Genetic transformation of sweet oranges with the D4E1 gene driven by the AtPP2 promoter

The objective of this work was to produce transgenic 'Pêra' and 'Valência' sweet orange plants using the D4E1 gene driven by the Arabidopsis thaliana phloem protein (AtPP2) promoter and to quantify transgene expression in different transformation events. Genetic transformation experiments were carried out with epicotyl segments co‑cultivated with Agrobacterium tumefaciens. Six plants from 'Pêra' sweet orange and seven plants from 'Valência' sweet orange were confirmed as different transgenic events by means of the polymerase chain reaction (PCR) and the Southern blot techniques. Transgene expression was quantified using real‑time quantitative PCR. D4E1 gene expression levels vary from 5 up to 50 times among different transformation events.

Regulation of the vitellogenin gene B1 promoter after transfer into hepatocytes in primary cultures.

The estrogen-dependent and tissue-specific regulation of the Xenopus laevis vitellogenin gene B1 promoter has been studied by lipid-mediated DNA transfer into Xenopus hepatocytes in primary culture. Hepatocytes achieve an efficient hormonal control of this promoter through a functional interaction between the estrogen responsive elements and a promoter proximal region upstream of the TATA box, which is characterized by a high density of binding sites for the transcription factors CTF/NF-1, C/EBP and HNF3. DNA accessibility to restriction enzymes within the chromosomal copy of the vitellogenin gene B1 promoter shows that the estrogen responsive unit and the promoter proximal region are sensitive to digestion in uninduced and estrogen-induced hepatocytes but not in erythrocyte nuclei. Together, these findings support the notion that chromatin configuration as well as the interplay of promoter elements mediate proper hormone-dependent and tissue-specific expression of the B1 vitellogenin gene.

A functional variant in the Stearoyl-CoA desaturase gene promoter enhances fatty acid desaturation in pork

There is growing public concern about reducing saturated fat intake. Stearoyl-CoA desaturase (SCD) is the lipogenic enzyme responsible for the biosynthesis of oleic acid (18:1) by desaturating stearic acid (18:0). Here we describe a total of 18 mutations in the promoter and 3′ non-coding region of the pig SCD gene and provide evidence that allele T at AY487830:g.2228T>C in the promoter region enhances fat desaturation (the ratio 18:1/18:0 in muscle increases from 3.78 to 4.43 in opposite homozygotes) without affecting fat content (18:0+18:1, intramuscular fat content, and backfat thickness). No mutations that could affect the functionality of the protein were found in the coding region. First, we proved in a purebred Duroc line that the C-T-A haplotype of the 3 single nucleotide polymorphisms (SNPs) (g.2108C>T; g.2228T>C; g.2281A>G) of the promoter region was additively associated to enhanced 18:1/18:0 both in muscle and subcutaneous fat, but not in liver. We show that this association was consistent over a 10-year period of overlapping generations and, in line with these results, that the C-T-A haplotype displayed greater SCD mRNA expression in muscle. The effect of this haplotype was validated both internally, by comparing opposite homozygote siblings, and externally, by using experimental Duroc-based crossbreds. Second, the g.2281A>G and the g.2108C>T SNPs were excluded as causative mutations using new and previously published data, restricting the causality to g.2228T>C SNP, the last source of genetic variation within the haplotype. This mutation is positioned in the core sequence of several putative transcription factor binding sites, so that there are several plausible mechanisms by which allele T enhances 18:1/18:0 and, consequently, the proportion of monounsaturated to saturated fat.

BDNF promoter I methylation correlates between post-mortem human peripheral and brain tissues.

Several psychiatric disorders have been associated with CpG methylation changes in CG rich promoters of the brain-derived neurotrophic factor (BDNF) mainly by extracting DNA from peripheral blood cells. Whether changes in peripheral DNA methylation can be used as a proxy for brain-specific alterations remains an open question. In this study we aimed to compare DNA methylation levels in BDNF promoter regions in human blood cells, muscle and brain regions using bisulfite-pyrosequencing. We found a significant correlation between the levels of BDNF promoter I methylation measured in quadriceps and vPFC tissues extracted from the same individuals (n = 98, Pearson, r = 0.48, p = 4.5 × 10(-7)). In the hippocampus, BDNF promoter I and IV methylation levels were strongly correlated (Pearson, n = 37, r = 0.74, p = 1.4 × 10(-7)). We found evidence for sex-dependent effect on BDNF promoter methylation levels in the various tissues and blood samples. Taken together, these data indicate a strong intra-individual correlation between peripheral and brain tissue. They also suggest that sex determines methylation patterns in BDNF promoter region across different types of tissue, including muscle, brain, and blood.

