Affinity comparison of different THCA synthase to CBGA using modeling computational approaches

The Δ^9-Tetrahydrocannabinol (THCA) is the primary psychoactive compound of Cannabis Sativa. It is produced by Δ^1- Tetrahydrocannabinolic acid synthase (THCA) which catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) the precursor of the THCA. In this study, we were interested by the three dimensional structure of THCA synthase protein. Generation of models were done by MODELLER v9.11 and homology modeling with Δ1-tetrahydrocannabinolic acid (THCA) synthase X ray structure (PDB code 3VTE) on the basis of sequences retrieved from GenBank. Procheck, Errat, and Verify 3D tools were used to verify the reliability of the six 3D models obtained, the overall quality factor and the Prosa Z-score were also used to check the quality of the six modeled proteins. The RMSDs for C-alpha atoms, main-chain atoms, side-chain atoms and all atoms between the modeled structures and the corresponding template ranged between 0.290 Å-1.252 Å, reflecting the good quality of the obtained models. Our study of the CBGA-THCA synthase docking demonstrated that the active site pocket was successfully recognized using computational approach. The interaction energy of CBGA computed in ‘fiber types’ proteins ranged between -4.1 95 kcal/mol and -5.95 kcal/mol whereas in the ‘drug type’ was about -7.02 kcal/mol to -7.16 kcal/mol, which maybe indicate the important role played by the interaction energy of CBGA in the determination of the THCA level in Cannabis Sativa L. varieties. Finally, we have proposed an experimental design in order to explore the binding energy source of ligand-enzyme in Cannabis Sativa and the production level of the THCA in the absence of any information regarding the correlation between the enzyme affinity and THCA level production. This report opens the doors to more studies predicting the binding site pocket with accuracy from the perspective of the protein affinity and THCA level produced in Cannabis Sativa.     

甜菜ATP合酶β亚基基因atpB的克隆、序列分析及进化

atpB基因编码ATP合酶β亚基,是光合作用中的重要基因。ATP合酶是生物体内能量代谢的关键酶,参与氧化磷酸化和光合磷酸化反应。利用植物叶绿体基因组在进化过程中高度保守的特点,根据已知植物烟草、水稻和菠菜等的叶绿体基因组全序列,设计并合成了一对引物,以甜菜叶绿体DNA为模板,PCR扩增得到包含atpB 完整基因（GenBank登录号为 DQ067451）在内的一段序列,测序与序列分析表明：该克隆片段全长2 293 bp,其中包括有1 497 bp的编码区序列,推测编码498个氨基酸。同源性比较,该克隆基因与烟草、菠菜、油菜、水稻atpB基因的核苷酸序列同源性分别为90.92%、95.79%、87.71%和86.37%,推测的氨基酸序列同源性分别为94.58%、97.19%、92.17%和91.97%。同时,建立了几种植物的氨基酸序列系统进化树。     

A new 2CTAB/PCI method improves DNA amplification success from faeces of Mediterranean (Barbary macaques) and tropical (lowland gorillas) primates

Animal genomic DNA extracts of sufficient quality to address questions about population biology or behavioural ecology can be obtained from faeces when adequate extraction procedures are used. The presence of PCR inhibitors in extract products appears generally the main factor limiting DNA amplification success. We compared DNA amplification success from faeces of a tropical primate (western lowland gorilla, Gorilla g. gorilla) and a Mediterranean primate (Barbary macaque, Macaca sylvanus) between a standardized extraction technique widely used in animals (QIAamp^® stool kit), a technique mainly used in plant species (CTAB) and a new protocol (2CTAB/PCI). Amplification success varied from 51% to 97%, the highest success being reached with the 2CTAB/PCI protocol in both species.     

