Putative TATA Box Research Articles

Unconventional features in the transcription and processing of spliceosomal small nuclear RNAs in the protozoan parasite Trichomonas vaginalis

Trichomonas vaginalis is a medically important protozoan parasite, and a deep-branching, evolutionarily divergent unicellular eukaryote that has conserved several key features of eukaryotic gene expression. Trichomonas vaginalis possesses a metazoan/plant-like capping apparatus, mRNAs with a cap 1 structure and spliceosomes containing the five small nuclear RNAs (snRNAs). However, in contrast to metazoan and plant snRNAs, the structurally conserved T. vaginalis snRNAs were initially identified as lacking the canonical guanosine cap nucleotide. To explain this unusual condition, we sought to investigate transcriptional and processing features of the spliceosomal snRNAs in this protist. Here, we show that T. vaginalis spliceosomal snRNA genes mostly lack typical eukaryotic promoters. In contrast to other eukaryotes, the putative TATA box in the T. vaginalis U6 snRNA gene was found to be dispensable for transcription or RNA polymerase selectivity. Moreover, U6 transcription in T. vaginalis was virtually insensitive to tagetitoxin compared with other cellular transcripts produced by the same RNA polymerase III. Most important and unexpected, snRNA transcription in T. vaginalis appears to bypass capping as we show that these transcripts retain their original 5′-triphosphate groups. In conclusion, transcription and processing of spliceosomal snRNAs in T. vaginalis deviate considerably from the conventional rules of other eukaryotes.

Ovine KRTAP36-2: A New Keratin-Associated Protein Gene Related to Variation in Wool Yield.

Keratin-associated proteins (KAPs) are structural components of wool fibres. High-glycine/tyrosine (HGT)-KAPs are a subset of the KAP family, and their abundance in fibres varies. In this study, we report the discovery of an ovine HGT-KAP gene to which we assigned the name KRTAP36-2. Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analyses revealed four variants of this gene in a screening population of 170 sheep from a variety of breeds. The DNA sequencing of the variants revealed four single-nucleotide polymorphisms (SNPs) and a dinucleotide deletion. Three of these SNPs were in the coding region, and one of these was non-synonymous and potentially led to the amino acid substitution p.Cys27Gly near the middle of the protein. The remaining SNP was located near the putative TATA box, and the di-nucleotide deletion was near the putative transcription initiation site. The effect of this variation in KRTAP36-2 was investigated in 274 Southdown × Merino lambs that were the progeny of five sires. Variation was only found to be associated with wool yield, that is, the proportion of the greasy fleece that remained as clean fleece upon scouring (expressed as a percentage). This may have some value in increasing wool production.

Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species

Conditional promoters allowing both induction and silencing of gene expression are indispensable for basic and applied research. The xylP promoter (pxylP) from Penicillium chrysogenum was demonstrated to function in various mold species including Aspergillus fumigatus. pxylP allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer. Here we structurally characterized and engineered pxylP in A. fumigatus to optimize its application. Mutational analysis demonstrated the importance of the putative TATA-box and a pyrimidine-rich region in the core promoter, both copies of a largely duplicated 91-bp sequence (91bpDS), as well as putative binding sites for the transcription factor XlnR and a GATA motif within the 91bpDS. In agreement, pxylP activity was found to depend on XlnR, while glucose repression appeared to be indirect. Truncation of the originally used 1643-bp promoter fragment to 725 bp largely preserved the promoter activity and the regulatory pattern. Integration of a third 91bpDS significantly increased promoter activity particularly under low inducer concentrations. Truncation of pxylP to 199 bp demonstrated that the upstream region including the 91bpDSs mediates not only inducer-dependent activation but also repression in the absence of inducer. Remarkably, the 1579-bp pxylP was found to act bi-bidirectionally with a similar regulatory pattern by driving expression of the upstream-located arabinofuranosidase gene. The latter opens the possibility of dual bidirectional use of pxylP. Comparison with a doxycycline-inducible TetOn system revealed a significantly higher dynamic range of pxylP. Taken together, this study identified functional elements of pxylP and opened new methodological opportunities for its application.

Hepatitis B Virus Variants with Multiple Insertions and/or Deletions in the X Open Reading Frame 3' End: Common Members of Viral Quasispecies in Chronic Hepatitis B Patients.

