Promoter Analysis

Mosquitoes that bite humans are vectors for a number of infectious diseases affecting millions of people per year. Insect repellents help to prevent and control the outbreak of insect-borne diseases such as malaria, Lyme disease, etc. The citronella oil has the repellency activity against Aedes aegypti mosquitoes. Maximum oil (4.68g) was extracted from 400g of citronella plant using organic solvent extraction method. The composition of the oil was analyzed by Gas Chromatography. The extracted oil was microencapsulated (1.5% gelatin and 1.5% Arabic gum) by complex coacervation method. This citronella oil was treated on cotton fabrics using gelatin and gum acacia microcapsules, by pad dry method. The effect of the repellents on cotton fabrics with 15 %, 30% and 50% against Aedes aegypti was studied by cage and field test with human arms covered with treated and untreated fabrics (at 26±2°C and 80±5 % RH). This same experiment was repeated with once and twice washed microencapsulated citronella oil treated fabric (MCF) and citronella oil treated fabric (CF). In this experiment 50% concentrated repellents give the best mosquito repellency than the other two in both MCF and CF. Despite the microencapsulated oil treated fabric shown best repellency effect than citronella oil treated fabric because of controlled release characteristics of cross linked natural polymers.

We have previously reported the isolation and characterization of a gene (Zm 13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm 13 5′ flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5′ regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the β-glucuronidase (GUS) gene under the control of various sized fragments of the Zm13 5′ flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from −100 to −54, while other sequences which amplify that expression reside between −260 and −100. The replacement of the normal terminator with a portion of the Zm13 3′ region containing the putative polyadenylation signal and site also increased GUS expression. While the −260 to −100 region contains sequences similar to other protein-binding domains reported for plants, the −100 to −54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression.

Coenzyme Q10 (CoQ10) is a quinine consisting of ten units of the isoprenoid side-chain. Because it limits the oxidative attack of free radicals to DNA and lipids, CoQ10 has been used as an antioxidant for foods, cosmetics and pharmaceuticals. Decaprenyl diphosphate synthase (DPS) is the key enzyme for synthesis of the decaprenyl tail in CoQ10 with isopentenyl diphosphate. The ddsA gene coding for DPS from Gluconobacter suboxydans was expressed under the control of an Escherichia coli constitutive promoter. Analysis of the cell extract in recombinant E. coli BL21/pACDdsA by high performance liquid chromatography and mass spectrometry showed that CoQ10 rather than endogenous CoQ8 was biologically synthesized as the major coenzyme Q. Expression of the ddsA gene with low copy number led to the accumulation of CoQ10 to 0.97 mg l−1 in batch fermentation. A high cell density (103 g l−1) in fed-batch fermentation of E. coli BL21/pACDdsA increased the CoQ10 concentration to 25.5 mg l −1 and its productivity to 0.67 mg l−1 h−1, which were 26.0 and 6.9 times higher than the corresponding values for batch fermentation.

A binary vector containing two reporter gene cassettes has been developed. This vector is ideal for optimising new plant transformation systems. Following optimisation, one of the reporter genes can be replaced with a gene of interest; the second can be used as a marker to confirm transgenic lines, and to estimate locus number and determine zygosity. This allows simple, efficient and economical screening for homozygous single-insert lines and azygous controls.

The presence of the endochitinase CHIT36 from Trichoderma harzianum TM was assessed in several antagonistic Trichoderma strains belonging to different molecular taxonomic groups. CHIT37 from T. harzianum CECT 2413 was sequenced and found to display 89% homology with CHIT36 at the amino acid level. Northern analysis showed that chit36Y from T. asperellum is regulated both by glucose and nitrogen levels. Stress conditions, colloidal chitin and N-acetyl-glucosamine are effective inducers of this gene. The promoter of chit36Y was cloned and was used to direct expression of a gfp reporter gene in Trichoderma transformants. Confrontation experiments with the plant pathogen Rhizoctonia solani revealed that direct contact between the fungi is not necessary for gfp expression. The R. solani-inducing factor appears to be a soluble molecule capable of diffusing through a dialysis membrane (Pichia pastoris was active against different phytopathogens, confirming the importance of this endochitinase in the mycoparasitic activity of Trichoderma antagonistic strains.

