Str Promoter Research Articles

Bioinformatic Analysis in Designing Mega-primer in Overlap Extension PCR Cloning (OEPC) Technique

Bioinformatics has developed into an application tool for basic and applied research in the biomedical and biotechnology field. Polymerase Chain Reaction (PCR) is a common technique in the molecular area that has always involved bioinformatics science. PCR cloning techniques such as TA cloning and PCR-mediated cloning exhibit complex processes with low success rates. One easy, effective, and practical solution is to use a mega-primer with the Overlap Extension PCR Cloning (OEPC) technique. The success of PCR cloning using the mega-primer design in the OEPC technique is strongly influenced by the characteristics of the mega-primer used. Knowledge of mega-primer characteristics is one of the important factors in the success of PCR cloning. The design process for the mega-primer str promoter was characterized based on the principle of a genetic algorithm using the web-based bioinformatics tools such as ClustalW, NetPrimer, and BLAST. The success of the mega-primer construction in producing recombinant pSB1C3 vector has been confirmed by the sequencing method and the function of the reporting protein (AmilCP). DNA analysis shows a 100 % homologous sequence on the str promoter, while E. coli colonies successfully express the purplish-blue color. Mega-primer characters can save costs and time of the research by maintaining the primer parameters that provide optimal values and increase the success value of PCR cloning via bioinformatics software. Hence, implications on biological problems, especially using DNA and amino acid sequences, could solve rapidly.

Revisiting the ORCA gene cluster that regulates terpenoid indole alkaloid biosynthesis in Catharanthus roseus

Transcription factor (TF) gene clusters in plants, such as tomato, potato, petunia, tobacco, and almond, have been characterized for their roles in the biosynthesis of diverse array of specialized metabolites. In Catharanthus roseus, three AP2/ERF TFs, ORCA3, ORCA4, and ORCA5, have been shown to be present on the same genomic scaffold, forming a cluster that regulates the biosynthesis of pharmaceutically important terpenoid indole alkaloids (TIAs). Our analysis of the recently updated C. roseus genome sequence revealed that the ORCA cluster comprises two additional AP2/ERFs, the previously characterized ORCA2 and a newly identified member designated as ORCA6. Transcriptomic analysis revealed that the ORCAs are highly expressed in stems, followed by leaves, roots and flowers. Expression of ORCAs was differentially induced in response to methyl-jasmonate and ethylene treatment. In addition, ORCA6 activated the strictosidine synthase (STR) promoter in tobacco cells. Activation of the STR promoter was significantly higher when ORCA2 or ORCA6 was coexpressed with the mitogen-activated protein kinase kinase, CrMPKK1. Furthermore, transient overexpression of ORCA6 in C. roseus flower petals activated TIA pathway gene expression and TIA accumulation. The results described here advance our understanding of regulation of TIA pathway by the ORCA gene cluster and the evolution for plant ERF gene clusters.

Isolation and Characterization of str Promoter from Bacteria Escherichia coli DH5α using Reporter Gene AmilCP (Acropora millepora)

In heat stress conditions, E. coli will produce EF-Tu protein abundantly under the regulation of str promoter. The str promoter from E. coli is known to have sequences that are recognized by two sigma factors (σ70 and σS). The aim of this research was to isolate and characterize str promoter from E. coli DH5a using reporter gene AmilCP. The str promoter from E. coli DH5α (289 bp) had been isolated by Colony PCR using specific primers. Bioinformatics analysis of str promoter showed high conserved areas (100%) for some kinds of E. coli strains. The str promoter was constructed in pSB1C3 vector by modifying OEPC to regulate reporter gene AmilCP. Qualitatively, the ability of str promoter constructs in regulating AmilCP gene was observed as purple-blue colonies of E. coli. Quantitative analysis was performed by measuring cell extract of E. coli at a wavelength of 588 nm after treated at different temperatures (4, 24, 37, 45, 50, 55 and 60 °C) for 1 hour. The ability of str promo ter to express reporter gene AmilCP was compared to one powerful promoter which is the lacUV5 promoter. The results show that both promoters are functional promoters and can be induced by temperature (50 °C).

