Limitation in use of luciferase reporter genes for 3′-untranslated region analysis

Luciferase reporter genes are widely used for the functional characterization of regulatory elements in 3′-untranslated region (3′-UTR). Using a transient expression assay system with pancreatic cell lines, we demonstrated that luciferase reporter gene constructs show not only the elements with special sequences in 3′-UTR that can affect luciferase activity, but also elements containing random sequences that were ligated into the same site. The extent of the decrease in luciferase activity was dependent on the length of the DNA fragments. Our findings strongly suggested a need to re-examine the 3′-UTR characterizations of many eukaryotic genes which have been studied to date with luciferase reporter genes. Lintao Wang and Jingjing Zhang are equal contributors.     

The Tall Fescue Turf Grass Class I Chitinase Gene <Emphasis Type="Italic">FaChit1</Emphasis> Is Activated by Fungal Elicitors,Dehydration, Ethylene,and Mechanical Wounding

The cDNA, genomic DNA, and promoter sequence of FaChit1, a class I chitinase gene from Festuca arundinacea, were isolated and characterized in the present work. The deduced amino acid sequence of FaChit1 contains the chitin binding, catalytic, and proline and glycine-rich domains characteristic for most class I chitinases, but no C-terminal extension region. FaChit1 is induced effectively by fungal elicitors, dehydration, and ethylene, but only slightly by mechanical wounding. To identify potential stress-related cis-acting elements, 5′ sequences 935, 651, and 233 bp upstream of the FaChit1 start codon were fused to the GUS reporter gene and analyzed in transgenic tobacco. The results indicated that the 935 bp fragment closely mirrored endogenous gene expression and that the 651 bp fragment was sufficient to direct reporter the gene expression in response to fungal elicitors, ethylene, dehydration, or mechanical wounding due to both known and presently uncharacterized cis-acting elements. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular cloning of a novel chimeric HMW glutenin subunit gene <Emphasis Type="Italic">1Dx5′</Emphasis> from a common wheat line W958

A novel chimeric high-molecular-weight (HMW) glutenin subunit gene from a new common wheat line W958 (2n = 6x = 42) was isolated and characterized. SDS–PAGE analysis revealed that this glutenin subunit has similar electrophoretic mobility to 1Dx5, so it was designated 1Dx5′. Genomic DNA from W958 was amplified and a 2,505-bp fragment was obtained. The 1Dx5′ subunit showed a chimeric primary structure of 1Dx5 and 1Dx2, with the 1Dx5 sequence in the 5′ and middle repetitive regions and the 1Dx2 sequence in the repetitive domain and 3′ region. MALDI-TOF-MS analysis demonstrated that 1Dx5′ had a molecular weight of 86815.1 Da, close to that of an x-type glutenin subunit. Secondary structure analysis showed that this subunit had six helixes and one strand, including four helixes in the repetitive domain which could enhance the dough properties. Additionally, the promoter of 1Dx5′ was obtained and showed the same sequence as 1Dx5 or 1Dx2 except for a few base conversions. The promoter analysis indicated that the cis-acting regulatory elements of 1Dx5′ were the same as those of 1Dx5 and/or 1Dx2. Previously, we have demonstrated that this novel glutenin subunit is associated with good bread-making quality and comprises a very large proportion of the F2 segregation population. Consequently, we suggest that the amino acid residue composition and the secondary structure of the subunit may contribute to the bread-making quality. In summary, the novel 1Dx5′ gene could have greater potential in wheat quality improvement.     

Stress responses in alfalfa (Medicago sativa L.). 8. Cis-elements and trans-acting factors for the quantitative expression of a bean chalcone synthase gene promoter in electroporated alfalfa protoplasts

A chimeric gene consisting of a bean (Phaseolus vulgaris L.) chalcone synthase (CHS) promoter fused to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed, and further induced by fungal elicitor, when electroporated into alfalfa (Medicago sativa L.) suspension cell protoplasts. Functional analysis of 5 deletions of the CHS promoter-CAT construct in these protoplasts indicated that the region between –326 and –130 contained both activator and silencer elements. Co-electroporation experiments confirmed that these cis-acting elements were binding sites for functionally active trans factors. In vitro DNase I footprinting revealed four potential binding sites for alfalfa suspension cell nuclear proteins between positions –326 and –130 of the CHS promoter. These sites mapped to regions shown to contain functional cis-acting elements on the basis of the deletion analysis. Three of these sites mapped to previously identified binding sites for bean nuclear proteins. Competition gel retardation analysis using oligonucleotide probes containing binding site sequences revealed sequence-specific binding of alfalfa nuclear proteins to an AT-rich element and a putative GT-1 factor consensus binding sequence. Our results define cis elements and their cognate trans factors functionally active in determining the quantitative expression of a defense response gene in a heterologous transient expression system.Abbreviations CAT chloramphenicol acetyltransferase - CHS chalcone synthase (EC 2.3.1.74) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

The mannopine synthase promoter contains vectorial cis-regulatory elements that act as enhancers and silencers