A zero erucic acid (C22:1) line of Brassica juncea (VH486), adapted to the agronomic conditions of Northern India, has been modified for its fatty acid composition in the seed oil with antisense constructs using the sequence of fad2 gene of B. rapa. The full-length B. rapa fad2 cDNA sequence was determined by 5’ and 3’ RACE of a partial sequence available in the EST database. Construct pASfad2.1 contained 315 to 1251 bp and construct pASfad2.2 contained 1 to 1251 bp fragment of the fad2 gene, both in antisense orientation, driven by a truncated napin promoter. Analysis of the levels of linoleic acid (C18:2) in the BC1 seeds of single-copy transgenics showed that the construct pASfad2.2 gave better suppression of the fad2 gene as compared to the construct pASfad2.1. The BC1 transgenic seeds containing the pASfad2.2 construct segregated into two distinct classes of C18:2>20% (putative null homozygotes) and C18:2<20% (putative heterozygotes) in a 1:1 ratio, while the T1 seeds segregated into three classes, C18:2>20%, C18:2 between 12% and 20%) and C18:2<12% (putative homozygotes) in a 1:2:1 ratio. Putative homozygous T1 seeds (C18:2<12% analyzed by the half-seed method) of four of the transgenic lines were grown to establish T2 homozygous lines. These had ca. 73% C18:1 and 8 to 9% each of C18:2 and C18:3 (α-linolenic acid) fractions in comparison to ca. 53% C18:1, 24% C18:2 and 16% C18:3 in the parental line VH486.

We dissected the regulatory region of the AVP1 gene encoding the vacuolar H+-pyrophosphatase (V-PPase) of Arabidopsis thaliana by using a GUS-reporter assay system. The cloned 1.4 kb 5&#39;-regulatory region in the GUS-reporter transgenic plants was sufficient for the light-induced repression. Furthermore, the 1.4 kb regulatory region was active in all tissues examined and its activity was especially enhanced in pollen, whereas the shorter 0.4 kb regulatory region was active only in pollen. Further detailed analyses revealed that the GUS activity in pollen was regulated by at least three cis-acting regions in an additive or synergetic manner. These findings establish a distinct mechanism of the tissue-specific regulation of V-PPase expression in developing pollen. and imply the biological significance of the V-PPase in pollen maturation.

The availability of sequence information for thousands of genes for many organisms is currently unmatched by functional studies. A cost-effective and high-throughput cloning system for PCR products was therefore adopted to enable the rapid assessment of coding and promoter sequences in functional assays in transgenic cells. Unlike other systems that involve initial cloning into a specialized PCR fragment cloning vector, this method describes a rapid and cost-effective procedure for the amplification of a DNA fragment by PCR, its phosphorylation and its direct insertion into the vector of choice. Restriction enzymes are only required once for the preparation of the recipient vector, which is blunt-ended and dephosphorylated. No special primer designs (e.g. restriction enzyme sites or flanking homologous sequences) or subcloning steps are required. The turn-around time from source organism genomic DNA to new recombinant DNA is 24 hrs. It is particularly suitable for functional genomics projects or the generation of libraries from PCR products where a large number of fragments need to be cloned into the same vector. We have used this method to rapidly clone 72 full-length genes (ranging from 0.8 to 6.4 kb) and putative promoters (2 kb each) from Arabidopsis thaliana into plant cell expression cassettes for subsequent direct functional analyses in transgenic cells.

The activating transcription factor 1 (AP-1) family of proteins consists of a large number of inducible factors that are implicated in many biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. Here, we investigated the role of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the JC virus (JCV) promoter in glial cells. DNA binding studies demonstrated the specific association of c-Jun with its DNA sequences corresponding to the AP-1 site within the JCV promoter. Functional analysis of the promoter showed that ectopic expression of c-Jun and c-Fos results in an additive activation of the JCV early and late promoters. Further functional assays indicated that the JCV AP-1 binding site is sufficient to confer responsiveness to both c-Jun/c-Fos- and UV-induced activation when transposed to a heterologous promoter. Analysis of c-Jun expression during the viral infection cycle by Western blotting ...

Industrial strains of Penicillium chrysogenum possess many genomic changes leading to higher levels of penicillin. In this work several production and wild-type strains of Penicillium chrysogenum were used in comparative nucleotide sequence analysis of the biosynthesis cluster. The alignments confirmed sequence conservation not only in promoter regions of the biosynthesis genes but also throughout the entire 44.7-kbp genomic fragment comprising the whole biosynthesis cluster with 15.5-kbp and 13.1-kbp flanking regions. As another titre-enhancing mechanism we subsequently examined gene dosage in two production strains introduced here, NMU2/40 and B14. Quantitative real-time PCR and Southern blot analysis showed the amplification of the biosynthesis genes in both these strains. Through the real-time PCR method the exact copy number was estimated for each of the pcbAB, pcbC and penDE genes. The equal pool of all three genes per genome was confirmed for the both production strains indicating that in these strains the entire penicillin cluster has been amplified as an intact element. Penicillium chrysogenum NMU2/40 was found to carry four copies of the cluster, while six copies were estimated for B14. This also proves the contribution of the additional titre-enhancing mechanisms in both strains, since the industrial data referred much higher production of these strains compared with the single copy reference strain NRRL 1951.

