Restriction Enzymes HpaII Research Articles

Highly sensitive detection of DNA methyltransferase1 triggered by methylation protection

Type 2 diabetes is on the rise year by year, and 80 % of patients with type 2 diabetes die from cardiovascular complications. Strict control of blood glucose can significantly reduce the occurrence and development of diabetic microvascular complications, but can not effectively prevent the occurrence of diabetic macrovascular lesions. DNA methyltransferase 1 (DNMT1) plays an important role in developing phenotypic switching and secretion of inflammatory factors in vascular smooth muscle cells(VSMC), leading to the deregulation of vascular function. In this work, a biosensor based on methylation protection was designed. In the presence of the target DNMT 1, the formed methylation sites protect the dsDNA from being digested by the HpaII restriction enzyme. At the same time, a large number of G-quadruplex are produced by the catalyzed hairpin assembly (CHA) reaction cycle, and the peroxidase-like activity of G-tetraplex and hemin is used to continuously catalyze the color change of substrate ABTS, thus realizing dual signal amplification. The binding of magnetic beads to dsDNA effectively reduces the background signal and enables a highly selective and sensitive analysis of the target DNMT 1 in complex biological samples, with a detection limit as low as 0.22 UmL−1, showing a good linear relationship in the range of 0.22–10 UmL−1. A new approach for the study of macrovascular damage is provided in type 2 diabetes.

Detection of Phytoplasma belonging to the 16SrXXI group in Scott pine and mountain pine

One of the reasons for the deterioration of coniferous stands condition in a number of European countries is associated with their infection with phytoplasmas (obligate intracellular pathogens) — bacteria lacking a cell wall. The aim of the work is to identify the presence of phytoplasma infection in samples of Scots pine (Pinus sylvestris L.) and mountain pine (Pinus mugo Turra) collected in the Moscow and Samara regions, which had characteristic symptoms of the disease, and to determine the taxonomic affiliation of the phytoplasm. Phytoplasma was detected using direct and nested PCR with primer pairs P1/16S-Sr and R16F2n/R16R2 respectively. Phytoplasma DNA was found in six of the seven pine specimens, including an asymptomatic one. Analysis of restriction fragment length’s polymorphism after digestion of DNA amplicons with restriction endonucleases AluI, MseI, HhaI, HpaII, HaeIII, RsaI, and TaqI indicated the similarity of Russian strains of Scotch pine and mountain pine phytoplasmas to Lithuanian strains of PineLRN and PineBLD of mountain pine phytoplasma (GenBank Accession Number MK089821 and MK089819, respectively) identified as ‘Candidatus Phytoplasma pini’ (subgroup 16SrXXI-A). A phytoplasma related to this species has been registered on the territory of the Russian Federation for the first time.

Molecular identification of Sarcocystis neurona in tissues of wild boars (Sus scrofa) in the border region between Brazil and Uruguay.

Sarcocystis neurona, owing to its clinical importance in domestic animals, is currently one of the most studied agents, presenting a wide range of intermediate hosts that have not yet been described, mainly in wild fauna. Thus, the aim of this study was to describe the detection and molecular detection of S. neurona by amplification of the 18S rRNA region in the tissues of wild boars killed by boar control program in border Brazil Uruguay. A total of 79 samples of DNA from wild boar tissues from the LADOPAR/UFSM sampling bank were used, with Nested-PCR reactions being performed for amplification of the 18S rRNA region and the expected final product of 290bp. Subsequently, the positive samples were subjected to restriction fragment length polymorphism (RFLP) technique with the restriction enzymes DdeI and HPAII. A second semi-Nested reaction was performed to obtain a larger sequence of nucleotides with amplification of the 18S region and the expected final product of 500bp for S. neurona and Nested amplification ITS1 with product final of 367 pb. In 32 samples, it was possible to detect S. neurona both by nested Nested-PCR reaction and RFLP, and the presence of the agent was confirmed by sequencing, corresponding to 40.51% of the total tissues evaluated. This is the first report of the occurrence of this species of Sarcocystis in wild boars, and further studies evaluating the role of these animals as intermediate hosts, and in the epidemiology of this protozoan are necessary, as well as verifying the risk factors for infection.

First Report of 16SrII Group Related Phytoplasma Associated with Areca Palm Yellow Leaf Disease on Areca catechu in China.