A 479-bp bi-directional promoter controls the expression of two genes (mas1′ and mas2′) that encode enzymes for the synthesis of the opine mannopine in plant tissues infected with Agrobacterium tumefaciens. This 5′ regulatory region (mas promoter) contains all the cis-acting elements involved in mediating the complex regulatory properties of these genes in plants. Using different mas promoter regions fused to a minimal 35S promoter (35SΔ108), we found that the regulatory properties of these divergent promoters result from the presence of orientation-dependent negative and positive regulatory regions. Some of these elements have the unusual property of acting as enhancers in one orientation and as silencers in the other. Using electrophoretic mobility shift analysis (EMSA), we showed that the functional mas promoter regions identified by fluorometric and histochemical assays for reporter gene activity in transgenic plants have the ability specifically to bind nuclear protein factors from Nicotiana tabacum, Phaseolus vulgaris, Solanum tuberosum, and Arabidopsis thaliana. Received: 7 May 1999 / Accepted: 5 August 1999     

Common Human 5′ Dopamine Transporter (SLC6A3) Haplotypes Yield Varying Expression Levels In Vivo

1. Individuals display significant differences in their levels of expression of the dopamine transporter (DAT; SLC6A3). These differences in DAT are strong candidates to contribute to individual differences in motor, mnemonic and reward functions. To identify “cis”-acting genetic mechanisms for these individual differences, we have sought variants in 5′ aspects of the human DAT gene and identified the haplotypes that these variants define.2. We report (i) significant relationships between 5′ DAT haplotypes and human individual differences in ventral striatal DAT expression assessed in vivo using [¹¹C] cocaine PET and (ii) apparent confirmation of these results in studies of DAT expression in postmortem striatum using [³H] carboxyflurotropane binding.3. These observations support the idea that cis-acting variation in 5′ aspects of the human DAT/SLC6A3 locus contributes to individual differences in levels of DAT expression in vivo. 5′ DAT variation is thus a good candidate to contribute to individual differences in a number of human phenotypes.These authors contributed equally to this article     

Common genetic variation in the 3′-untranslated region of gonadotropin-releasing hormone receptor regulates gene expression <Emphasis Type="Italic">in cella</Emphasis> and is associated with thyroid function,insulin secretion as well as insulin sensitivity in polycystic ovary syndrome patients

Gonadotropin-releasing hormone receptor (GNRHR) is a member of the G protein-coupled Ca(2+)-dependent family of receptors. It interacts with GnRH, whose signaling plays an important role in thyroid-stimulating hormone (TSH) secretion and insulin activity. There has been no study on the genetic effect of GNRHR on TSH secretion and insulin action in polycystic ovary syndrome (PCOS). We decided to investigate whether naturally occurring genetic variation at the human GNRHR locus is associated with thyroid function, insulin secretion and insulin sensitivity in PCOS. We undertook a systematic search for polymorphisms in GNRHR by resequencing the gene and then genotyped common single-nucleotide polymorphisms across the locus in 261 PCOS patients well-phenotyped for several metabolic traits to determine associations. A test for association of common genetic variants with susceptibility to PCOS was carried out in a large cohort of 948 subjects. Finally, we experimentally validated the marker-on-trait associations using GNRHR 3′-UTR region/reporter analysis in 293T cells. The 3′-UTR variant rs1038426 was associated with serum thyroid concentration (P = 0.007), change of insulin levels during oral glucose tolerance test (P = 0.004) and insulin sensitivity index (P = 0.014). In a functional study, 3′-UTR variant T allele increased reporter expression by a transfected luciferase reporter/GNRHR 3′-UTR expression plasmid. In conclusion, our results strongly suggest that common genetic variant in GNRHR contributes to the phenotypic expression of PCOS. The findings suggest novel pathophysiological links between the GNRHR locus and thyroid function and insulin secretion in PCOS.     

Abundant conserved microRNA target sites in the 5′-untranslated region and coding sequence

Recent studies have shown that miRNAs can target the promoter and CDS region. Thus, we predicted miRNA target sites in the 5′-UTR, CDS and 3′-UTR of Homo sapiens, Mus musculus and Drosophila melanogaster using miRanda and TargetScan. Target-site densities normalized with the average region length were higher in the 5′-UTR than 3′-UTR in all three organisms but were lower in the negative data set. Interestingly, the putative target sites were more conserved than non-target regions in both the 5′-UTR and 3′-UTR, implying that target sites in the 5′-UTR are subject to high selective pressure and might be functional. In Drosophila, 48 of 78 (61.5%) miRNAs showed high similarities with predicted siRNAs. Based on the results of previous experimental studies and a large-scale statistical analysis, we conclude that miRNA-mediated regulation is not limited to the 3′-UTR. However, the functionality of target sites in the 5′-UTR and CDS requires thorough investigation.     

