Structure,variation and expression analysis of glutenin gene promoters from Triticum aestivum cultivar Chinese Spring shows the distal region of promoter 1Bx7 is key regulatory sequence

In this study, ten glutenin gene promoters were isolated from model wheat (Triticum aestivum L. cv. Chinese Spring) using a genomic PCR strategy with gene-specific primers. Six belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoters, and four to low-molecular-weight glutenin subunit (LMW-GS). Sequence lengths varied from 1361 to 2554 bp. We show that the glutenin gene promoter motifs are conserved in diverse sequences in this study, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show that HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (> − 700 bp) compared with LMW-GS. The y-type HMW-GS gene promoters possess unique motifs including RY repeat and as-2 box compared to the x-type. We also identified important motifs in the distal region of HMW-GS gene promoters including the 5′-UTR Py-rich stretch motif and the as-2 box motif. We found that cis-acting elements in the distal region of promoter 1Bx7 enhanced the expression of HMW-GS gene 1Bx7. Taken together, these data support efforts in designing molecular breeding strategies aiming to improve wheat quality. Our results offer insight into the regulatory mechanisms of glutenin gene expression.     

Coordinate regulation of bovine prion protein gene promoter activity by two Sp1 binding site polymorphisms

Relationships between insertion/deletion (Ins/Del) polymorphisms of the bovine prion protein gene (PRNP) promoter and bovine spongiform encephalopathy (BSE) susceptibility have been reported. Our previous study has shown that polymorphisms of −6C → T included in the specific protein 1 (Sp1) site in the 5′-flanking region of bovine PRNP influence the promoter activity of bovine PRNP. The present study shows that 12 and 23 bp Ins/Del polymorphisms in the upstream region and an additional polymorphism (−47C → A) in the Sp1 binding site coordinately affect the promoter activity. Reporter gene assays demonstrated that the bovine PRNP promoter containing −47A and 23 bp Del/12 bp Ins or 23 bp Ins/12 bp Ins showed lower promoter activity compared with other haplotypes (23 bp Del/12 bp Ins or 23 bp Ins/12 bp Del with −47C) or the wild-type haplotype (23 bp Del/12 bp Del with −47C). Furthermore, gel shift assays showed that the binding activity of Sp1 to the PRNP promoter was influenced by both polymorphisms with corresponding effects on the promoter activity. The coordinate regulation of the bovine PRNP promoter suggests the two Sp1 binding site polymorphisms control Sp1 binding to the PRNP promoter and its activity.     

Single-nucleotide polymorphisms and activity analysis of the promoter and enhancer of the pig lactase gene

Lactose intolerance in northern Europeans is strongly associated with a single-nucleotide polymorphism (SNP) located 14 kb upstream of the human lactase gene: − 13,910*C/T. We examined whether SNPs in the 5′ flanking region of the pig lactase gene are similar to those in the human gene and whether these polymorphisms play a functional role in regulating pig lactase gene expression. The 5′ flanking region of the lactase gene from several different breeds of pigs was cloned and analyzed for gene regulatory activity of a luciferase reporter gene. One SNP was found in the enhancer region (− 797*G/A) and two were found in the promoter region (− 308*G/C and − 301*A/G). The promoter C_− 308,G_− 301(Pro-CG) strongly promotes the expression of the lactase gene, but the promoter G_− 308,A_− 301(Pro-GA) does not. The enhancer A_− 797(Enh-A) genotype for Pro-GA can significantly enhance promoter activity, but has an inhibitory effect on Pro-CG. The Enhancer G_− 797(Enh-G) has a significant inhibitory effect on both promoters. In conclusion, the order of effectiveness on the pig lactase gene is Enh-A + Pro-GA > Enh-A/G + Pro-CG > Enh-G + Pro-GA.     

Analysis of the promoter region of the gene LIP1 encoding triglyceride lipase from Fusarium graminearum

Triglyceride lipases catalyze the reversible degradation of glycerol esters with long-chain fatty acids into fatty acids and glycerol. In silico analysis of 5′-end flanking sequence of the gene LIP1 encoding a triglyceride lipase from the wheat head blight pathogen Fusarium graminearum revealed the presence of several cis-regulatory elements. To delineate the function of these regulatory elements, we constructed a series of deletion mutants in the LIP1 promoter region fused to the open reading frame of a green fluorescent protein (GFP) and assayed the promoter activity. Analysis of GFP expression levels in mutants indicated that a 563-bp promoter sequence was sufficient to drive the expression of LIP1 and regulatory elements responsible for the gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements spanned within the 563-372 bp region were mutated using a targeted mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be required for the basal expression of LIP1, glucose suppression and fatty acid induction, respectively.     

Validation of a novel in vitro assay using ultra performance liquid chromatography–mass spectrometry (UPLC/MS) to detect and quantify hydroxylated metabolites of BDE-99 in rat liver microsomes

The purpose of this study was to develop and validate an ultra performance liquid chromatography–mass spectrometry (UPLC/MS) method to investigate the hepatic oxidative metabolism of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99), a widely used flame retardant and ubiquitous environmental contaminant. Hydroxylated metabolites were extracted using liquid-to-liquid extraction, resolved on a C₁₈ column with gradient elution and detected by mass spectrometry in single ion recording mode using electrospray negative ionization. The assay was validated for linearity, accuracy, precision, limit of quantification, range and recovery. Calibration curves were linear (R² ≥ 0.98) over a concentration range of 0.010–1.0 μM for 4-OH-2,2′,3,4′,5-pentabromodiphenyl ether (4-OH-BDE-90), 5′-OH-2,2′,4,4′,5-pentabromodiphenyl ether (5′-OH-BDE-99) and 6′-OH-2,2′,4,4′,5-pentabromodiphenyl ether (6′-OH-BDE-99), and a concentration range of 0.0625–12.5 μM for 2,4,5-tribromophenol (2,4,5-TBP). Inter- and intra-day accuracy values ranged from −2.0% to 6.0% and from −7.7% to 7.3%, respectively, and inter- and intra-day precision values ranged from 2.0% to 8.5% and from 2.2% to 8.6% (n = 6), respectively. The limits of quantification were 0.010 μM for 4-OH-BDE-90, 5′-OH-BDE-99 and 6′-OH-BDE-99, and 0.0625 μM for 2,4,5-TBP. Recovery values ranged between 85 and 100% for the four analytes. The validated analytical method was applied to identify and quantify hydroxy BDE-99 metabolites formed in vitro. Incubation of BDE-99 with rat liver microsomes yielded 4-OH-BDE-90 and 6′-OH-BDE-99 as major metabolites and 5′-OH-BDE-99 and 2,4,5-TBP as minor metabolites. To our knowledge, this is the first validated UPLC/MS method to quantify hydroxylated metabolites of PBDEs without the need of derivatization.