenavirus, lymphocytic choriomeningitis virus (LCMV). The Z protein is a small zinc-binding RING protein with an unknown function which is required for the viral life cycle. Here, we demonstrate an association between Z and the host cell translation factor, eukaryotic initiation factor 4E (eIF-4E) in infected and transfected cells. Z's association with both ribosomal proteins and this translation factor led us to investigate whether Z could modulate host cell translation. In cell culture, Z selectively represses protein production in an eIF-4E-dependent manner. Specifically, we see reduction in cyclin D1 protein production with no effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells transfected with Z. Previous reports indicate that cyclin D1 is sensitive to eIF-4E levels, whereas GAPDH is not. Consistent with this, we observe preferential downregulation of cyclin D1 during infection and no effect on GAPDH. Further, no changes in RNA levels were observed for cyclin D1 or GAPDH transcripts. The interaction between eIF-4E and Z may provide a mechanism for slower growth observed in infected cells and a viral strategy for establishing chronic infection.

Relative quantitative RT-PCR protocol for TrkB expression in neuroblastoma using GAPD as an internal control.

An RT-PCR protocol for the relative quantitative measurement of TrkB transcripts using glyceraldehyde-3-phosphate-dehydrogenase (GAPD) transcripts as an internal control is described. Both TrkB and GAPD sequences were co-amplified in the exponential phase of amplification using 5'-biotinylated primers. The PCR products were subjected to PAGE, electro-transferred to nylon membrane and detected by a chemiluminescent procedure using alkaline phosphatase conjugated with avidin. Signals detected on X-ray film were analyzed by densitometry. The ratio between TrkB and GAPD expression levels was determined to normalize the expression levels of TrkB transcripts. Initially, strong signals of GAPD transcripts led to overexposure of X-ray film compared to those of TrkB, which causes difficulties in the accurate determination of the TrkB/GAPD ratio. To circumvent this problem, uniformly biotinylated GAPD primers were replaced with a mixture of biotinylated and non-biotinylated GAPD primers of the same sequence and concentration. GAPD signals detected on X-ray film were proportionally decreased as the amount of biotin-labeled primers was reduced in the total GAPD primers. This modification enabled both GAPD and TrkB signals to be analyzed within the linear range of X-ray film detection without affecting the amplification efficiency of TrkB sequence. Use of composite primers may have a wide range of applicability in quantitative analysis of nucleic acids.

Several novel transcripts of glyceraldehyde-3-phosphate dehydrogenase expressed in adult chicken testis.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in addition to being a classic glycolytic enzyme, is a multifunctional protein involved in relevant cell functions such as DNA replication, DNA repair, translational control of gene expression, and apoptosis. Although the multifunctional nature of GAPDH suggests versatility in the mechanisms regulating its expression, no major qualitative changes and few quantitative changes in the GAPDH transcripts have been reported. While studying the expression of GAPDH during spermatogenesis, we detected alternative initiations to TATA box and alternative splicings in the 5' region of the pre-mRNA, resulting in at least six different types of mRNAs. The amount and the polyadenylation of the GAPDH transcripts increased in mature testis in relation to immature testis and further increased when cell suspensions from mature testis were exposed to heat shock. These results suggest that alternative initiation, alternative splicing, and polyadenylation could provide the necessary versatility to the regulation of the expression of this multifunctional protein during spermatogenesis.

Transcriptional Regulation of Glyceraldehyde-3-Phosphate Dehydrogenase by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

The action of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on transcription and steady-state mRNA levels, protein expression, and enzymatic activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cultured human keratinocytes was examined. GAPDH transcription increased as a function of time of exposure to TCDD, reaching a maximum of 23-fold after 8 days. Northern blot analysis showed a 4-fold increase in mRNA after 8 days exposure. Protein levels were increased 4-fold, and dehydrogenase activity by 5-fold, under the same exposure conditions. GAPDH is commonly thought of as a constitutive housekeeping gene and is often used as a loading control for northern blot analysis. Our data suggest that caution should be used when using GAPDH as an RNA control, especially in studies examining cell proliferation and carcinogenesis.

Riboprobe expression cassettes for measuring IGF-I, β-actin and glyceraldehyde 3-phosphate dehydrogenase transcripts

The development of riboprobe expression cassettes for phosphorimager-based quantitation of steady-state transcripts for three different genes using solution hybridization, RNase protection assays is described. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin genes are widely used as reporter genes to estimate the amount and integrity of RNA as well as for comparing gene expression among different tissues. To directly compare expression of these two genes in lymphoid tissue and liver, cDNA fragments of β-actin and GAPDH from both mice and rats were generated by RT-PCR and cloned together into pGEM1 under control of the T7 RNA polymerase promoter. Antisense transcripts from this fusion construct protected the appropriate-sized fragments of β-actin (115 nt) and GAPDH (214 nt) in RNA isolated from rat spleen, thymus and liver. Expression of GAPDH transcripts was less variable across tissues because this mRNA was only two-fold lower in liver as compared to either thymus or spleen, whereas expression of β-actin transcripts was eight-fold lower in liver than in these tissues. Two other riboprobe expression cassettes (IGF-I/ actin) were constructed by ligating a cDNA fragment of mouse or rat β-actin that would protect 115 nt to either a mouse or rat IGF-I genomic DNA fragment containing 182 bp of exon 4. These mouse and rat IGF-I/ actin riboprobes were used to conclusively demonstrate that rat CSF-1-derived bone marrow macrophages, mouse elicited peritoneal macrophages and the murine PU5-1R macrophage cell line synthesize abundant transcripts for both IGF-I and β-actin. However, the mouse M1 progenitor myeloid cell line does not express RNA for IGF-I, as demonstrated by the absence of protected transcripts for IGF-I in the presence of abundant protected transcripts for β-actin. Phosphorimager scanning of the gels revealed that macrophages of both mice and rats express IGF-I transcripts at a level of 60–100% of those found in liver. These data show that a single riboprobe can be developed to generate multigene antisense RNAs that can then be used to quantitatively compare IGF-I transcripts in macrophages and other tissues to an internal standard, with GAPDH transcripts being less variable among tissues than those for β-actin. This approach should be broadly applicable for measuring a variety of markers of cellular activation.