Simple and efficient genomic DNA extraction protocol for molecular characterisation of Phytophthora colocasiae causing taro leaf blight

Genomic DNA extraction protocol with relatively high quantity and purity is prerequisite for the successful molecular identification and characterisation of plant pathogens. Conventional DNA extraction methods are often time-consuming and yield only very poor quantity of genomic DNA for samples with higher mycelial age. In our laboratory, we have aimed at establishing an efficient DNA isolation procedure, exclusively for the oomycete pathogen Phytophthora colocasiae causing serious leaf blight disease in taro. For this a phenol free protocol was adopted, which involves SDS/Proteinase K-based inactivation of protein contaminants, extraction of nucleic acids using chloroform: isoamyl alcohol and later precipitation of genomic DNA using isopropanol and sodium acetate. The purity of the isolated DNA was analysed by A_260/280 and A_260/230 spectrophotometric readings and confirmed by restriction digestion with restriction enzyme Eco RI. In this study, a comparative assessment was done with CTAB method and the commercial genomic DNA purification kit (Thermo Fisher Scientific, Fermentas, EU). The extracted DNA was found to be suitable for further downstream applications like ITS amplification of the rDNA ITS region and PCR amplification with species-specific primers.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp^® DNA Stool Mini Kit.     

Cannabinoids act as necrosis-inducing factors in Cannabis sativa

Cannabis sativa is well known to produce unique secondary metabolites called cannabinoids. We recently discovered that Cannabis leaves induce cell death by secreting tetrahydrocannabinolic acid (THCA) into leaf tissues. Examinations using isolated Cannabis mitochondria demonstrated that THCA causes mitochondrial permeability transition (MPT) though opening of MPT pores, resulting in mitochondrial dysfunction (the important feature of necrosis). Although Ca²⁺ is known to cause opening of animal MPT pores, THCA directly opened Cannabis MPT pores in the absence of Ca²⁺. Based on these results, we conclude that THCA has the ability to induce necrosis though MPT in Cannabis leaves, independently of Ca²⁺. We confirmed that other cannabinoids (cannabidiolic acid and cannabigerolic acid) also have MPT-inducing activity similar to that of THCA. Moreover, mitochondria of plants which do not produce cannabinoids were shown to induce MPT by THCA treatment, thus suggesting that many higher plants may have systems to cause THCA-dependent necrosis.Key words: cannabinoid, Cannabis sativa, cylophilin D, mitochondrial permeability transition, necrosisCannabis sativa produces unique secondary metabolites consisting of alkylresorcinol and monoterpene groups.¹ These metabolites called cannabinoids are well known to show a variety of interesting pharmacological activities including psychoactive effect and analgesic effect. Therefore, cannabinoids have attracted a great deal of attention, whereas why C. sativa produces such metabolites has long remained unclear. However, we have recently obtained evidences indicating the physiological function of THCA in Cannabis leaves.²We discovered that THCA is stored in capitate-sessile glands on Cannabis leaves and that secretion of this cannabinoid into leaf tissues causes cell death. When the properties of THCA were examined using cultured Cannabis cells, this cannabinoid induced plasmamembrane shrinkage and DNA degradation. These responses are regarded as the features of apoptotic cells, but were not suppressed by apoptosis inhibitors. In contrast, the necrosis inhibitor cyclosporine A significantly inhibited both plasmamembrane shrinkage and DNA degradation in Cannabis cells. Therefore, we assumed that THCA induces necrotic cell death in Cannabis cells and leaves.Necrosis in plants and animals is usually triggered by MPT though opening of MPT pores.