The areca palm, Areca catechu L., family Arecaceae is an important herbal medicine which has potential for the treatment of parasitic diseases, digestive function disorders and depression (Peng et al. 2015). Yellow leaf disease (YLD), caused by phytoplasma, is a destructive disease of Areca catechu. In 1981, the YLD was first discovered in Tunchang, Hainan, China. According to the investigation in 2020, the occurrence area of YLD was 32 102.38 hm2 in Hainan, China, resulting in 50%-60% yield loss. Previous researchers based on 16S rDNA gene PCR amplification analysis showed that YLD in Hainan was caused by 16SrI group phytoplasma (Che et al. 2010). In August, 2022, yellow leaf symptoms were observed on middle and lower leaves of Areca catechu. Forty symptomatic plants and three asymptomatic samples were collected in Wenchang, Hainan, China (19°33'9″N, 110°48'5″E). Forty-three samples (0.1g each) were used to extract total DNA (TIANGEN plant genomic DNA extraction kit). Phytoplasma universal primers named P1/P7 (Schneider et al. 1995) and R16F2n/R16R2 (Gundersen and Lee 1996) for 16Sr DNA and primers named fTuf1/rTuf1 and fTufu/rTufu (Schneider et al. 1997) for tuf genes were used for amplifying phytoplasma sequences from isolated DNA samples by nested PCR. No fragment was amplified in asymptomatic plants and four out of forty symptomatic samples could amplify target fragment. R16F2n/R16R2 amplicons (1 248 bp) and fTufu/rTufu amplicons (845 bp) from four symptomatic Areca catechu samples were sequenced in BGI (https://genomics.cn/). The 16Sr DNA GenBank accession numbers of four positive strains (named HNWC5, HNDZ1, HNDZ3 and HNDZ6) were OQ586072, OQ586085, OQ586086, OQ586087, respectively and the tuf GenBank accession numbers were OQ595209, OQ595210, OQ595211, OQ595212, respectively. Sequence alignment showed that the 16S rDNA and tuf sequence of HNDZ1, HNDZ3 and HNDZ6 were 100% consistent. 16S rDNA of HNWC5 was 99.96% consistent with HNDZ1 and tuf of HNWC5 was 98.31% consistent with HNDZ1. Interestingly, blast search based on 16S rDNA gene of HNWC5 showed 100% sequence identity with that of 16SrII group phytoplasma such as 'Eclipta prostrata' phytoplasma strain Ep1(MH144204.1), 'Aeschynomene americana' phytoplasma isolate AA1(MH231157.1) and 'Acacia confusa' witches'-broom phytoplasma isolate HK6(ON408364.1). Blast search based on tuf gene of HNWC5 showed 98.7% sequence identity with that of bamboo witches'-broom phytoplasma (FJ853160.1) and 91.02% sequence identity with that of 'podocarpus nagi' fasciation phytoplasma (KR633146) and 90.78% sequence identity with that of 'Musa acuminata' elephantiasis disease phytoplasma (MF983708). The phylogenetic tree was constructed based on 16Sr DNA gene by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value (Kumar et al. 2016). The result indicated that the HNWC5, HNDZ1, HNDZ3 and HNDZ6 phytoplasma strains clustered a subclade in 16SrII group. The virtual RFLP analysis based on the 16Sr DNA gene sequence was performed by the online phytoplasma classification tool iPhyClassifier (Zhao et al. 2009) using restriction endonucleases of AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI. The result indicated that HNWC5 was most similar to the reference pattern of peanut witches'-broom phytoplasma (16SrII-A subgroup, GenBank accession: L33765) and the pattern similarity coefficient of HNWC5 is 1.00. However, the HpaII restriction endonuclease pattern of HNDZ1, HNDZ3 and HNDZ6 was different from L33765 and the similarity coefficient was 0.97, which indicated this strain may represent a new subgroup within the 16SrII group. To our knowledge, this is the first report of 16SrII group related phytoplasma associated with YLD on Areca catechu in China. Our study contributes to understanding the polymorphism of phytoplasma causing YLD and provides an important reference for pathogen specific detection.