Zinc Finger Proteins Act as Transcriptional Repressors of Alkaloid Biosynthesis Genes in Catharanthus roseus

In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus.

Catharanthus roseus G-box binding factors 1 and 2 act as repressors of strictosidine synthase gene expression in cell cultures.

The enzyme encoded by the strictosidine synthase (Str) gene catalyses a key step in the biosynthesis of therapeutically valuable terpenoid indole alkaloids. In Catharanthus roseus the Str gene was shown to be regulated by a wide variety of signals including auxin, methyl jasmonate and fungal elicitors in cell suspension cultures and by tissue-specific control in plant organs. The Str promoter contains a functional G-box (CACGTG) cis-regulatory sequence. In order to understand better the mechanisms involved in the regulation of Str gene expression, we isolated the C. roseus cDNAs encoding G-box binding factors Crgbf1 and Crgbf2. The binding specificity of their protein products CrGBF1 and CrGBF2 was analysed by competitive electrophoresis mobility and saturation binding assays. CrGBF1 had a high binding specificity for class I G-boxes including the Str G-box. CrGBF1 showed a lower affinity for class II G-boxes and for the G-box-like element (AACGTG) found in the tryptophan decarboxylase (Tdc) gene which encodes another enzyme involved in TIA biosynthesis. CrGBF2 showed a high affinity for all types of G-boxes tested and to a lesser extent for the Tdc G-box-like element. Transient bombardment experiments demonstrated that both CrGBF1 and CrGBF2 can act in vivo as transcriptional repressors of the Str promoter via direct interaction with the G-box. These data indicate that GBFs may play functional role in the regulation of expression of the terpenoid indole alkaloid biosynthetic gene Str.

A Catharanthus roseus BPF-1 homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic gene Str and is induced by elicitor via a JA-independent signal transduction pathway.

Plants respond to pathogen attack by induction of various defence responses, including the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the elicitor-induced expression of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str) is mediated via the plant stress hormonejasmonate. In the promoters of several defence-related genes, cis-acting elements have been identified that are important for transcriptional regulation upon stress signals. Here we show that an upstream region in the Str promoter confers responsiveness to partially purified yeast elicitor and jasmonate. Yeast one-hybrid screening with this element as a bait identified a MYB-like protein, which shows high homology to parsley box P-binding factor-1 (PcBPF-1). In vitro analyses showed that the Str promoter fragment contained a novel binding site for BPF-1-like proteins with higher binding affinity than the previously described box P. CrBPF-1 mRNA accumulated rapidly in elicitor-treated C. roseus suspension cells, whereas no induction was observed with jasmonate. Inhibitor studies indicated that CrBPF-1 plays a role in an elicitor-responsive but jasmonate-independent signal transduction pathway, acting downstream of protein phosphorylation and calcium influx.

A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2.

Jasmonate (JA) is an important plant stress hormone that induces various plant defense responses, including the biosynthesis of protective secondary metabolites. The induction of the secondary metabolite biosynthetic gene Strictosidine synthase (Str) in Catharanthus roseus (periwinkle) cells by elicitor requires JA as a second messenger. A 42 bp region in the Str promoter is both necessary and sufficient for JA- and elicitor-responsive expression. This region is unlike other previously identified JA-responsive regions, and contains a GCC-box-like element. Yeast one-hybrid screening identified cDNAs encoding two AP2-domain proteins. These octadecanoid-derivative responsive Catharanthus AP2-domain (ORCA) proteins bind in a sequence-specific manner the JA- and elicitor-responsive element. ORCA2 trans-activates the Str promoter and its expression is rapidly inducible with JA and elicitor, whereas Orca1 is expressed constitutively. The results indicate that a GCC-box-like element and ORCA2 play key roles in JA- and elicitor-responsive expression of the terpenoid indole alkaloid biosynthetic gene Str.