^3,4 MPT is known to cause mitochondrial dysfunction by mitochondrial swelling and loss of mitochondrial membrane potential (ΔΨm),^5,6 and we also confirmed that THCA induces mitochondrial swelling and ΔΨm reduction in mitochondria isolated from Cannabis cells and that pretreatment with cyclosporine A inhibits both responses. Based on these evidences, we concluded that THCA has the activity to induce MPT-dependent necrosis.As described above, MPT pores play an important role in necrosis induction, whereas the mechanism of their opening in higher plants has not been fully understood. However, binding of cyclophilin D (a protein present in mitochondrial matrix) to MPT pores is shown to be essential for their opening in plants as well as animal.^7–9 In animal mitochondria, Ca²⁺ mediates this binding reaction, leading to opening of MPT pores. Wheat mitochondria are also shown to undergo swelling through opening of MPT pores in response to Ca²⁺,⁹ whereas MPT pores of oats,¹⁰ Arabidopsis thaliana¹¹ and C. sativa² do not open by Ca²⁺ treatment. In contrast, THCA catalyzed opening of Cannabis MPT pores in the absence of Ca²⁺, suggesting that THCA directly mediates binding of cyclophilin D to MPT pores (Fig. 1). In addition, we have now confirmed that THCA causes dysfunction though MPT in mitochondria of plants (rice, soybean, A. thaliana and Scutellaria baicalensis) lacking cannabinoid-producing ability (data not shown). Therefore, many higher plants may have the systems to induce THCA-dependent necrosis.Open in a separate windowFigure 1A model depicting the opening mechanism of MPT pores in mitochondria. CYD, cyclophilin D; CN, cannabinoid.Furthermore, we investigated whether other cannabinoids and their related compounds can mediate MPT in Cannabis mitochondria. When the MPT-inducing activity of each sample was measured by monitoring both ΔΨm reduction (Fig. 2) and mitochondrial swelling (data not shown), we confirmed that cannabinoids tested here (cannabidiolic acid and cannabigerolic acid) possess the activities similar to those of THCA. On the other hand, olivetolic acid (the akylresorcinol moiety of cannabinoid) and geraniol (the monoterpene moiety of cannabigerolic acid) showed neither ΔΨm reduction nor mitochondrial swelling (Fig. 2). These results suggested that the structures (cannabinoid skeleton) where monoterpene and olivetolic acid are coupled to each other seem essential for opening of MPT pores. Therefore, we assumed that plant cyclophilin D and MPT pores have the cannabinoid-binding site.Open in a separate windowFigure 2Change of ΔΨm by treatment with various compounds (A) and their chemical structures (B). The isolated mitochondria were stained with the ΔΨm-indicating reagent (tetramethylrhodamine methylester, TMRM) and then incubated with 200 µM of each compound for 60 min. The intensity of TMRM fluorescence was measured using a fluorescence microplate reader. A decrease of the fluorescence intensity indicates ΔΨm reduction. CBDA, cannabidiolic acid; CBGA, cannabigerolic acid; OLA, olivetolic acid.Plant cell death is shown to participate in important physiological responses such as leaf senescence, somatic embryogenesis and defense against microbial pathogens.^12,13 Based on its induction mechanism, plant cell death is largely classified into apoptosis and necrosis. Although the molecular mechanism of apoptosis has been extensively investigated, there is little precise information on plant necrosis. However, our study would provide important insight into necrosis-inducing mechanisms in higher plants.     

Single Fish Egg DNA Extraction for PCR Amplification

Modern stock researches on marine biomass are basically genetic and rely increasingly on PCR-based manipulations of informative DNA markers for detecting the genetic diversity. This study developed a simple and rapid single tube method for DNA extraction from a single fish egg. The 15 min protocol was based on the use of Chelex 100 resin and urea to breakdown membrane and connective tissue of eggs. From various sizes of a single egg of walleye pollack (Theragra chalcogramma), the amounts of total nucleic acids were reproducibly obtained to be 18.25 ± 1.92 μg per egg. Using DNA templates diluted ranging 1/10⁰–1/10⁵, PCR amplification for the mitochondrial cytochrome b (Cytb) gene was successfully performed, and the 1/10² diluted template yielded the best result in PCR amplification for three different DNA marker genes. This method is quite simple and economical, and enables to provide the high throughput often demanded by the stock identification of marine biomass, in which large numbers of specimens of single fish eggs must be analyzed.     

Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle amplification

Many techniques in molecular biology require the use of pure nucleic acids in general and circular DNA (plasmid or mitochondrial) in particular. We have developed a method to separate these circular molecules from a mixture containing different species of nucleic acids using rolling circle amplification (RCA). RCA of plasmid or genomic DNA using random hexamers and bacteriophage Phi29 DNA polymerase has become increasingly popular for the amplification of template DNA in DNA sequencing protocols. Recently, we reported that the mutant single-stranded DNA binding protein (SSB) from Thermus thermophilus (TthSSB) HB8 eliminates nonspecific DNA products in RCA reactions. We developed this method for separating circular nucleic acids from a mixture having different species of nucleic acids. Use of the mutant TthSSB resulted in an enhancement of plasmid or mitochondrial DNA content in the amplified product by approximately 500×. The use of mutant TthSSB not only promoted the amplification of circular target DNA over the background but also could be used to enhance the amplification of circular targets over linear targets.     

A Simple Method for the Purification of Organelle DNA of Plants

We report here a simple procedure for the purification of the organelle DNA. Mitochondrial DNA from Sorghum and the chloroplast DNA from Populus and spinach were purified using this protocol. The method utilizes a quick centrifugation of the isolated organelle DNA through a two step CsCl density gradient for removal of small molecular weight nucleic acids which pose a major problem for getting clean restriction patterns. This method of purification can be adopted with any isolation procedure for organelle DNA.     

A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide

We describe a modification of the DNA extraction method, in which cetyltrimethylammonium bromide (CTAB) is used to extract nucleic acids from plant tissues. In contrast to the original method, the modified CTAB procedure is faster, omits the selective precipitation and CsCl gradient steps, uses less expensive and toxic reagents, requires only inexpensive laboratory equipment and is more readily adapted to high-throughput DNA extraction. This protocol yields approximately 5-30 microg of total DNA from 200 mg of tissue fresh weight, depending on plant species and tissue source. It can be completed in as little as 5-6 h.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and beta-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp DNA Stool Mini Kit.     

Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids

Here we report efficient and selective postsynthesis labeling strategies, based on an advanced phosphoramidation reaction, for nucleic acids of either synthetic or enzyme-catalyzed origin. The reactions provided phosphorimidazolide intermediates of DNA or RNA which, whether reacted in one pot (one-step) or purified (two-step), were directly or indirectly phosphoramidated with label molecules. The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated labeling efficacy by their F/N ratio values (number of fluorophores per molecule of nucleic acid) of 0.02–1.2 which are comparable or better than conventional postsynthesis fluorescent labeling methods for DNA and RNA. Yet, PCR and UV melting studies of the one-step phosphoramidation-prepared FITC-labeled DNA indicated that the reaction might facilitate nonspecific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific labeling nucleic acids at the 5′-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. The two-step phosphoramidation-based, effective, and site-specific labeling method has the potential to expedite critical research including visualization, quantification, structural determination, localization, and distribution of nucleic acids in vivo and in vitro.     

RAPD analysis in flax: Optimization of yield and reproducibility using klenTaq 1 DNA polymerase,chelex 100, and gel purification of genomic DNA

We have developed an optimized RAPD analysis approach using the unusually heat-stable KlenTaq1 DNA polymerase. This enzyme is used in conjunction with a genomic DNA isolation method that includes a modified CTAB DNA isolation protocol, ethanol re-precipitation of resuspended nucleic acids from 2M NaCl, and Chelex 100 treatment. When needed, additional gel purification and isolation of high molecular weight DNA for use as a template in RAPD analysis is shown to remove amplification product ambiguity from within isolates of the same line as well as from between lines. This optimized RAPD analysis was used to define polymorphisms in lines of flax nearly isogenic for rust resistance at theL locus. It should also be useful for any plant species.     

