Φ29 DNA Research Articles

Field-effect transistor biosensor with signal amplification by ternary initiation complexes for detection of wide-range RNA concentration

Electrical detection of RNAs using transistor-based biosensors has attracted attention as a strategy for medical diagnosis and environmental monitoring. Herein, we demonstrated a proof-of-concept for specific, sensitive, and label-free RNA detection using a field-effect transistor (FET) biosensor with signal amplification by ternary initiation complexes (SATIC), which is an isothermal one-step nucleic acid amplification initiated by the combination of target RNA, circular DNA template and DNA primer. The SATIC system-applied FET biosensor specifically and quantitatively detected the target RNA with a single-nucleotide difference via the negative charges derived from the amplification products formed by a nucleic acid amplification reaction with φ29 DNA polymerase on the gate surface. In particular, the control of the amplification time allowed the detection of target RNA molecules over a wide concentration range, resulting in a detection limit of up to 6 copies/μL. Therefore, a transistor-based bioassay using the SATIC system could be useful for simple and sensitive nucleic acid analysis.

A primer-independent DNA polymerase-based method for competent whole-genome amplification of intermediate to high GC sequences.

Multiple displacement amplification (MDA) has proven to be a useful technique for obtaining large amounts of DNA from tiny samples in genomics and metagenomics. However, MDA has limitations, such as amplification artifacts and biases that can interfere with subsequent quantitative analysis. To overcome these challenges, alternative methods and engineered DNA polymerase variants have been developed. Here, we present new MDA protocols based on the primer-independent DNA polymerase (piPolB), a replicative-like DNA polymerase endowed with DNA priming and proofreading capacities. These new methods were tested on a genomes mixture containing diverse sequences with high-GC content, followed by deep sequencing. Protocols relying on piPolB as a single enzyme cannot achieve competent amplification due to its limited processivity and the presence of ab initio DNA synthesis. However, an alternative method called piMDA, which combines piPolB with Φ29 DNA polymerase, allows proficient and faithful amplification of the genomes. In addition, the prior denaturation step commonly performed in MDA protocols is dispensable, resulting in a more straightforward protocol. In summary, piMDA outperforms commercial methods in the amplification of genomes andmetagenomes containing high GC sequences and exhibits similar profiling, error rateand variant determination as the non-amplified samples.

DNA Polymerases for Whole Genome Amplification: Considerations and Future Directions.

In the same way that specialized DNA polymerases (DNAPs) replicate cellular and viral genomes, only a handful of dedicated proteins from various natural origins as well as engineered versions are appropriate for competent exponential amplification of whole genomes and metagenomes (WGA). Different applications have led to the development of diverse protocols, based on various DNAPs. Isothermal WGA is currently widely used due to the high performance of Φ29 DNA polymerase, but PCR-based methods are also available and can provide competent amplification of certain samples. Replication fidelity and processivity must be considered when selecting a suitable enzyme for WGA. However, other properties, such as thermostability, capacity to couple replication, and double helix unwinding, or the ability to maintain DNA replication opposite to damaged bases, are also very relevant for some applications. In this review, we provide an overview of the different properties of DNAPs widely used in WGA and discuss their limitations and future research directions.

Characterization of a recombinant tomato leaf curl New Delhi Virus (ToLCNDV) in a perennial medicinal climber host (Ipomoea cairica (L.) Sweet).

During the year 2020-2021, a disease syndrome very commonly observed in railway creepers (Ipomoea cairica(L.) Sweet) was taken into consideration from Gorakhpur Province (UP East region). Whitefly, a common vector for plant-related viral diseases was observed for wide transmission of disease. DNA from 17 infected leaf samples was isolated and analyzed through PCR using specific primers designed for the detection of Begomoviruses. Further amplification of isolated DNA fragments supporting a begomovirus infection with an estimated size of (2.7kb). RCA of the isolated DNA sample was carried out using ϕ29 DNA polymerase by digesting it through a set of restriction endonucleases (such as BamHI, Kpn1, HindIII, EcoRI) obtaining the best result through KpnI. The amplified segment was cloned into pUC 18vectors. The obtained sequences were matched using the NCBI BLAST database showing the highest sequence similarity of 98.7% with ToLCNDV of snake gourd (Accession no.KY780199), isolates of CP genes sequence in Varanasi. ToLCNDV, a begomovirus of bipartite nature was first reported to be from Tomato (Solanaceae), later affecting certain members of the Cucurbitaceae family in India and adjacent countries. The obtained DNA sequence was submitted at NCBI with the name ToLCNDV-IP (GenBank Accession no. OM777194). The phylogenetic analysis clubbed the current isolate ToLCNDV-IP with Asian isolates. All European isolates were clubbed in separate clades indicating two distinct groups of ToLCNDV isolates. ToLCNDV-IP isolate was found to be an intralineage recombinant between two Asian isolates viz ToLCNDV isolate from Papaya (India) and ToLCNDV isolate from Tomato (Pakistan). This study shows the association of recombinant ToLCNDV infection in a new hostIpomoea cairicafor the first time in India.