A G-box element from the Catharanthus roseus strictosidine synthase (Str) gene promoter confers seed-specific expression in transgenic tobacco plants.

The enzyme encoded by the strictosidine synthase (Str) gene from Catharanthus roseus catalyses a key step in the biosynthesis of the pharmaceutically important terpenoid indole alkaloids. Str cDNA and genomic clones have already been isolated, allowing us to study the regulation of Str gene expression. Here we focus on the role of a putative cis-acting element, CACGTG, in the Str promoter. This sequence is known as a G-box, and functions as a transcription-regulating sequence in a number of other promoters. By means of electrophoretic mobility shift assays it was demonstrated that the Str G-box is capable of interacting with nuclear factors in tobacco and with the cloned tobacco G-box-binding factor TAF-1. Disruption of the Str G-box sequence by two single-nucleotide mutations prevented binding of factors, thereby demonstrating the specificity of the observed interactions. Functional analysis in transgenic tobacco plants demonstrated that these mutations also reduced the transcriptional activity of constructs containing tetramers of the Str G-box sequence. Expression directed by a tetramer of the Str G-box fused to a truncated promoter containing only a TATA box was confined to seeds and was found to increase during seed maturation. Thus, the Str G-box tetramer is able to direct seed-specific expression independently of other regulatory sequences. G-box-directed expression in leaves required the presence of an enhancer region from the cauliflower mosaic virus (CaMV) 35S promoter. The results indicate that the G-box needs to interact with other elements to drive expression in leaf, and that it can by itself confer seed-specific expression as a multimer. The fact that only some of the G-boxes found in different promoters serve as seed-specific elements indicates that sequences flanking the G-box determine the transcriptional activity in different tissues. Based on sequence comparisons we propose that the nucleotides at positions -4, -3, -2 and/or +4 are important in determining seed-specific expression.

The human stromelysin promoter contains a previously unreported 1.0-kb sequence

Cloning and characterization of the promoter region controlling the gene encoding human stromelysin (Str) has been previously reported [Quinones et al J. Biol. Chem. 264 (1989) 8339–8344]. We have characterized independently isolated genomic clones of the STR promoter, designated pSKStrB and 682, that are considerably different from the published sequence. Although the sequences up to an XbaI site at −480 of the 5' regions are identical, a novel 1.0-kb segment exists upstream from − 480. This sequence is absent from the published clone, but its presence in the genomic DNA from twelve individuals has been confirmed by both PCR analysis and restriction mapping. Upstream of the novel 1-kb segment, the sequence of the published clone reappears, but in pSKStrB exists in inverse orientation.

Micrococcus luteus, a bacterium with a high genomic G+C content, contains Escherichia coli-type promoters

The G + C content of Micrococcus luteus DNA is 74%, which is much higher than that of Escherichia coli (about 50%). In order to understand the influence of GC-directed mutation pressure (GC pressure) on the promoter structure, the initiation sites for both in vitro and in vivo transcription of M. luteus streptomycin (str) and spectinomycin (spc) operons were identified by the reverse transcriptase mapping method. The promoter sequences of M. luteus are similar to those of E. coli, but have significantly higher G + C contents. In an in vitro run-off transcription assay using truncated DNA templates, RNA polymerases from both M. luteus and E. coli were able to transcribe correctly from both the str promoter and the spc major promoter of M. luteus.