Nonrandom X Chromosome Inactivation Detection

X chromosome inactivation patterns may be clinically useful in assessing tumor clonality, determining carrier status for certain X-linked disorders, and evaluating the pathogenicity of a genetic variant identified in an X-linked gene. The protocols in this article utilize the highly polymorphic trinucleotide repeat within the first exon of the human androgen receptor gene (AR) and the methylation-sensitive restriction enzyme HpaII to distinguish between the maternal and paternal alleles and simultaneously determine their methylation status. The data obtained from these protocols can be used to calculate the ratio of inactivation between the two alleles that ultimately reflects whether a female has a random or nonrandom pattern of X chromosome inactivation. © 2023 Wiley Periodicals LLC. Basic Protocol 1: X chromosome inactivation assay Basic Protocol 2: PCR amplification and labeling of digested and undigested DNA templates.

Molecular detection of Toxoplasma gondii, Neospora caninum and Sarcocystis spp in tissues of Sus scrofa slaughtered in southern Brazil.

The aim of this study was to determine the presence of deoxyribonucleic acid (DNA) from Toxoplasma gondii, Sarcocystis spp. and Neospora caninum, in tissues of wild boars slaughtered in southern Brazil. A total of 156 samples were collected from different organs of 25 wild boars, and DNA from at least one of the protozoa investigated was detected in 79 samples. To differentiate between infectious agents, restriction fragment length polymorphism was performed using the restriction enzymes DdeI and HpaII. For N. caninum, conventional PCR was performed with specific primers. The DNA of at least one of the studied pathogens was detected in each animal: 26.58% for T. gondii, 68.36% for Sarcocystis spp. and 5.06% for N. caninum. Coinfection between T. gondii and Sarcocystis spp. occurred in 14 animals, between T. gondii and N. caninum in only one male animal, between Sarcocystis spp. and N. caninum in a female, while co-infection with the three agents was equally observed in only one male animal. Considering the high frequency of detection and its zoonotic risk, especially T. gondii, it appears that wild boars can be potential sources of transmission of infectious agents and the adoption of monitoring measures in these populations should be prioritized.

Methylation Profile of 18S rDNA Gene in Brain and Muscle of Tambaqui Exposed to Parasiticide Trichlorfon

Until recently, there were no reports on the methylation profile of Amazonian fish specimens employing restriction enzymes. The 18S rDNA methylation status of samples of captive tambaqui (Colossoma macropomum) exposed to the antiparasitic Trichlorfon was examined in the current study. The method performed makes use of the CCGG site's sensitivity to the presence of methylation in palindrome cytosines for the restriction enzymes HpaII and MspI. The cleavage occurs by the detection or non-detection of the methyl radical in the inner (C5mCGG) or outer (5mCCGG) cytosine, which makes it possible to compare the DNA methylation profile. When compared to the specimens examined as the control group, the results obtained revealed an increase in methylated fragments of the 18S rDNA gene at LC concentrations of 30% and 50%.

Characterization of Plant Growth Promoting Endophytic Bacteria from Castor Plants [Ricinus communis (L.)

The present study was carried out in June–Janauary (2021–2022) at the Laboratory of the Department of Microbiology, Chimanbhai Patel College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University (SDAU), Sardarkrushinagar, Gujarat, India. 15 promising endophytic bacterial isolates from castor plants showing higher dry matter accumulation in seedlings of wheat were subjected to detailed characterization for their molecular and plant growth promoting abilities. The bacterial isolate Erc7 performed best for plant growth promoting activities viz., ammonia and IAA production, phosphate solubilization, nitrogen fixation, HCN production and antagonistic activity against Fusarium oxysporum f. sp. ricini in-vitro which cause wilt in castor plant. Pot trials involving wheat seed inoculation (rabi 2021–22) with bacterial isolates showed that the treatment with endophytic bacterial isolate Erc7 showed maximum shoot and root length (47.65 cm, 10.50 cm), shoot fresh and dry weight (824.90 mg, 308.83 mg), root fresh and dry weight (122.37 mg, 36.20 mg), spike length (09.76 cm) and number of seeds in central tiller plant-1 (15.33). PCR amplification of 16S rRNA genes and ARDRA profiling using restriction enzymes AluI, TaqI and HpaII grouped the 15 isolates in 2 clusters (A and B) at a Jaccard’s similarity co-efficient of 0.54. The best PGPR isolate Erc7 was gene sequenced for 16S rRNA gene. The sequence was submitted to the NCBI Genbank (Accession number ON514555). Upon BlastN It showed maximum similarity to Bacillus cereus. The isolates Erc7 showed presence of diplobacillus endospores which is a typical characteristic of Bacillus cereus.