Molecular Cloning of MYMV Genome and Infectivity of Yellow Mosaic Virus in Green Gram Using Different Viral Transmission Tools

Mungbean yellow mosaic virus (MYMV) causes massive crop losses in green gram. MYMV is a member of begomovirus with bipartite genome comprising DNA-A and DNA-B components, which is transmitted by whiteflies. Cloning and preparation of infectious clone is very much essential for screening germplasm or transgenic material of pulse crops since viruliferous whiteflies may not be available throughout the year. In the current work, we have amplified rolling circle mediated viral genome of MYMV using Φ29 DNA polymerase. The amplified products was digested and cloned into the plant expression vector pCAMBIA2301.The cloned constructs was then transformed into Agrobacterium LBA4404 through freeze thaw method. Further, three viral transmission techniques including mechanical rubbing, Agroinfiltration and Agroinoculation, were employed for assessing the mosaic symptoms in green gram. The molecular confirmation through polymerase chain reaction (PCR) indicated that the yellow mosaic symptoms were formed due to infectivity of MYMV in the green gram.

Viral packaging ATPases utilize a glutamate switch to couple ATPase activity and DNA translocation

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors.

Structural Insights into gp16 ATPase in the Bacteriophage ϕ29 DNA Packaging Motor.

Biological motors, ubiquitous in living systems, convert chemical energy into different kinds of mechanical motions critical to cellular functions. Gene product 16 (gp16) in bacteriophage ϕ29 is among the most powerful biomotors known, which adopts a multisubunit ring-shaped structure and hydrolyzes ATP to package double-stranded DNA (dsDNA) into a preformed procapsid. Here we report the crystal structure of the C-terminal domain of gp16 (gp16-CTD). Structure-based alignment and molecular dynamics simulations revealed an essential binding surface of gp16-CTD for prohead RNA, a unique component of the motor complex. Furthermore, our simulations highlighted a dynamic interplay between the N-terminal domain and the CTD of gp16, which may play a role in driving movement of DNA into the procapsid. Lastly, we assembled an atomic structural model of the complete ϕ29 dsDNA packaging motor complex by integrating structural and experimental data from multiple sources. Collectively, our findings provided a refined inchworm-revolution model for dsDNA translocation in bacteriophage ϕ29 and suggested how the individual domains of gp16 work together to power such translocation.

Engineering Micrometer-Sized DNA Tracks for High-Speed DNA Synthesis and Biosensing.

φ29 DNA polymerase (Polφ29) is capable of synthesizing long-chain single-stranded (ss) DNA molecules by copying the sequence of a small ss circular DNA template (ssCDT) in a process known as rolling circle amplification (RCA). The use of a ssCDT in RCA, however, comes with a key drawback: the rate of DNA synthesis is significantly reduced. We hypothesize that this issue can be overcome using a very long linear ssDNA template with a repeating sequence. To test this idea, we engineered a DNA assembly, which we denote "micrometer-sized DNA track" (μDT). This μDT, with an average length of ≈13.5 μm, is made of a long chain DNA with a primer-binding domain at its 3' end and ≈1000 repeating sequence units at its 5' end, each carrying a DNA anchor. We find that Polφ29 copies μDT at a speed ≈5-time faster than it does a related ssCDT. We use this to design a simple all-in-one printed paper device for rapid and sensitive detection of microRNA let-7. This paper sensor is capable of detecting 1 pM let-7a in 10 minutes.