Deletions in the 3′ end region of the hepatitis B virus (HBV) X open reading frame (HBX) may affect the core promoter (Cp) and have been frequently associated with hepatocellular carcinoma (HCC). The aim of this study was to investigate the presence of variants with deletions and/or insertions (Indels) in this region in the quasispecies of 50 chronic hepatitis B (CHB) patients without HCC. We identified 103 different Indels in 47 (94%) patients, in a median of 3.4% of their reads (IQR, 1.3–8.4%), and 25% (IQR, 13.1–40.7%) of unique sequences identified in each quasispecies (haplotypes). Of those Indels, 101 (98.1%) caused 44 different altered stop codons, the most commonly observed were at positions 128, 129, 135, and 362 (putative position). Moreover, 39 (37.9%) Indels altered the TATA-like box (TA) sequences of Cp; the most commonly observed caused TA2 + TA3 fusion, creating a new putative canonical TATA box. Four (8%) patients developed negative clinical outcomes after a median follow-up of 9.4 (8.7–12) years. In conclusion, we observed variants with Indels in the HBX 3′ end in the vast majority of our CHB patients, some of them encoding alternative versions of HBx with potential functional roles, and/or alterations in the regulation of transcription.

Dysfunction of CYC2g is responsible for the evolutionary shift from radiate to disciform flowerheads in the Chrysanthemum group (Asteraceae: Anthemideae).

Evolutionary shifts among radiate, disciform and discoid flowerheads have occurred repeatedly in a number of major lineages across the Asteraceae phylogeny; such transitions may also appear within evolutionarily young groups. Although several studies have demonstrated that CYC2 genes partake in regulating floral morphogenesis in Asteraceae, the evolution of capitulum forms within a recently diverging lineage has remained poorly understood. Here, we study the molecular regulation of the shift from a radiate to a disciform capitulum within the Chrysanthemum group. This is a recently radiating group mainly comprising two genera, Chrysanthemum and Ajania, that are phylogenetically intermingled but distinct in flowerhead morphology: Chrysanthemumspp. with radiate capitula and Ajaniaspp. with disciform capitula. We found that the morphogenesis of zygomorphy in the marginal floret in Ajania was disrupted soon after floral primordium emergence; CYC2g, one of the CYC2 copies that was expressed prominently in the ray floret of Chrysanthemum was not expressed in flowerheads of Ajania. Weakening the expression of ClCYC2g in Chrysanthemum lavandulifolium led to the gradual transition of a ray flower toward the disc-like form. Molecular evolutionary analyses indicated that the disciform capitulum might have evolved only once, approximately 8Mya, arising from dysfunction of the CYC2g orthologs. A 20-nt deletion, including a putative TATA-box of the Ajania-type CYC2g promoter, appeared to inhibit the expression of the gene. Considering the divergent habitats of Chrysanthemum and Ajania, we propose that the shift from radiate to disciform capitulum must have been related to changes in pollination strategies under selective pressure.

Regulatory effect of heat shock transcription factor-1 gene on heat shock proteins and its transcriptional regulation analysis in small abalone Haliotis diversicolor

BackgroundThe effects of diverse stresses ultimately alter the structures and functions of proteins. As molecular chaperones, heat shock proteins (HSPs) are a group of highly conserved proteins that help in the refolding of misfolded proteins and the elimination of irreversibly damaged proteins. They are mediated by a family of transcription factors called heat shock factors (HSFs). The small abalone Haliotis diversicolor is a species naturally distributed along the southern coast of China. In this study, the expression of HdHSF1 was inhibited by RNAi in hemocytes in order to further elucidate the regulatory roles of HdHSF1 on heat shock responsive genes in abalone. Meanwhile, to understand the transcriptional regulation of the HdHSF1 gene, the 5′-upstream regulatory region of HdHSF1 was characterized, and the relative promoter activity was examined by dual-luciferase reporter gene assay system in HEK293T cell lines.ResultsAfter the inhibition of the H. diversicolor HSF1 gene (HdHSF1) by dsRNA (double-stranded RNA), the expression of most heat shock related-genes was down-regulated (p < 0.05). It indicated the importance of HdHSF1 in the heat shock response of H. diversicolor. Meanwhile, 5′-flanking region sequence (2633 bp) of the HdHSF1 gene was cloned; it contained a putative core promoter region, TATA box, CAAT box, CpG island, and many transcription elements. In HEK293T cells, the 5′-flanking region sequence can drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. Exposure of cells to the high-temperature (39 °C and 42 °C) resulted in the activation of HdHSF1 promoter activity, which may explain why the expression of the HdHSF1 gene participates in heat shock response. Luciferase activity of different recombinant plasmids, which contained different truncated promoter fragments of the HdHSF1 gene in HEK293T cells, revealed the possible active regions of the promoter. To further identify the binding site of the critical transcription factor in the region, an expression vector with the site-directed mutation was constructed. After being mutated on the GATA-1 binding site, we found that the luciferase activity was significantly increased, which suggested that the GATA-1 binding site has a certain weakening effect on the activity of the HdHSF1 promoter.ConclusionsThese findings suggest that GATA-1 may be one of the transcription factors of HdHSF1, and a possible signaling pathway mediated by HdHSF1 may exist in H. diversicolor to counteract the adverse effects of heat shock stress.

