Absolute Gene Expression Patterns of Thioredoxin and Glutaredoxin Redox Systems in Mouse

Abstract

This work provides the first absolute expression patterns of genes coding for all known components of both thioredoxin (Trx) and glutaredoxin (Grx) systems in mouse: Trx1, Trx2, Grx1, Grx2, TrxR1, TrxR2, thioredoxin/glutathione reductase, and glutathione reductase. We devised a novel assay that, combining the advantages of multiplex and real-time PCR, streamlines the quantitation of the actual mRNA copy numbers in whole-animal experiments. Quantitations reported establish differences among adult organs and embryonic stages, compare mRNA decay rates, explore the significance of alternative mRNA isoforms derived from TrxR1 and Grx2 genes, and examine the time-course expression upon superoxide stress promoted by paraquat. Collectively, these quantitations show: i) unique expression profiles for each transcript and mouse organ examined, yet with some general trends like the higher amounts of mRNA species coding for thioredoxins than those coding for the reductases that control their redox states and activities; ii) continuous expression during embryogenesis with outstanding up-regulations of Trx1 and TrxR1 mRNAs in specific temporal sequences; iii) drastic differences in mRNA stability, liver decay rates range from 2.8 h (thioredoxin/glutathione reductase) to > 35 h (Trx1 and Trx2), and directly correlate with mRNA steadystate values; iv) testis-specific differences in the amounts (relative to total isoforms) of transcripts yielding the mitochondrial Grx2a and 67-kDa TrxR1 variants; and v) coordinated up-regulation of TrxR1 and glutathione reductase mRNAs in response to superoxide stress in an organ-specific manner. Further insights into in vivo roles of these redox systems should be gained from more focused studies of the mechanisms underlying the vast differences reported here at the transcript level.

Mammalian Antioxidant Defenses Are Not Inducible by H2O2

“…A systematic global gene microarray analysis of different tissues of mice under H 2 O 2 stress should answer this question. Nevertheless, limited data of two in vivo studies that monitored the expression of a few antioxidants in mice injected into the peritoneum with the superoxide anion-generating drug paraquat also failed to detect induction (23,24). Based on our results, we thus propose that human cells in culture do not carry an H 2 O 2 -inducible but rather a constitutive transcriptional antioxidant response, which might also apply to the in vivo situation.…”

“…cDNAs were synthesized from 10 g of total RNA by using the Superscript choice kit (Invitrogen) with a T7-(dT) 24 primer incorporating a T7 RNA polymerase promoter. cRNAs were generated and biotin-labeled by T7-dependent in vitro transcription (Enzo Biochemical, New York, NY).…”

Spermatocyte/Spermatid-specific Thioredoxin-3, a Novel Golgi Apparatus-associated Thioredoxin, Is a Specific Marker of Aberrant Spermatogenesis

“…Moreover, TrxR-1 and TrxR-2 mRNA levels in mouse testis are relatively high compared with other tissues such as brain, heart, or lung (24).…”

New Methodologies for Assessing the Presence and Ecological Effects of Pesticides in Doñana National Park (SW Spain)

“…Absolute quantification (real quantification) gives the molecule copy number of each transcript in each of the samples under study using a calibration curve. Although relative quantification is much easier to perform because a calibration curve is not necessary, relative data are much less informative than absolute data (Jurado et al, 2003;Prieto-Álamo et al, 2003;Jiménez et al, 2005;RuizLaguna et al, 2005RuizLaguna et al, , 2006. The commercialization of real-time PCR equipments allows for absolute expression data with precision levels unattainable for those generated by conventional end-time PCR.…”

“…Studies reporting absolute transcript abundance of genes are infrequent, even though it is the only adequate procedure to accurately assess the expression of a gene (Prieto-Alamo et al, 2003). We used this rigorous approach to provide the first comprehensive and absolute quantification analysis of the overall expression patterns of transcript coding for several ecotoxicological interesting protective and detoxificant enzymes in M. spretus.…”