Development of PCR-RFLP Technique for Identify Several Members of Fusarium incarnatum-equiseti Species Complex and Fusarium fujikuroi Species Complex.

Fusarium incarnatum-equiseti species complex (FIESC) contain over 40 members. The primer pair Smibo1FM/Semi1RM on the RPB2 partial gene has been reported to be able to identify Fusarium semitectum. The F. fujikuroi species complex (FFSC) contains more than 50 members. The F. verticillioides as a member of this complex can be identified by using VER1/VER2 primer pair on the CaM partial gene. In this research, the Smibo1FM/Semi1RM can amplify F. sulawesiense, F. hainanense, F. bubalinum, and F. tanahbumbuense, members of FIESC at 424 bp. The VER1/VER2 can amplify F. verticillioides, F. andiyazi, and F. pseudocircinatum, members of FFSC at 578 bp. Polymerase chain reaction-restriction fragment length polymorphism by using the combination of three restriction enzymes EcoRV, MspI, and HpyAV can differentiate each species of FIESC used. The two restriction enzymes HpaII and NspI can distinguish each species of FFSC used. The proper identification process is required for pathogen control in the field in order to reduce crop yield loss.

Повреждение ДНК в тканях белых крыс при воздействии дыма лесных пожаров

Wildfire smoke affects the health of the population of the entire planet and each year the situation is getting worse. Fire smoke has mutagenic, carcinogenic and other long-term effects. The aim of this paper was to study the genotoxic effect of wildfire smoke on male white rats and the transgenerational effect of wildfire smoke on their offspring. Smoke exposure simulation was performed on 40 male white rats in 200-litre exposure chambers. Forest litter, branches, fallen bark, and the upper layer of soil served as a combustible substrate. The exposure was carried out for 4 hours 5 days a week in the course of 4 weeks. Offspring of both sexes were obtained from the exposed animals and intact females. We analysed DNA fragmentation and global DNA methylation in the gonadal tissue and blood cells of the exposed animals immediately after the exposure and global DNA methylation in the blood cells of the offspring upon reaching sexual maturity. The research was performed using the comet assay with modifications to study global DNA methylation with MspI and HpaII restriction enzymes. The exposed animals showed an increased level of global DNA methylation in their blood cells. In male offspring, a decrease in the level of global DNA methylation in the blood cells was revealed, compared with the controls, while females showed no differences from the controls. The established facts of changes in global DNA methylation after exposure to wildfire smoke require further in-depth research, since the mechanism of the development of this phenomenon remains rather unclear.

Construction of a Dye-Sensitized and Gold Plasmon-Enhanced Cathodic Photoelectrochemical Biosensor for Methyltransferase Activity Assay.

DNA methyltransferases may function as important biomarkers of cancers and genetic diseases. Herein, we develop a dye-sensitized and gold plasmon-enhanced cathodic photoelectrochemical (PEC) biosensor on the basis of p-type covalent organic polymers (COPs) for the signal-on measurement of M.SssI methyltransferase (M.SssI MTase). The cathodic PEC biosensor is constructed by the in situ growth of p-type COP films onto a glass coated with indium tin oxide and the subsequent assembly of biotin- and HS-labeled double-stranded DNA (dsDNA) probes onto the COP film via biotin-streptavidin interaction. The dsDNA probe contains the recognition sequence of M.SssI MTase. The COP thin films possess a porous ultrathin nanosheet structure with abundant active sites, facilitating the generation of a high photocurrent compared with the hydrothermally synthesized ones. The presence of DNA methyltransferases can prevent the digestion of restriction endonuclease HpaII, consequently inducing the introduction of gold nanoparticles (AuNPs) to the dsDNA probes via the S-Au bond and the intercalation of rhodamine B (RhB) into the DNA grooves to produce a high photocurrent due to the dye-photosensitized enhancement and AuNP-mediated surface plasmon resonance. However, in the absence of M.SssI MTase, HpaII digests the dsDNA probes, and neither AuNPs nor RhB can be introduced onto the electrode surface, leading to a low photocurrent. This cathodic PEC biosensor possesses high sensitivity and good selectivity, and it can screen the inhibitors and detect M.SssI MTase in serum as well.