Analysis of a new begomovirus unveils a composite element conserved in the CP gene promoters of several Geminiviridae genera: Clues to comprehend the complex regulation of late genes.

A novel bipartite begomovirus, Blechum interveinal chlorosis virus (BleICV), was characterized at the genome level. Comparative analyses revealed that BleICV coat protein (CP) gene promoter is highly divergent from the equivalent region of other begomoviruses (BGVs), with the single exception of Tomato chino La Paz virus (ToChLPV) with which it shares a 23-bp phylogenetic footprint exhibiting dyad symmetry. Systematic examination of the homologous CP promoter segment of 132 New World BGVs revealed the existence of a quasi-palindromic DNA segment displaying a strongly conserved ACTT-(N7)-AAGT core. The spacer sequence between the palindromic motifs is constant in length, but its sequence is highly variable among viral species, presenting a relaxed consensus (TT)GGKCCCY, which is similar to the Conserved Late Element or CLE (GTGGTCCC), a putative TrAP-responsive element. The homologous CP promoter region of Old World BGVs exhibited a distinct organization, with the putative TATA-box overlapping the left half of the ACTT-N7 composite element. Similar CP promoter sequences, dubbed “TATA-associated composite element” or TACE, were found in viruses belonging to different Geminiviridae genera, hence hinting unsuspected evolutionary relationships among those lineages. To get cues about the TACE function, the regulatory function of the CLE was explored in distinct experimental systems. Transgenic tobacco plants harboring a GUS reporter gene driven by a promoter composed by CLE multimers expressed high beta-glucuronidase activity in absence of viral factors, and that expression was increased by begomovirus infection. On the other hand, the TrAP-responsiveness of a truncated CP promoter of Tomato golden mosaic virus (TGMV) was abolished by site-directed mutation of the only CLE present in it, whereas the artificial addition of one CLE to the -125 truncated promoter strongly enhanced the transactivation level in tobacco protoplasts. These results indicate that the CLE is a TrAP-responsive element, hence providing valuable clues to interpret the recurrent association of the CLE with the TACE. On the basis of the aforesaid direct evidences and the insights afforded by the extensive comparative analysis of BleICV CP promoter, we propose that the TACE might be involved in the TrAP-mediated derepression of CP gene in vascular tissues.

Molecular characteristics of rhesus macaque interleukin-22: cloning, invitro expression and biological activities.

Interleukin-22 (IL-22) is a potential therapeutic agent for diseases driven by epithelial injury. To characterize the IL-22 expressed by rhesus macaques, animals that are irreplaceable for human disease research, rhesus macaque IL-22 (rhIL-22) was cloned and expressed, and its biological activity and invivo distribution were examined. It was found that the rhIL-22 gene consists of five introns and six exons, including a short non-coding exon starting 22bp downstream of a putative TATA box. The amino acid sequence of rhIL-22 showed 95·5% identity to that of humans, and it shared two conserved disulphide bonds, three N-glycosylation sites and all the critical residues for binding to IL-22R1. High levels of IL-22 mRNA were observed in the liver, pancreas, lymphoid tissues and especially in the outer-body barriers such as the intestinal tract of rhesus macaques. Functionally, purified rhIL-22 has a similar but a little earlier effect on signal transducer and activator of transcription 3 phosphorylation at Tyr705 compared with that of commercial human IL-22. The expression of the antibacterial proteins β-defensin-2, S100A8, S100A9, RegIIIα and Muc1 by HT-29 cells was largely upregulated after stimulation with rhIL-22. Recombinant rhIL-22 could also significantly promote the proliferation of human intestinal epithelial cells without affecting cell apoptosis. These data indicate that rhesus macaque IL-22 is highly similar to that of humans in both structure and function, and tests of therapeuticeffects of human IL-22 on human diseases in rhesus macaques are warranted.