To examine the genetic controls of endosperm (ES) specificity, several cereal seed storage protein (SSP) promoters were isolated and studied using a transient expression analysis system. An oat globulin promoter (AsGlo1) capable of driving strong ES-specific expression in barley and wheat was identified. Progressive 5′ deletions and cis element mutations demonstrated that the mechanism of specificity in the AsGlo1 promoter was distinct from that observed in glutelin and prolamin promoters. A novel interrupted palindromic sequence, ACATGTCATCATGT, was required for ES specificity and substantially contributed to expression strength of the␣AsGlo1 promoter. This sequence was termed the endosperm specificity palindrome (ESP) element. The GCN4 element, which has previously been shown to be required for ES specificity in cereal SSP promoters, had a quantitative role but was not required for tissue specificity. The 960-bp AsGlo1 promoter and a 251-bp deletion containing the ESP element also drove ES-specific expression in stably transformed barley. Reporter gene protein accumulated at very high levels (10% of total soluble protein) in ES tissues of plants transformed with an AsGlo1:GFP construct. Expression strength and tissue specificity were maintained over five transgenic generations. These attributes make the AsGlo1 promoter an ideal promoter for biotechnology applications. In conjunction with previous findings, our data demonstrate that there is more than one genetically distinct mechanism by which ES specificity can be achieved in cereal SSP promoters, and also suggest that there is redundancy between transcriptional and post-transcriptional tissue specificity mechanisms in cereal globulin genes.

Two highly similar genes encoding unique extracellular, glycosylated glutathione S-transferases (GSTs) of the human-pathogenic nematode, Onchocerca volvulus (Ov-GST1a and Ov-GST1b), have been isolated and characterised. The genes are ≈3 kb in length and consist of seven exons interrupted by introns of ≈100 bp in length, with the exception of intron II, which is ≈1.6 kb in length. Interestingly, exon I and II encode a signal peptide and an N-terminal extension before sequence homology to other GSTs begins. The 5′ flanking region was sequenced and analysed for transcription factor binding sites. Consistent with the lack of a TATA box, analysis of the mRNAs by primer extension showed multiple transcription start sites spread over a 60 bp region. To examine the activity and specificity of the Ov-GST1a gene promoter, we have exploited Caenorhabditis elegans as a heterologous transformation system. To analyse whether transgenic C. elegans are able to carry out processing and post-transcriptional modifications of the Ov-GST1a correctly, the protein was ectopically overexpressed in C. elegans. The parasite-derived Ov-GST1a gene product was correctly processed in transgenic C. elegans and posttranslational modifications, such as signal peptide cleavage and N-glycosylation, were performed successfully. This further demonstrates the potential of C. elegans as a host for expression of candidate vaccine antigens from O. volvulus and affirms the role of C. elegans as a model for parasitic nematodes.

Coenzyme Q10 (CoQ10) is a quinine consisting of ten units of the isoprenoid side-chain. Because it limits the oxidative attack of free radicals to DNA and lipids, CoQ10 has been used as an antioxidant for foods, cosmetics and pharmaceuticals. Decaprenyl diphosphate synthase (DPS) is the key enzyme for synthesis of the decaprenyl tail in CoQ10 with isopentenyl diphosphate. The ddsA gene coding for DPS from Gluconobacter suboxydans was expressed under the control of an Escherichia coli constitutive promoter. Analysis of the cell extract in recombinant E. coli BL21/pACDdsA by high performance liquid chromatography and mass spectrometry showed that CoQ10 rather than endogenous CoQ8 was biologically synthesized as the major coenzyme Q. Expression of the ddsA gene with low copy number led to the accumulation of CoQ10 to 0.97 mg l−1 in batch fermentation. A high cell density (103 g l−1) in fed-batch fermentation of E. coli BL21/pACDdsA increased the CoQ10 concentration to 25.5 mg l −1 and its productivity to 0.67 mg l−1 h−1, which were 26.0 and 6.9 times higher than the corresponding values for batch fermentation.

Starch, the main component of the wheat grain, is the product of a complex biochemical pathway. The sbeIIa gene plays a key role in controlling the synthesis of starch, in particular, the biosynthesis of amylopectin, in maturing wheat grain. To investigate its regulatory mechanisms and endosperm-specific expression pattern, the sbeIIa promoter (3094 bp in length) was cloned using APCR and sequenced. The effect of a series of deletions was studied using a GUS transient assay system. Results showed that the 3094 bp sequence (sbe.g construct) exhibited full stable promoting activity and that the activities of 5′ or 3′ deletions reduced levels of GUS expression. Some constructs with internal deletions showed only weak activity, however, sbe.e, with a deletion from −1579 – −1210 bp resulted in higher levels of expression than the full-length promoter sequence, sbe.g. This indicates that motifs such as the −300 bp element, G-box and/or P-box act as positive elements and are necessary in determining the promoter’s endosperm-specific pattern and that negative repressor elements or motifs may also be present within the −1579 – −1210 bp sequence. The age of wheat endosperm tissue used in the GUS-transient assay system is shown to be of significant importance.