Functional Analysis and Randomization of the Nisin-Inducible Promoter for Tuning Gene Expression in Lactococcus lactis

Three putative promoter regions were identified preceding the nisZ gene in Lactococcus lactis HSM-22. To investigate their function in the control of nisZ biosynthesis, green fluorescence protein (GFP) was adopted as probe to determine activities of the three promoters. The results showed that PnisZ-0 containing two sets of the ?35 and ?10 regions exhibited the same maximum activity as promoter PnisZ-2 containing the putative promoter region near the start codon. However, the GFP expression level directed by PnisZ-0 was twofold higher than that found with PnisZ-2 under low-dose nisin, indicating that promoter PnisZ-1 distant from the start codon could be important in response to the inducer nisin. Then, Pnis-2 was randomized to develop functional promoters through the degenerate oligonucleotide approach in L. lactis. 35 inducible promoters and 14 constitutive promoters were obtained, covering 3–5 logs of expression levels in small increments of activity. Sequence analysis revealed that base changes in both consensus sequence and spacing sequence resulted in remarkable decrease of promoter activity, while the sequence outside ?35 and ?10 regions would influence the promoter function radically. The functional promoters were evaluated for the efficiency and stability to control β-galactosidase (Gal) expression in L. lactis. High correlation was obtained between the Gal activity and promoter strength, suggesting that promoters developed here have the potential for fine tuning gene expression in L. lactis.     

Gene expression assays for actin, ubiquitin, and three microsatellite-encoding genes in Helianthus annuus (Asteraceae)

? Premise of the study: The "tuning knob" model of King et al. (Endeavor 21: 36-40, 1997) postulates that microsatellite mutations can alter phenotypes in a stepwise fashion. Some proposed mechanisms involve regulation of gene expression. To study the effect of microsatellites harbored in untranslated regions on gene expression in Helianthus annuus, we have developed TaqMan assays for three microsatellite-encoding genes, and two constitutively expressed genes, actin and ubiquitin, to serve as standards. ? Methods and Results: All five TaqMan assays yielded strong log-linear relationships between cycle threshold (C(T)) values and cDNA concentrations (R(2) = 0.98-0.99). Standard curves were based on five concentrations for each of five individuals. Efficiencies ranged from 0.83 to 1.03. ? Conclusions: The developed tools will allow for relative quantification of gene expression across individuals. Genotyping these loci will allow for testing the "tuning knob" hypothesis. Further, the actin and ubiquitin assays should be generally applicable to gene expression studies in H. annuus.     

Identification of functional DNA variants in the constitutive promoter region of MDM2

Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare, MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 and is p53-responsive, and an upstream constitutive promoter (P1), which is p53-independent. Both promoter regions contain DNA variants that could influence the expression levels of MDM2, including the well-studied single nucleotide polymorphism (SNP) SNP309, which is located in the promoter P2; i.e., upstream of exon 2. In this report, we screened the promoter P1 for DNA variants and assessed the functional impact of the corresponding SNPs. Using the dbSNP database and genotyping validation in individuals of European descent, we identified three common SNPs (?1494?G?>?A; indel 40?bp; and ?182?C?>?G). Three major promoter haplotypes were inferred by using these three promoter SNPs together with rs2279744 (SNP309). Following subcloning into a gene reporter system, we found that two of the haplotypes significantly influenced MDM2 promoter activity in a haplotype-specific manner. Site-directed mutagenesis experiments indicated that the 40?bp insertion/deletion variation is causing the observed allelic promoter activity. This study suggests that part of the variability in the MDM2 expression levels could be explained by allelic p53-independent P1 promoter activity.     

Curcumin causes promoter hypomethylation and increased expression of FANCF gene in SiHa cell line

Curcumin and resveratrol were evaluated for their potential to cause reversal of promoter hypermethylation and associated gene expression of FANCF in SiHa cell line. Methylation specific PCR along with bisulphite sequencing revealed the demethylation of 12 CpG sites out of 15 CpG sites spanning ?280 to ?432 region of FANCF promoter after treatment with curcumin and fivefold up regulation of FANCF gene expression as shown by qRT-PCR. In vitro methylation assay also showed that M.SssI an analogue of DNMT1 was effectively inhibited at 50 lM concentration of curcumin. Resveratrol was not found to be effective in causing reversal of promoter hypermethylation of FANCF gene when used at 20 lM for 4 days in SiHa cell line.     

基因捕捉及其在植物基因分离和功能基因组学上的应用

基因捕捉是一种报告基因的随机整合技术。基因捕捉系统已成为分离基因、鉴定基因功能的重要手段。基因捕捉(gene traps)包括增强子捕捉(enhancer trap)、启动子捕捉(promoter trap)和基因捕捉(gene trap),通称为基因捕捉(gcne traps)。在增强子捕捉中,报告基因与一个基本启动子融合,这个启动子不能使报告基因表达,但可被临近的增强子激活。在启动子捕捉和基因捕捉中,报告基因的启动子被去除,融合基因只有以正确的方向插入到转录单元内才能表达。对基因捕捉系统的结构特征、构建方法、应用范围、研究现状和应用前景等作了系统论述,并对有关问题进行了讨论。     

Cloning of a new LEA1 gene promoter from soybean and functional analysis in transgenic tobacco