MGMT Promoter Methylation Is a Strong Prognostic Biomarker for Benefit from Dose-Intensified Temozolomide Rechallenge in Progressive Glioblastoma: The DIRECTOR Trial.

PURPOSE: Rechallenge with temozolomide (TMZ) at first progression of glioblastoma after temozolomide chemoradiotherapy (TMZ/RT→TMZ) has been studied in retrospective and single-arm prospective studies, applying temozolomide continuously or using 7/14 or 21/28 days schedules. The DIRECTOR trial sought to show superiority of the 7/14 regimen. EXPERIMENTAL DESIGN: Patients with glioblastoma at first progression after TMZ/RT→TMZ and at least two maintenance temozolomide cycles were randomized to Arm A [one week on (120 mg/m(2) per day)/one week off] or Arm B [3 weeks on (80 mg/m(2) per day)/one week off]. The primary endpoint was median time-to-treatment failure (TTF) defined as progression, premature temozolomide discontinuation for toxicity, or death from any cause. O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation was prospectively assessed by methylation-specific PCR. RESULTS: Because of withdrawal of support, the trial was prematurely closed to accrual after 105 patients. There was a similar outcome in both arms for median TTF [A: 1.8 months; 95% confidence intervals (CI), 1.8-3.2 vs. B: 2.0 months; 95% CI, 1.8-3.5] and overall survival [A: 9.8 months (95% CI, 6.7-13.0) vs. B: 10.6 months (95% CI, 8.1-11.6)]. Median TTF in patients with MGMT-methylated tumors was 3.2 months (95% CI, 1.8-7.4) versus 1.8 months (95% CI, 1.8-2) in MGMT-unmethylated glioblastoma. Progression-free survival rates at 6 months (PFS-6) were 39.7% with versus 6.9% without MGMT promoter methylation. CONCLUSIONS: Temozolomide rechallenge is a treatment option for MGMT promoter-methylated recurrent glioblastoma. Alternative strategies need to be considered for patients with progressive glioblastoma without MGMT promoter methylation.

Two cilengitide regimens in combination with standard treatment for patients with newly diagnosed glioblastoma and unmethylated MGMT gene promoter: results of the open-label, controlled, randomized phase II CORE study.

BACKGROUND: Survival outcomes for patients with glioblastoma remain poor, particularly for patients with unmethylated O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter. This phase II, randomized, open-label, multicenter trial investigated the efficacy and safety of 2 dose regimens of the selective integrin inhibitor cilengitide combined with standard chemoradiotherapy in patients with newly diagnosed glioblastoma and an unmethylated MGMT promoter. METHODS: Overall, 265 patients were randomized (1:1:1) to standard cilengitide (2000 mg 2×/wk; n = 88), intensive cilengitide (2000 mg 5×/wk during wk 1-6, thereafter 2×/wk; n = 88), or a control arm (chemoradiotherapy alone; n = 89). Cilengitide was administered intravenously in combination with daily temozolomide (TMZ) and concomitant radiotherapy (RT; wk 1-6), followed by TMZ maintenance therapy (TMZ/RT→TMZ). The primary endpoint was overall survival; secondary endpoints included progression-free survival, pharmacokinetics, and safety and tolerability. RESULTS: Median overall survival was 16.3 months in the standard cilengitide arm (hazard ratio [HR], 0.686; 95% CI: 0.484, 0.972; P = .032) and 14.5 months in the intensive cilengitide arm (HR, 0.858; 95% CI: 0.612, 1.204; P = .3771) versus 13.4 months in the control arm. Median progression-free survival assessed per independent review committee was 5.6 months (HR, 0.822; 95% CI: 0.595, 1.134) and 5.9 months (HR, 0.794; 95% CI: 0.575, 1.096) in the standard and intensive cilengitide arms, respectively, versus 4.1 months in the control arm. Cilengitide was well tolerated. CONCLUSIONS: Standard and intensive cilengitide dose regimens were well tolerated in combination with TMZ/RT→TMZ. Inconsistent overall survival and progression-free survival outcomes and a limited sample size did not allow firm conclusions regarding clinical efficacy in this exploratory phase II study.

Sterol regulatory element binding protein-1a transactivates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene promoter

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB)catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a)binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription.

Sterol regulatory element binding protein-1a transactivates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene promoter

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB)catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a)binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription.