Regulation of the synthesis of E. coli elongation factor TU

Rapidly growing E. coli with two active genes (tufA, tufB) for elongation factor (EF) Tu contains three times as much EF-Tu (tuf) mRNA as EF-G (fus) mRNA on a molar basis, but about seven times as much EF-Tu as EF-G or ribosomes. The concentration and translational efficiency of fus (EF-G) mRNA is about that for ribosomal protein mRNAs. The high molar concentration of EF-Tu relative to EF-G or ribosomes is achieved in part by translating tuf mRNA more efficiently than these other mRNAs. In a tufA + tufB -::Mu strain, the tuf:fus mRNA ratio is 1, but the concentration of EF-Tu and tuf mRNA is the same as in the wild-type strain. Thus cells with only an active tufA gene increase the concentration but not the translational efficiency of tuf mRNA. In such cells the concentration of fus mRNA is almost three times that in the wild-type strain. Because the tufA gene is distal to but cotranscribed with the fus gene as part of the four gene str operon, the wildtype concentration of tuf mRNA in these tufB - cells must be produced by increasing the concentration of transcript corresponding to the entire str operon. Thus transcription of the tufA gene can only proceed from the str promoter. Extracts of the tufBcells contain tuf transcripts that correspond not only in size to the entire 4.5 kb str operon, but also to the size (≈1 kb) of a tuf gene. Our evidence suggests that this 1 kb tuf transcript is derived by posttranscriptional modification of the primary str operon transcript and that this modification could in part explain the high translational efficiency of tuf mRNA.

Isolation and characterization of stable hybrid mRNA molecules transcribed from ribosomal protein promoters in E. coli

The promoters from the str and spc operons of ribosomal proteins from E. coli were inserted into the Hind II cleavage site of mini-Col E1 (pVH51) plasmid. For both promoters, strains with the hybrid plasmid accumulated a small RNA species not present in strains carrying the vector. These RNAs were analyzed by RNA sequencing techniques and compared to DNA sequences. In both cases, synthesis of the new RNA species is initiated by the cloned r protein promoter at the site predicted by previous in vitro experiments. The RNAs extend across the Hind II site used for cloning and terminate specifically in the vector sequences. The termination site was localized to six consecutive thymine nucleotides preceded by a sequence with dyad symmetry. We found that the RNA from the str promoter was 205 (± 3) nucleotides long and that from the spc promoter was 177 (±3) nucleotides long. These “hybrid mRNAs” are much more stable than ordinary mRNA. The str hybrid mRNA has a half-life of about 8 min, and the spc hybrid mRNA has a half-life of about 18 min at 37°C. These hybrid mRNAs provide an in vivo system with which to examine directly the discrete transcription products from ribosomal protein promoters, and to study promoter function and mRNA metabolism in vivo.

DNA sequences of promoter regions for the str and spc ribosomal protein operons in E. coli

The DNA sequences have been determined for promoter regions of two ribosomal protein operons in E. coli, the str operon and the spc operon. The site of in vitro transcription initiation within each of these promoter regions has been determined. The start site of the str operon occurs 69 bases upstream from the initiation codon of the S12 gene. The start site of the spc operon occurs 72 bases upstream from the L14 gene, and only 91 bases downstream from the termination codon of the S17 gene (which is in the preceding S10 operon). Both promoters are similar to other sequenced promoters in that they each have an identifiable “Pribnow box” sequence 5 bases upstream from the transcription start site. The spc promoter has a long sequence of 2 fold symmetry centered within the Pribnow box; the str promoter has a shorter but similar symmetry. At positions −69 through −40 in the spc operon, another long region of symmetry is present which may be the termination signal of the preceding S10 operon. Extensive sequence similarity between the str and spc promoter regions is found downstream from the Pribnow box-that is, in a transcribed region preceding the translation start sites.

Isolation and characterization of a promoter mutant in the str ribosomal protein operon in E. coli

The λ fus3 transducing phage carries several operons for ribosomal proteins of E. coli, including the str operon. A mutant transducing phage with a promoter mutation in this operon has been isolated. This mutant shows reduced stimulation of synthesis of proteins encoded by the operon, S12, S7, and elongation factors G and Tu, in ultraviolet-irradiated cells. This mutation also abolishes in vitro transcription from the str promoter. The DNA sequence of the mutant promoter shows that it is a point mutation 6 bases upstream from the in vitro transcription start site, changing the “Pribnow box” sequence from TAAAATT to TAAAACT. These results indicate that the site altered by the mutation, which is in the region just preceding the transcription start site, is important for the expression of the str operon.