Transcriptional regulation of follicle-stimulating hormone β-subunit in marmoset by an alternate distal promoter

Follicle-stimulating hormone (FSH) is essential for mammalian folliculogenesis and spermatogenesis. Common marmoset (Callithrix jacchus) is a New World primate which exhibits an unusual FSH profile across the ovarian cycle with a mid-follicular FSH peak that is not observed in Catarrhini primates like humans. Since transcription of FSH β-subunit gene (FSHβ) is a rate-limiting step in the production of mature FSH, this study aimed to investigate the regulation of marmoset FSHβ gene expression in comparison to human. In silico analysis of the FSHβ promoter sequences identified a TATA box element upstream of the conventional TATA box element in marmoset but not in human sequence. FSHβ mRNA transcript longer than the conventional transcript was detected in marmoset pituitary implying presence of a distal transcription start site. In luciferase reporter assays, the marmoset putative distal promoter had higher activity than the corresponding human region even in absence of the conventional proximal promoter. Indeed higher affinity binding of TATA box-binding protein to the putative distal TATA box element was obtained in electrophoretic mobility shift assay. This suggests existence of a differential regulation of FSHβ transcription in marmoset compared to humans.

Assessment of a functional marker for bacterial blight resistance gene locus Xa 27 in rice.

The bacterial blight gene locus Xa 27 in rice has two striking differences in the presumed promoter-raised resistant/susceptible allele expression. We used a functional marker targeting the 25 deletion at 18 bp upstream of the putative TATA box and assayed the allelic status at Xa 27 in 572 rice genotypes. The alleles evaluation obtained from 191 cultivars tested may contribute to little information of the resistance specificity associated with allele diversity. The allele frequency associated with complete resistance in the indica was different from that associated with complete resistance in the japonica.

Widespread and evolutionary analysis of a MITE family Monkey King in Brassicaceae.

BackgroundMiniature inverted repeat transposable elements (MITEs) are important components of eukaryotic genomes, with hundreds of families and many copies, which may play important roles in gene regulation and genome evolution. However, few studies have investigated the molecular mechanisms involved. In our previous study, a Tourist-like MITE, Monkey King, was identified from the promoter region of a flowering time gene, BnFLC.A10, in Brassica napus. Based on this MITE, the characteristics and potential roles on gene regulation of the MITE family were analyzed in Brassicaceae.ResultsThe characteristics of the Tourist-like MITE family Monkey King in Brassicaceae, including its distribution, copies and insertion sites in the genomes of major Brassicaceae species were analyzed in this study. Monkey King was actively amplified in Brassica after divergence from Arabidopsis, which was indicated by the prompt increase in copy number and by phylogenetic analysis. The genomic variations caused by Monkey King insertions, both intra- and inter-species in Brassica, were traced by PCR amplification. Genomic sequence analysis showed that most complete Monkey King elements are located in gene-rich regions, less than 3kb from genes, in both the B. rapa and A. thaliana genomes. Sixty-seven Brassica expressed sequence tags carrying Monkey King fragments were also identified from the NCBI database. Bisulfite sequencing identified specific DNA methylation of cytosine residues in the Monkey King sequence. A fragment containing putative TATA-box motifs in the MITE sequence could bind with nuclear protein(s) extracted from leaves of B. napus plants. A Monkey King-related microRNA, bna-miR6031, was identified in the microRNA database. In transgenic A. thaliana, when the Monkey King element was inserted upstream of 35S promoter, the promoter activity was weakened.ConclusionMonkey King, a Brassicaceae Tourist-like MITE family, has amplified relatively recently and has induced intra- and inter-species genomic variations in Brassica. Monkey King elements are most abundant in the vicinity of genes and may have a substantial effect on genome-wide gene regulation in Brassicaceae. Monkey King insertions potentially regulate gene expression and genome evolution through epigenetic modification and new regulatory motif production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0490-9) contains supplementary material, which is available to authorized users.