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops

Cytosine-and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34 -bases long cytosine-rich sequence and its complementary guanin e-rich strand corresponding to the first intron of the n-mycgene were studied. Both sequences , not yet studied, contain a 12 - base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively ...

A chromosomal insertion toolbox for promoter probing, mutant complementation and pathogenicity studies in Ralstonia solanacearum

We describe here the construction of a delivery system for stable and directed insertion of gene constructs in a permissive chromosomal site of the bacterial wilt pathogen Ralstonia solanacearum. The system consists of a collection of suicide vectors the Ralstonia chromosome (pRC) series that carry an integration element flanked by transcription terminators and two sequences of homology to the chromosome of strain GMI1000, where the integration element is inserted through a double recombination event. Unique restriction enzyme sites and a GATEWAY cassette enable cloning of any promoter::gene combination in the integration element. Variants endowed with different selectable antibiotic resistance genes and promoter::gene combinations are described. We show that the system can be readily used in GMI1000 and adapted to other R. solanacearum strains using an accessory plasmid. We prove that the pRC system can be employed to complement a deletion mutation with a single copy of the native gene, and to measure transcription of selected promoters in monocopy both in vitro and in planta. Finally, the system has been used to purify and study secretion type III effectors. These novel genetic tools will be particularly useful for the construction of recombinant bacteria that maintain inserted genes or reporter fusions in competitive situations (i.e., during plant infection).

The promoter activity of human Mfn2 depends on Sp1 in vascular smooth muscle cells

AIMS: Mitofusin-2 (Mfn2) expression is dysregulated in vascular proliferative disorders and its overexpression attenuates the proliferation of vascular smooth muscle cells (VSMCs) and neointimal lesion development after balloon angioplasty. We sought to gain insight into the mechanisms that control Mfn2 expression in VSMCs. METHODS AND RESULTS: We cloned and characterized 2 kb of the 5'-flanking region of the human Mfn2 gene. Its TATA-less promoter contains a CpG island. In keeping with this, 5'-rapid amplification of cDNA ends revealed six transcriptional start sites (TSSs), of which TSS2 and TSS5 were the most frequently used. The strong CpG island was found to be non-methylated under conditions characterized by large differences in Mfn2 gene expression. The proximal Mfn2 promoter contains six putative Sp1 motifs. Sp1 binds to the Mfn2 promoter and its overexpression activates the Mfn2 promoter in VSMCs. Chemical inhibition of Sp1 reduced Mfn2 expression, and Sp1 silencing reduced transcriptional activity of the Mfn2 promoter. In keeping with this view, Sp1 and Mfn2 mRNA levels were down-regulated in the aorta early after an atherogenic diet in apolipoprotein E-knockout mice or in VSMCs cultured in the presence of low serum. CONCLUSION: Sp1 is a key factor in maintaining basal Mfn2 transcription in VSMCs. Given the anti-proliferative actions of Mfn2, Sp1-induced Mfn2 transcription may represent a mechanism for prevention of VSMC proliferation and neointimal lesion and development.

The promoter activity of human Mfn2 depends on Sp1 in vascular smooth muscle cells

AIMS: Mitofusin-2 (Mfn2) expression is dysregulated in vascular proliferative disorders and its overexpression attenuates the proliferation of vascular smooth muscle cells (VSMCs) and neointimal lesion development after balloon angioplasty. We sought to gain insight into the mechanisms that control Mfn2 expression in VSMCs. METHODS AND RESULTS: We cloned and characterized 2 kb of the 5'-flanking region of the human Mfn2 gene. Its TATA-less promoter contains a CpG island. In keeping with this, 5'-rapid amplification of cDNA ends revealed six transcriptional start sites (TSSs), of which TSS2 and TSS5 were the most frequently used. The strong CpG island was found to be non-methylated under conditions characterized by large differences in Mfn2 gene expression. The proximal Mfn2 promoter contains six putative Sp1 motifs. Sp1 binds to the Mfn2 promoter and its overexpression activates the Mfn2 promoter in VSMCs. Chemical inhibition of Sp1 reduced Mfn2 expression, and Sp1 silencing reduced transcriptional activity of the Mfn2 promoter. In keeping with this view, Sp1 and Mfn2 mRNA levels were down-regulated in the aorta early after an atherogenic diet in apolipoprotein E-knockout mice or in VSMCs cultured in the presence of low serum. CONCLUSION: Sp1 is a key factor in maintaining basal Mfn2 transcription in VSMCs. Given the anti-proliferative actions of Mfn2, Sp1-induced Mfn2 transcription may represent a mechanism for prevention of VSMC proliferation and neointimal lesion and development.