Simplified DNA Extraction and Improved PCR Based Detection of Greening Bacterium in Citrus

Citrus greening disease caused by a fastidious bacterium is an important graft transmissible disease in commercial citrus in India and other parts of the world. Polymerase chain reaction (PCR) is a sensitive and convenient method for detection of greening bacterium. A non-phenol chloroform method of DNA extraction was evaluated for DNA quality and PCR based detection of greening bacterium. The method was comparable with a commercial DNA extraction kit (Qiagen) and better than a CTAB based DNA extraction method. To improve the reliability, three primer sets (primers A, B, and C yielding amplicons of 1160 bp, 703 bp and 451 bp, respectively) and two polymerase enzymes (Taq polymerase and Klen Taq polymerase) were evaluated. The primer set C provided better amplification when compared to primer sets A and B. Primer C in combination with Taq polymerase provided amplification band at a DNA template concentration of 100 pg but good amplification band was obtained at still lower DNA template concentration of 0.1 pg when Klen Taq polymerase was used. The standardized PCR protocol combining non-phenol chloroform method of DNA isolation, primer set C and Klen Taq polymerase enzyme was found very effective in detecting greening bacterium in citrus trees. The sequence of cloned amplicon from 16S ribosomal RNA gene had 89–100 % sequence identity with corresponding sequence of Candidatus Liberibacter asiaticus from China, Brazil, Japan and Pune isolate of India, C. Liberibacter americnus from Brazil and C. Liberibacter africanus from Africa.     

Subcellular localization defines modification and production of Δ<Superscript>9</Superscript>-tetrahydrocannabinolic acid synthase in transiently transformed <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Objective

Through heterologous expression of the tetrahydrocannabinolic acid synthase (THCAS) coding sequence from Cannabis sativa L. in Nicotiana benthamiana, we evaluated a transient plant-based expression system for the production of enzymes involved in cannabinoid biosynthesis.

Results

Thcas was modularized according to the GoldenBraid grammar and its expression tested upon alternative subcellular localization of the encoded catalyst with and without fusion to a fluorescent protein. THCAS was detected only when ER targeting was used; cytosolic and plastidal localization resulted in no detectable protein. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification might have an influence on the stability of the protein. Activity assays with cannabigerolic acid as a substrate showed that the recombinant enzyme produced not only THCA (123 ± 12 fkat g _FW ^?1 activity towards THCA production) but also cannabichromenic acid (CBCA; 31 ± 2.6 fkat g _FW ^?1 activity towards CBCA production).

Conclusion

Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.

     

Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8

Quantitative PCR is becoming the method of choice for the detection of pathogenic microorganisms and other targets in the environment. A major obstacle when amplifying DNA is the presence of inhibiting substances like humic acids that decrease the efficiency of PCR. We combined the polymeric adsorbent Supelite™ DAX-8 with a large-volume (10 mL) nucleic acid extraction method to decrease the humic acid content prior to qPCR quantification in water samples. The method was tested by spiking with humic acid standards and the bacterial surrogate Acinetobacter baylyi ADP1. Improvements in qPCR detection of ADP1 after application of DAX-8 resin (5 and 10 w/v%) were compared with the effects of added bovine serum albumin (BSA) (50, 100 and 200 ng/μL). Both additions improved detection of ADP1 by counteracting inhibitory effects. There were no changes in mean cycle threshold difference (ΔC_T) after application of DAX-8 compared to the control despite some loss of DNA, whereas significant increases occurred for BSA, irrespective of BSA concentration applied. The use of DAX-8 leads to an increase in qPCR amplification efficiency in contrast to BSA. The commonly used method to calculate genomic sample concentrations by comparing measured CT values relative to standard curves is only valid if amplification efficiencies of both are sufficiently similar. DAX-8 can provide this efficiency by removing humic acids permanently from nucleic acid extracts and has the potential to significantly increase the reliability of reported non-detects and measured results obtained by qPCR in environmental monitoring.     

Structure and Function of ∆1-Tetrahydrocannabinolic Acid (THCA) Synthase, the Enzyme Controlling the Psychoactivity of Cannabissativa

?1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ?1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75? resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.