Characterization of a Thioredoxin-1 Gene from Taenia solium and Its Encoding Product.

Taenia solium thioredoxin-1 gene (TsTrx-1) has a length of 771 bp with three exons and two introns. The core promoter gene presents two putative stress transcription factor binding sites, one putative TATA box, and a transcription start site (TSS). TsTrx-1 mRNA is expressed higher in larvae than in adult. This gene encodes a protein of 107 amino acids that presents the Trx active site (CGPC), the classical secondary structure of the thioredoxin fold, and the highest degree of identity with the Echinococcus granulosus Trx. A recombinant TsTrx-1 (rTsTrx-1) was produced in Escherichia coli with redox activity. Optimal activity for rTsTrx-1 was at pH 6.5 in the range of 15 to 25°C. The enzyme conserved activity for 3 h and lost it in 24 h at 37°C. rTsTrx-1 lost 50% activity after 1 h and lost activity completely in 24 h at temperatures higher than 55°C. Best storage temperature for rTsTrx-1 was at −70°C. It was inhibited by high concentrations of H2O2 and methylglyoxal (MG), but it was inhibited neither by NaCl nor by anti-rTsTrx-1 rabbit antibodies that strongly recognized a ~12 kDa band in extracts from several parasites. These TsTrx-1 properties open the opportunity to study its role in relationship T. solium-hosts.

Characterization and expression analysis of Toll-like receptor 3 cDNA from Atlantic salmon (Salmo salar).

Innate pathway activation is fundamental for early anti-viral defense in fish, but currently there is insufficient understanding of how salmonid fish identify viral molecules and activate these pathways. The Toll-like receptor (TLR) is believed to play a crucial role in host defense of pathogenic microbes in the innate immune system. In the present study, the full-length cDNA of Salmo salar TLR3 (ssTLR3) was cloned. The ssTLR3 cDNA sequence was 6071 bp long, containing an open reading frame of 2754 bp and encoding 971 amino acids. The TLR group motifs, such as leucine-rich repeat (LRR) domains and Toll-interleukin-1 receptor (TIR) domains, were maintained in ssTLR3, with sixteen LRR domains and one TIR domain. In contrast to descriptions of the TLR3 in rainbow trout and the murine (TATA-less), we found a putative TATA box in the proximal promoter region 29 bp upstream of the transcription start point of ssTLR3. Multiple-sequence alignment analysis of the ssTLR3 protein-coding sequence with other known TLR3 sequences showed the sequence to be conserved among all species analyzed, implying that the function of the TLR3 had been sustained throughout evolution. The ssTLR3 mRNA expression patterns were measured using real-time PCR. The results revealed that TLR3 is widely expressed in various healthy tissues. Individuals challenged with infectious pancreatic necrosis virus and immunostimulated with polyinosinic:polycytidylic acid exhibited increased expression of TLR3 at the mRNA level, indicating that ssTLR3 may be involved in pathogen recognition in the early innate immune system.

The Stripe Rust Resistance Gene Yr10 Encodes an Evolutionary-Conserved and Unique CC–NBS–LRR Sequence in Wheat

The first seedling or all-stage resistance (R) R gene against stripe rust isolated from Moro wheat (Triticum aestivum L.) using a map-based cloning approach was identified as Yr10. Clone 4B of this gene encodes a highly evolutionary-conserved and unique CC-NBS-LRR sequence. Clone 4E, a homolog of Yr10, but lacking transcription start site (TSS) and putative TATA-box and CAAT-box, is likely a non-expressed pseudogene. Clones 4B and 4E are 84% identical and divergent in the intron and the LRR domain. Gene silencing and transgenesis were used in conjunction with inoculation with differentially avirulent and virulent stripe rust strains to demonstrate Yr10 functionality. The Yr10 CC-NBS-LRR sequence is unique among known CC-NBS-LRR R genes in wheat but highly conserved homologs (E = 0.0) were identified in Aegilops tauschii and other monocots including Hordeum vulgare and Brachypodium distachyon. Related sequences were also identified in genomic databases of maize, rice, and in sorghum. This is the first report of a CC-NBS-LRR resistance gene in plants with limited homologies in its native host, but with numerous homologous R genes in related monocots that are either host or non-hosts for stripe rust. These results represent a unique example of gene evolution and dispersion across species.