Biomineralized Metal-Organic Framework Nanoparticles Enable Enzymatic Rolling Circle Amplification in Living Cells for Ultrasensitive MicroRNA Imaging.

The enzymatic amplification strategy in living cells faces challenges of highly efficient intracellular codelivery of amplification reagents including DNA polymerase. In this work, we develop biomineralized metal-organic framework nanoparticles (MOF NPs) as a carrier system for intracellular codelivery of ϕ29 DNA polymerase (ϕ29DP) and nucleic acid probes and realize a polymerization amplification reaction in living cells. A pH-sensitive biodegradable MOF NP of zeolitic imidazolate framework-8 (ZIF-8) is utilized to encapsulate ϕ29DP and adsorb nucleic acid probes. After uptake into cells, the encapsulated ϕ29DP and surface-adsorbed DNA probes are released and escaped from endolysosomes. In the presence of ϕ29DP and deoxyribonucleotide triphosphates (dNTPs), the intracellular miRNA-21 triggers a rolling circle amplification (RCA) reaction and the autonomous synthesized Mg2+-dependent DNAzyme cleaves the fluorogenic substrate, providing a readout fluorescence signal for the monitoring of miRNA-21. This is the first example of the intracellular RCA reaction in living cells. Therefore, the proposed method provides new opportunities for achieving enzymatic amplification reaction in living cells.

Tyrosines involved in the activity of φ29 single-stranded DNA binding protein.

The genome of Bacillus subtilis phage ϕ29 consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5’ end (TP-DNA). ϕ29 DNA polymerase is the enzyme responsible for viral DNA replication, due to its distinctive properties: high processivity and strand displacement capacity, being able to replicate the entire genome without requiring the assistance of processivity or unwinding factors, unlike most replicases. ϕ29 single-stranded DNA binding protein (SSB) is encoded by the viral gene 5 and binds the ssDNA generated in the replication of the ϕ29 TP-DNA. It has been described to stimulate the DNA elongation rate during the DNA replication. Previous studies proposed residues Tyr50, Tyr57 and Tyr76 as ligands of ssDNA. The role of two of these residues has been determined in this work by site-directed mutagenesis. Our results showed that mutant derivative Y57A was unable to bind to ssDNA, to stimulate the DNA elongation and to displace oligonucleotides annealed to M13 ssDNA, whereas mutant Y50A behaved like the wild-type SSB.

Metagenomics Approaches in Discovery and Development of New Bioactive Compounds from Marine Actinomycetes.

Marine actinomycetes are prolific sources of marine drug discovery system contributing for several bioactive compounds of biomedical prominence. Metagenomics, a culture-independent technique through its sequence- and function-based screening has led to the discovery and synthesis of numerous biologically significant compounds like polyketide synthase, Non-ribosomal peptide synthetase, antibiotics, and biocatalyst. While metagenomics offers different advantages over conventional sequencing techniques, they also have certain limitations including bias classification, non-availability of quality DNA samples, heterologous expression, and host selection. The assimilation of advanced amplification and screening methods such as φ29 DNA polymerase, Next-Generation Sequencing, Cosmids, and recent bioinformatics tools like automated genome mining, anti-SMASH have shown promising results to overcome these constrains. Consequently, functional genomics and bioinformatics along with synthetic biology will be crucial for the success of the metagenomic approach and indeed for exploring new possibilities among the microbial consortia for the future drug discovery process.

New insights into the coordination between the polymerization and 3\u2032-5\u2032 exonuclease activities in \u03d529 DNA polymerase

Bacteriophage ϕ29 DNA polymerase has two activities: DNA polymerization and 3′-5′ exonucleolysis governed by catalytic sites present in two structurally distant domains. These domains must work together to allow the correct replication of the template and to prevent the accumulation of errors in the newly synthesized DNA strand. ϕ29 DNA polymerase is endowed with a high processivity and strand displacement capacity together with a high fidelity. Previous studies of its crystallographic structure suggested possible interactions of residues of the exonuclease domain like the Gln180 with the fingers subdomain, or water mediated and direct hydrogen bond by the polar groups of residues Tyr101 and Thr189 that could stabilize DNA binding. To analyse their functional importance for the exonuclease activity of ϕ29 DNA polymerase we engineered mutations to encode amino acid substitutions. Our results confirm that both residues, Tyr101 and Thr189 are involved in the 3′-5′ exonuclease activity and in binding the dsDNA. In addition, Tyr101 is playing a role in processivity and Thr189 is an important determinant in the fidelity of the DNA polymerase. On the other hand, the biochemical characterization of the mutant derivatives of residue Gln180 showed how the mutations introduced enhanced the 3′-5′ exonuclease activity of the enzyme. A potential structural conformation prone to degrade the substrate is discussed.