Evolution of cis-Acting Elements in 5′ Flanking Regions of Vertebrate Actin Genes

Regulation of the vertebrate actin multigene family involves the recognition of various regulatory sequences (cis-acting elements) that specify the distinct tissue type and developmental program of expression for each actin paralogue, which implies that the distribution of cis-acting elements may be unique for each paralogue gene. To elucidate the evolution of these unique distribution patterns, we improved a method to scan for cis-acting elements in the 5′ flanking regulatory region of genes and used it to analyze five cis-acting elements (SRE, MyoD binding site, Elk-1 binding site, positive and negative YY1 binding sites) of six actin paralogue genes (β and γ cytoplasmic actins, α and γ smooth muscle actins, and α skeletal and α cardiac actins) among various vertebrates. It was shown that although an element(s) may exist in all paralogue genes of the same species, its numbers, compositions, and distribution patterns or even sequences vary remarkably among paralogues, which contributes to their different tissue- and developmental-specific expression. However, each pair of coexpressed paralogues has some certain similarity in distribution patterns. Furthermore, among various orthologues of actin genes derived from diverse vertebrates, the sequences, numbers, and distribution patterns of these cis-acting elements are highly conserved or even identical in the long run of phylogeny of vertebrates. Taken together, the results described above strongly indicate that not only the structures of actins but also their expression patterns are essential in both the phylogeny and the physiology of vertebrates. The distribution patterns of cis-acting elements of various actin genes can be regarded as indicators of both horizontal (paralogous) and vertical (orthologous) evolution of actins. Received: 1 March 1999 / Accepted: 6 August 1999     

Regulation of zinc-responsive Slc39a5 (Zip5) translation is mediated by conserved elements in the 3′-untranslated region

Translation of the basolateral zinc transporter ZIP5 is repressed during zinc deficiency but Zip5 mRNA remains associated with polysomes and can be rapidly translated when zinc is repleted. Herein, we examined the mechanisms regulating translation of Zip5. The 3′-untranslated region (UTR) of Zip5 mRNA is well conserved among mammals and is predicted by mFOLD to form a very stable stem-loop structure. Three algorithms predict this structure to be flanked by repeated seed sites for miR-328 and miR-193a. RNAse footprinting supports the notion that a stable stem-loop structure exists in this 3′-UTR and electrophoretic mobility shift assays detect polysomal protein(s) binding specifically to the stem-loop structure in the Zip5 3′-UTR. miR-328 and miR-193a are expressed in tissues known to regulate Zip5 mRNA translation in response to zinc availability and both are polysome-associated consistent with Zip5 mRNA localization. Transient transfection assays using native and mutant Zip5 3′-UTRs cloned 3′ to luciferase cDNA revealed that the miRNA seed sites and the stem-loop function together to augment translation of Zip5 mRNA when zinc is replete.     

Identification and characterization of interferon regulatory factor-1 from orange-spotted grouper (<Emphasis Type="Italic">Epinephelus coioides</Emphasis>)

Interferon-regulatory factor 1 (IRF-1) is the first member of IRF family, which is involved in many biological processes such as immune response, antiviral defense, cell growth regulation, and apoptosis. In this study, an IRF-1 gene, EcIRF-1, was isolated and characterized from orange-spotted grouper (Epinephelus coioides). The full-length cDNA of EcIRF-1 is 1,730 bp, including an open reading frame of 906 bp, a 5′-terminal untranslated region (5′-UTR) of 153 bp, and a 3′-UTR of 671 bp. The EcIRF-1 gene consists of 10 exons and 9 introns, spanning over approximate 4.3 kb of genomic sequence. The 5′-UTR sequence contains an exon and an intron, and the 3′-UTR sequence is included in the last exon. Expression analysis by real-time PCR reveals that the EcIRF-1 gene is ubiquitously expressed in various healthy fish tissues, whereas its expression is upregulated in vivo in response to polyinosinic–polycytidylic acid or lipopolysaccharide stimulation. Subcellular localization analysis shows the EcIRF-1 is an intranuclearly localized and immobile protein in the cultured fish cells. Data presented in this paper provide an important base to further understand EcIRF-1 gene function and its regulation associated with interferon immune system in orange-spotted grouper.     

Molecular mechanisms of cAMP-regulated gene expression

The ability of many genes to be induced by cAMP is dependent on the presence of enhancers located in the regions of DNA upstream of the start sites to the genes. The two best characterized enhancers are the CRE (5′-TGACGTCA-3′) and the AP-2 site (5′-CCCCAGGC-3′). The activity of the CRE is modulated by sequences adjacent to the consensus sequence as well as by promoter context and cell type. The complex control of the CRE is reflected in the large number of cloned CRE binding proteins that arise both from unique genes and from splice variants. These factors are leucine zipper proteins that must dimerize before binding to DNA. Although all of the factors isolated can form active homodimers, many, are also able to form heterodimers. The, amino termini of these proteins contain consensus phosphorylation sites through which these factorstrans-activate their cognate promoters. The diversity of thetrans-acting factors and theircis-acting sequences reflects the precise control that cells require in the modulation of gene expression by cAMP.