Cloning and analysis of a functional promoter of fungal immunomodulatory protein from Flammulina velutipes

Fugal immunomodulatory protein from Flammulina velutipes (FIP-fve) belongs to FIPs family, which has precious pharmaceutical value. To understand the regulatory mechanism of FIP-fve expression, we have cloned a 900 bp genomic DNA fragment from the transcriptional start site of the FIP-fve gene using genomic walker technology. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs in the 900 bp of upstream region of the FIP-fve gene, which contains one putative TATA-boxes, four possible CAAT-boxes, one ABRE, one ARE, three CGTCA-motifs, two TGA-elements and four Skn-1 motifs. The eukaryotic expression vector pfveP:: GUS-GFP was transferred into tobacco via an agrobacterium-mediated leaf disc transformation. The results showed that the FIP-fve promoter could induce the reporter gene GUS or GFP expression in different tissues of tobaccos. This study would lay a foundation for expression regulation of FIP-fve and development of genetic-modified plant products.

Cloning, characterization, and heterologous expression of a dextranase gene from Penicillium pinophilum SMCU3-14

A DNA fragment encoding dextranase was cloned fromPenicillium pinophilum SMCU3-14 using the genome walking approach. Sequence analysis of the gene (SMCU-DEX) revealed a putative CAAT box at 165 (CCAAT), a putative TATA box at 93 (TATAA) in the 5 0 -noncoding region, and a polyadenylation signal (AATAAG) in the 3 0 -noncoding region. A cDNA sequence analysis revealed no evidence of introns. The deduced open reading frame is 1824 bp in length and encodes a predicted protein of 608 amino acids (molecular weight (MW) of 66 kDa), with a putative N-terminal 20-amino acid signal peptide, giving a predicted mature protein of 588 amino acids (MW of 64 kDa) that belongs to glycosyl hydrolase family 49, as with other fungal dextranases. This is the first report of a dextranase gene sequence from P. pinophilum. The cDNA was cloned and expressed in Escherichia coli, and the transformants showed dextranase activity on a dextran-containing agar medium. Crude extracts from the transformants analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis containing blue dextran revealed a distinct specific band of dextranase activity at an MW of approximately 66 kDa. This recombinant dextranase is likely to have valuable and cost-effective applications in medicine and industry.

Epigenetic control of gonadal aromatase (cyp19a1) in temperature-dependent sex determination of red-eared slider turtles.

In the red-eared slider turtle (Trachemys scripta), a species with temperature-dependent sex determination (TSD), the expression of the aromatase gene during gonad development is strictly limited to the female-producing temperature. The underlying mechanism remains unknown. In this study, we identified the upstream 5′-flanking region of the aromatase gene, gonad-specific promoter, and the temperature-dependent DNA methylation signatures during gonad development in the red-eared slider turtle. The 5′-flanking region of the slider aromatase exhibited sequence similarities to the aromatase genes of the American alligator, chicken, quail, and zebra finch. A putative TATA box was located 31 bp upstream of the gonad-specific transcription start site. DNA methylation at the CpG sites between the putative binding sites of the fork head domain factor (FOX) and vertebrate steroidogenic factor 1 (SF1) and adjacent TATA box in the promoter region were significantly lower in embryonic gonads at the female-producing temperature compared the male-producing temperature. A shift from male- to female-, but not from female- to male-, producing temperature changed the level of DNA methylation in gonads. Taken together these results indicate that the temperature, particularly female-producing temperature, allows demethylation at the specific CpG sites of the promoter region which leads the temperature-specific expression of aromatase during gonad development.