A Novel Strain of Pepper Leafroll Virus Infecting Common Bean and Soybean in Ecuador

Symptoms resembling those caused by begomoviruses (Geminiviridae: Begomovirus) have been observed in common bean and other leguminous crops in Ecuador in the last few years. The only begomovirus species found infecting legumes (common bean and cowpea) in the country to date is cabbage leaf curl virus (CabLCV) (Fiallo-Olive et al. 2018). During May to June 2017, a survey of leguminous plants was conducted in the Ecuadorian localities of Santa Elena (Santa Elena province), Milagro (Guayas province), and Pallatanga (Chimborazo province). Twenty-four plants showing leaf deformation and/or mosaic symptoms were sampled, including common bean (Phaseolus vulgaris, n = 21), soybean (Glycine max, n = 2), and pigeon pea (Cajanus cajan, n = 1). Most common bean plants were infested with whiteflies (Hemiptera: Aleyrodidae) that were also collected. Total DNA was extracted from leaf samples and used as template in polymerase chain reaction (PCR) using specific primers for CabLCV (MA2540 [5′-GATGTTGAAGCATCTGCAAACATTC-3′] and MA2541 [5′-CTAGGAACATCAGGGCTTCTCAAC-3′]) (Fiallo-Olive et al. 2018). All samples were negative for the presence of CabLCV. DNA extracts were then used as templates in rolling-circle amplification (RCA) using φ29 DNA polymerase (TempliPhi kit, GE Healthcare). Digestion of RCA products with a set of restriction enzymes yielded identical restriction patterns for six samples (five common bean and one soybean), suggesting the presence of a bipartite begomovirus. BamHI and NcoI fragments of approximately 2.6 kbp from a common bean plant were cloned in pBluescript II SK(+), and two selected clones were sequenced (Macrogen, Seoul, South Korea). BLAST analysis showed that the clones corresponded to begomoviral DNA-A (BamHI, 2,580 nt, GenBank accession no. MH481901) and DNA-B (NcoI, 2,561 nt, MH481902). Pairwise identity scores with isolates selected after BLAST analysis were calculated with SDT (Muhire et al. 2014). Cloned DNA-A and DNA-B showed the highest identity (93 and 92%, respectively) with pepper leafroll virus (PepLRV) (KC769819 and KC769820), a bipartite begomovirus identified infecting pepper, common bean, and tomato in Peru (Martinez-Ayala et al. 2014). To confirm the identity of the begomovirus present in the rest of the samples, PCR was performed with specific primers whose design was based on the cloned PepLRV DNA-A (MA2602 [5′-GTTAATGGACAATGAGGGTATGTG-3′] and MA2603 [5′-CACGAGGAGAATAATTGGCGTTAC-3′]) using Taq DNA polymerase (BIOTAQ, Bioline) and thermocycler conditions consisting of 35 cycles with 45 s annealing steps at 57°C. DNA fragments of the expected size (452 bp) were amplified from the six samples, and direct sequencing showed them to be identical to the cloned PepLRV DNA-A, supporting the presence of this virus. Based on the species/strain demarcation threshold for the viruses in the genus Begomovirus (Brown et al. 2015), the isolate characterized here belongs to a new strain of PepLRV. DNA was extracted from individual whiteflies (n = 42) using a Chelex-based method, and PCR was carried out using mitochondrial cytochrome oxidase I gene primers, C1-J-2195 (5′-TTGATTTTTTGGTCATCCAGAAGT-3′) and Btab-uni-PrimerR (5′-CTTAAATTTACTGCACTTTCTGCCAYATTAG-3′). Direct sequencing of PCR products showed that seven sequences (one deposited in GenBank with accession no. MH481903) were identical to numerous sequences of Bemisia tabaci Middle East-Asia Minor 1 (MEAM1, formerly B biotype), known to be an efficient vector of begomoviruses. To our knowledge, this is the first report of PepLRV in Ecuador and of soybean as a host for the virus, which had been identified only in Peru to date. Also, this is the first molecular evidence of the presence of B. tabaci MEAM1 in the country.