LEA1 gene from Glycine max can be expressed in late-embryo stage of plants, and respond to salinity and dehydration stress. To elucidate the mechanism for stress tolerance and high expression in seeds, we isolated and characterized the promoter of LEA1 gene (EQ, 1997 bp) starting the 5′LEA1 coding region. A deletion mutant of EQ promoter (ED) and the full length promoter (EQ) were fused to GUS reporter gene and transformed into the tobacco leaf discs. The results indicated that expression of the reporter gene (GUS) could be regulated by EQ promoter, and was stronger than the mutant under the stress conditions. Also, the expression level of GUS gene driven by EQ promoter in transgenic tobacco seeds was significantly higher than that by the mutant promoter, which meant that it had a better tissue-specificity. Therefore, the active domain for the promoter was located between ?1997 and ?1000 bp. Additionally, the activity of EQ promoter was 2.1-, 3.3- and 0.4- times stronger than the activity of promoter CaMV35S under salt (24 h), drought (10 h) or ABA (24 h), respectively. Meanwhile, the GUS activity of EQ promoter in seeds was 1.8-fold stronger compared to the promoter CaMV35S. In summary, the new promoter (EQ) is bi-functional, stress-inducible and seed-specific. These findings provide a further understanding for the regulation of LEA1gene expression, and suggest a new way for improving seed quality under saline and alkaline land.     

Genotype of an individual single nucleotide polymorphism regulates DNA methylation at the TRPC3 alternative promoter

A fundamental challenge in the post-genomics era is to understand how genetic variants can influence phenotypic variability and disease. Recent observations from a number of studies have highlighted a mechanism by which common genetic polymorphisms can influence DNA methylation, a major epigenetic silencing mechanism. We report that the alternative promoter of the human TRPC3 gene is regulated by allelic DNA methylation, dictated by the genotype of a single base pair polymorphism, rs13121031 located within the promoter CpG island. The common G allele is associated with high levels of methylation, while the less prevalent C allele is unmethylated. This methylation profile is observed in many tissue types, despite the expression of?TRPC3?being restricted to brain and heart.?TRPC3?is prominently expressed in the hindbrain, and a heterozygous brain sample showed modest skewing according to the allelic methylation, with preferential expression from the C allele. The?TRPC3?gene encodes a transient receptor potential channel that has been implicated in cerebellar ataxia and heart hypertrophy. The genotype-frequencies of rs13121031 were determined in cohorts of ataxia patients and in individuals with cardiac hypertrophy. These analyses revealed a statistical trend for the rare unmethylated homozygous C genotype to be present at a higher frequency in idiopathic ataxia patients (Fisher's test p=0.06), but not in those patients with known mutations (Fisher's test p=0.55) or in individuals with heart disease (Fisher's test p=0.807), when compared to a control population. Our results suggest that the?TRPC3?alternative promoter is a methylation quantitative-trait locus that may be involved in modulating the ataxia phenotype.     

Analysis of banana fruit-specific promoters using transient expression in embryogenic cells of banana cultivar Robusta (AAA Group)

The study reports the transient expression of gusA gene in embryogenic cells using three banana derived fruit-specific promoters. Banana embryogenic cells were transformed with a pCAMBIA-1301 derived plasmid construct harboring gusA gene driven by either chitinase, glucanase or expansin promoters derived from banana. The transient expression of ??-glucuronidase was studied 5?days after co-cultivation with Agrobacterium harboring the expression plasmids. The transformed embryogenic cells were treated with different inducers of ethylene such as ethephon, methyl jasmonate, methyl salicylate, abscisic acid and indole acetic acid. The maximum expression of 64099.78?pmoles 4-MU/h/mg total protein was noted with expansin promoter when the cells were treated with the combination of ethephon (0.25?mM) and MJ (10?mM). The results suggest that these promoters can be used to achieve fruit-specific expression of useful transgenes in banana. The results should prove to be an important guide for short term expression studies for promoter validation and gene screening.     

Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice

Previous studies indicated that a DNA fragment comprising nucleotides (nt) ?164 to +45 of the RTBV promoter is sufficient to drive phloem-specific expression of a reporter gene in transgenic rice plants. In addition, two potential cis elements, Box I (nt ?3 to +5) and Box II (nt ?53 to ?39) were identified by DNA-protein interaction assays. In this study, the results of further in vivo studies involving mutagenesis of selected DNA sequences and analysis of expression of a reporter gene in transgenic rice plants revealed that, in addition to Box I and Box II, other elements are required for phloem-specific gene expression, among which are a direct repeat (ASL Box, nt ?98 to ?79) and a GATA motif (nt ?143 to ?135). All the these elements bind rice nuclear factors specifically, and mutations of the elements were identified that resulted in loss-of-competition in electrophoretic mobility shift assays. A DNA fragment comprising nt ?164 to ?32, which contains Box II, the ASL Box and the GATA motif, conferred phloem-specific reporter gene expression independent of Box I when fused to a heterologous CaMV 35S minimal promoter and introduced to transgenic rice plants. Studies that introduced point mutations suggested that in the context of the ?103 to +45 promoter fragment, Box II and the ASL Box act synergistically to confer tissue-specific gene expression. Similar studies in the ?164 to +45 promoter fragment indicated that the ?164 to ?103 region, which includes the GATA motif, contains sequences that are functionally redundant with those in the ?103 to ?32 region, including the ASL Box and Box II. It is concluded that these regions act additively to direct phloem-specific gene expression.