Molecular cloning and overexpression of the tyrosine aminotransferase (TAT) gene leads to increased rosmarinic acid yield in Perilla frutescens

Perilla frutescens is a medicinal plant that produces various bioactive compounds, including unsaturated fatty acids and phenolic compounds such as rosmarinic acid (α-O-caffeoyl-3, 4 -dihydroxyphenylacetic acid, RA). Tyrosine aminotransferase (TAT) catalyzes the first step in the tyrosine-derived branch of RA biosynthesis, and TAT is presumed to have a role in RA accumulation. Here, we report the isolation of full-length TAT cDNA (designated PfTAT) from P. frutescens. Sequence analysis revealed that PfTAT contained 1,535 bp long and an open reading frame of 1,233 bp encoding 411 amino acid residues. Analysis of PfTAT genomic DNA revealed 7 exons and 5 introns. The 5′ flanking sequence of PfTAT was also cloned, and a group of putative cis-acting elements such TATA box, CAAT box, TC-rich repeats and G box were identified. Quantitative real-time PCR analysis indicated that constitutive expression of PfTAT in leaves was much higher than in roots and stems. A vector was constructed containing the PfTAT gene derived by the cauliflower mosaic virus 35S promoter. Transgenic P. frutescens overexpressing PfTAT was obtained with an Agrobacterium tumefaciens-mediated transformation system and overexpression was confirmed by PCR and Southern blot. PfTAT mRNA expression in transgenic plant lines measured by real-time quantitative PCR was 2–3 times greater than PfTAT expression in the untransformed plant line. Also, enhanced gene expression corresponded to significantly increased RA in PfTAT-transgenic lines, as quantified by HPLC. Our data emphasize the importance of PfTAT in the production of RA in P. frutescens.

Cloning, characterization and promoter analysis of S-RNase gene promoter from Chinese pear (Pyrus pyrifolia)

The 5′‐flanking region of the S12-, S13-, S21-RNase with a length of 854bp, 1448bp and 1137bp were successfully isolated by TAIL-PCR from genomic DNA from ‘Jinhua’, ‘Maogong’ (Pyrus pyrifolia) and ‘Yali’ (Pyrus bretschneideri) genomic DNA. Sequence alignment and analysis of S13-, S12-, S21-RNase gene promoter sequences with S2-, S3-, S4-, S5-RNase 5′-flanking sequences indicated that a homology region of about 240bp exists in the regions just upstream of the putative TATA boxes of the seven Chinese/Japanese pear S-RNase genes. Phylogenetic tree suggests that the homology region between the Chinese/Japanese pear and apple S-RNase gene promoter regions reflects the divergence of S-RNase gene was formed before the differentiation of subfamilies. Full length and a series of 5′-deletion fragments-GUS fusions were constructed and introduced into Arabidopsis thaliana plants. GUS activity were detected in S12-pro-(1 to 5)-GUS‐pBll01.2 transgenic pistils and progressively decreased from S12-pro-1-GUS-pBI l01.2 to S12-pro-5-GUS-pBll01.2. No GUS activity was detected in S12-pro-6-GUS-pBll01.2 transgenic pistil and other tissues of non-transformants and all transgenic plants. The result suggested S12-RNase promoter is pistil specific expression promoter.

Molecular analysis of a sunflower gene encoding an homologous of the B subunit of a CAAT binding factor

A genomic DNA fragment containing the complete LEAFY COTYLEDON1-LIKE (HaL1L) gene was retrieved by chromosome walking. Its sequence was confirmed and elongated by screening a sunflower genomic DNA BAC Library. HaL1L, whose cDNA had already been sequenced and characterized, encodes a NF-YB subunit of a CCAAT box-binding factor (NF-Y) involved in the early stages of zygotic and somatic embryogenesis in the Helianthus genus. In the HaL1L 5'-flanking region, elements specific to a putative TATA-box promoter and two "CG isles" were identified. An investigation of the methylation status of these CG rich DNA regions showed that differentially methylated cytosines were recognizable in the DNA of embryos on the fifth day after pollination in comparison to leaf DNA suggesting that during plant development epigenetic regulation of HaL1L transcription was achieved by methylating cytosine residues. We also searched the HaL1L nucleotide sequence for cis-regulatory elements able to interact with other transcription factors (TFs) involved in the HaL1L regulation. Of the elements identified, one of the most intriguing is WUSATA, the target sequence for the WUSCHEL (WUS) TF, which may be part of a complex regulation network controlling embryo development. In this article, we show that the WUSATA target site, located in the intron of HaL1L, is able to bind the TF WUS. Interestingly, we found auxin and abscisic acid responsive motifs in the HaL1L promoter region suggesting that this gene may additionally by under hormonal control. Finally, the presence of a cytoplasmic polyadenylation signal downstream to the coding region indicates that this gene may also be controlled at the translation level by a temporarily making the pre-synthesized HaL1L mRNA unavailable for protein synthesis.