Tracking DNA Synthesis with Single-Molecule Strand Displacement.

We have previously shown that double-stranded DNA labeled with a periodic series of fluorescent dyes can be used to track a single helicase. Here we demonstrate how this technique can be adapted to follow processive DNA synthesis. By monitoring strand displacement, we track the motion of a single ϕ29 DNA polymerase without labeling or altering the enzyme or the template strand, and without applying any force. We observe a wide range of speeds, with the highest exceeding by several times those observed in earlier in vitro single-molecule experiments. Because this method enables repeated observations of the same polymerase traversing identical segments of DNA, it should prove useful for determining the effects of sequence on DNA replication and transcription. In addition, future measurements of this type may allow us to examine in detail the interactions of individual DNA polymerases with other components of the replisome.

Molecular switch-like regulation enables global subunit coordination in a viral ring ATPase

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP-ATP exchange and ATP hydrolysis of the motor's biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit's catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP-ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.

A Novel Strain of the Begomovirus Tomato Leaf Curl Sudan Virus Infecting Datura stramonium in Sudan.

HomePlant DiseaseVol. 102, No. 9A Novel Strain of the Begomovirus Tomato Leaf Curl Sudan Virus Infecting Datura stramonium in Sudan PreviousNext DISEASE NOTES OPENOpen Access licenseA Novel Strain of the Begomovirus Tomato Leaf Curl Sudan Virus Infecting Datura stramonium in SudanH. S. Mohammed, M. A. El Siddig, A. A. El Hussein, J. Navas-Castillo, and E. Fiallo-OlivéH. S. MohammedSearch for more papers by this author, M. A. El SiddigSearch for more papers by this author, A. A. El HusseinSearch for more papers by this author, J. Navas-CastilloSearch for more papers by this author, and E. Fiallo-Olivé†Corresponding author: E. Fiallo-Olivé; E-mail: E-mail Address: [email protected]http://orcid.org/0000-0002-2460-1199Search for more papers by this authorAffiliationsAuthors and Affiliations H. S. Mohammed , Botany Department, Faculty of Science, University of Khartoum, 11115 Khartoum, Sudan, and Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga – Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), 29750 Algarrobo-Costa, Málaga, Spain M. A. El Siddig A. A. El Hussein , Botany Department, Faculty of Science, University of Khartoum, 11115 Khartoum, Sudan J. Navas-Castillo E. Fiallo-Olivé † , IHSM-UMA-CSIC, 29750 Algarrobo-Costa, Málaga, Spain. Published Online:6 Jul 2018https://doi.org/10.1094/PDIS-01-18-0195-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Devil’s trumpet (Datura stramonium) plants were frequently observed showing yellowing and leaf curl symptoms in Khartoum State (Sudan) in the last few years. Leaf samples from 14 symptomatic plants were collected in Ajaily (one in 2016 and seven in 2017) and Soba Al-Hilla (six in 2017) and dried using silica gel until analysis. To investigate the presence of begomoviruses (genus Begomovirus, family Geminiviridae), total nucleic acids were extracted from all leaf samples and used as template for polymerase chain reaction (PCR) with generic primers (AV494 and AC1048) targeting a core region of the begomoviral coat protein gene (Wyatt and Brown 1996). All samples yielded the expected DNA fragment of 550 bp. All 14 PCR products were directly sequenced at Macrogen (Seoul, South Korea) and showed a nucleotide identity of 99.2 to 100% between them. Blast analysis evidenced the highest nucleotide identity (98%) with an isolate of tomato leaf curl Sudan virus (ToLCSDV) found infecting tomato in Sudan (GenBank accession no. KC763630) (Fiallo-Olivé et al. 2013), suggesting that the symptomatic plants were infected by ToLCSDV. Rolling-circle amplification (RCA) using φ29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, UK) was carried out with extracts of all samples, and subsequent digestion with HpaII yielded identical restriction patterns for all of them. RCA products were also digested with a set of six-nucleotide restriction enzymes (BamHI, HindIII, EcoRI, and SalI). BamHI yielded an amplicon of ∼2.7 kbp from all the samples, which was cloned from one of them (sample Ajaily-2016) into pBluescript II SK(+) (Stratagene, La Jolla, CA). Two selected clones were sequenced (Macrogen) and were identical (2,768 nt, one deposited in GenBank under accession no. MG868938). Sequence was aligned with those selected after BLAST analysis using MUSCLE, and identity percentages were calculated with Sequence Demarcation Tool (Muhire et al. 2014). Cloned full-length viral genome showed the highest nucleotide identity (91%) with the abovementioned ToLCSDV isolate (KC763630) (Fiallo-Olivé et al. 2013). In accordance with the current taxonomic criteria for begomovirus species (cutoff value <91%) and strain (cutoff value <94%) demarcation (Brown et al. 2015), the begomovirus isolate characterized in this investigation represents a novel strain of ToLCSDV that we name strain Datura. To our knowledge, this is the first record of a species of the genus Datura as a natural host of ToLCSDV. Tomato leaf curl disease is a major limiting factor of tomato production in Sudan, at times resulting in up to 75% yield losses (Yassin 1983). The widespread presence of D. stramonium in areas where tomato is grown in Sudan poses an additional threat to the cultivation of this important crop in the region.

Allostery and molecular machines.

Machines undergo repeated energy (fuel)-driven cycling between various functional and structural states. Such cycling involves motions of the machine parts, which take place in a highly coordinated manner in time and space. This description applies not only to man-made machines but also to the molecular machines that mediate many of the key processes in all forms of life [1]. Examples include protein synthesis by the ribosome, DNA unwinding by helicases during replication, protein degradation by the proteasome and protein folding by chaperones. The coordinated movements of most biomolecular machines are achieved by allosteric regulation of ATP binding and hydrolysis. Consequently, a molecular level understanding of how the essential machines of life work requires invoking and further developing allosteric theory. Hence, the motivation for the Royal Society Discussion Meeting entitled ‘Allostery and molecular machines' that took place in June 2017. The meeting brought together scientists interested in allostery, in general, with others who are studying specific biomolecular machines of interest. The meeting led to many interesting discussions and resulted in this special issue of the Philosophical Transactions B . In 1965, Monod, Wyman & Changeux published a seminal paper [2] that described a so-called ‘concerted model' (referred to here and generally as the MWC model) for cooperative ligand binding by oligomeric proteins. This issue contains a paper by one of the co-authors of this landmark paper in which the MWC model is explained in brief and its application to the nicotinic acetylcholine receptor is then described [3]. The MWC model remains important but its elegance has come at the price of several restrictive assumptions. One key assumption is that cooperativity is due to a shift in equilibrium between different quaternary …

Limited reverse transcriptase activity of phi29 DNA polymerase.

Phi29 (Φ29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.

Efficient construction of xenogeneic genomic libraries by circumventing restriction-modification systems that restrict methylated DNA

An efficient method to construct xenogeneic genomic libraries with low errors and bias by circumventing restriction-modification systems that restrict methylated DNA was developed. Un-methylated genomic DNA of Escherichia coli prepared by ϕ29 DNA polymerase was introduced to Corynebacterium glutamicum R after ligation with un-methylated vector plasmids.

First Report of Sweet potato leaf curl virus and Sweet potato leaf curl deltasatellite 1 Infecting Blue Morning Glory in Portugal

Blue morning glory (Ipomoea indica) is a neotropical plant commonly used as ornamental and naturalized in tropical, subtropical and temperate regions worldwide, including the coastal areas of southern Europe. Vein yellowing, a symptom suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection, has been sporadically observed in I. indica plants in southern Portugal since 2006. In August 2017, leaf samples of I. indica were collected in southern Portugal (Faro municipality, n=8; Albufeira municipality, n=2). To investigate the possible presence of begomoviruses in the samples, total nucleic acids were extracted and used as template for rolling-circle amplification (RCA) using φ29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, UK). Subsequent digestion with a set of restriction endonucleases (BamHI, EcoRI, NheI, NcoI, PstI and SalI) yielded a similar restriction pattern for five samples collected in Faro, although only one of them showed vein yellowing in some leaves. Using...