Tyrosinase and laccase-producing Bacillus aryabhattai TFG5 and its role in the polymerization of phenols

BackgroundTyrosinases and laccases are oxidoreductase enzymes that are used widely in the food, feed, textile, and biofuel industries. The rapidly growing industrial demand for bacterial oxido-reductases has encouraged research on this enzyme worldwide. These enzymes also play a key role in the formation of humic substances (HS) that are involved in controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants besides increasing carbon sequestration and mitigating greenhouse gas emission in the environment. The present study aimed to screen and characterize extracellular tyrosinase and laccase-producing soil bacteria that could be utilized in the polymerization of phenols.ResultsTwenty isolates from different soil samples collected from forest ecosystems were characterized through ARDRA using restriction digestion with AluI, HpaII, and HaeIII restriction enzymes. The results of Hierarchical Cluster Analysis (HCA) revealed a 60 % similarity coefficient among 13 out of 20 isolates, of which, the isolate TFG5 exhibited only 10 % similarity when compared to all the other isolates. The isolate TFG5 exhibited both tyrosinase (1.34 U.mL− 1) and laccase (2.01 U.mL− 1) activity and was identified as Bacillus aryabhattai. The increased polymerization activity was observed when B. aryabhattai TFG5 was treated with phenols. The monomers such as catechol, p-Hydroxy benzoic acid, ferulic acid, and salicylic acid were polymerized efficiently, as evidenced by their FT-IR spectra depicting increased functional groups compared to the standard mushroom tyrosinase.ConclusionsThe polymerization ability of B. aryabhattai TFG5 could be applied to phenol-rich wastewater treatment for efficient precipitation of phenols. Furthermore, tyrosinases can be used for enhancing the synthesis of HS in soil.

Profiling system of oral microbiota utilizing polymerase chain reaction-restriction fragment length polymorphism analysis

ObjectivesProfiling of oral microbiota has traditionally been performed using conventional methods. These methods are relatively time-consuming and labor-intensive. Metagenomic analysis of oral microbiota using high-speed next-generation sequencing is a highly promising technology. However, it is expensive. This study sought to develop a simple and cost-effective profiling method for oral microbiota using 16S rRNA gene polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of PCR-amplified 16S ribosomal RNA genes. MethodsOral isolates of 59 bacterial species from human saliva, including Streptococcus, Actinomyces, and Veillonella, were cultured anaerobically on CDC Anaerobe 5% sheep blood agar plates. Genomic DNA was extracted from single colonies and 16S rRNA genes were PCR-amplified using the 27F and 1492R universal primers. The PCR products were purified and characterized by single digestion with HpaII restriction endonuclease. 16S rRNA gene sequences were obtained from the GenBank database, and the expected restriction profiles were compared with the RFLP patterns obtained from agarose gel electrophoresis. ResultsSixty-five RFLP patterns were obtained from 27 genera and 59 species. The expected fragment sizes of these species were calculated based on GenBank 16S rRNA gene sequences. Fifty-nine patterns were obtained from the analysis of GenBank sequences. The RFLP patterns produced with HpaII distinguished many oral bacterial species. RFLP patterns enabling identification of oral bacteria were generated. The 16S rRNA gene PCR-RFLP analysis did not require expensive equipment and reagents and was cost-effective. ConclusionPCR-RFLP analysis based on 16S rRNA genes could be an alternative method for oral microbiota analysis in smaller laboratories.

Digital Loop-Mediated Isothermal Amplification-Based Absolute Methylation Quantification Revealed Hypermethylated DAPK1 in Cervical Cancer Patients.

The aberrant methylation of many genes has been reported to be associated with various carcinomas. Accurate detection of the methylation level could provide critical insights into the diagnostic analysis of diseases. Here, a sensitive HpaII-edited absolute droplet loop-mediated isothermal amplification (HEADLAMP) method based on methylation-sensitive restriction enzyme (MSRE) HpaII was developed for the digital quantification of DNA methylation. Methylation levels of the death-associated protein kinase 1 (DAPK1) gene that is associated with many cancers were studied using β-actin as an internal reference. DAPK1 (2.5 pM) with 0.01% methylation (250 aM) can be detected with the conventional HpaII-edited LAMP assay. Using HEADLAMP, as low as 1% methylation level can be distinguished with an estimated limit of detection of 5 aM (ca. 3 copies/μL). Moreover, HEADLAMP can detect low levels of methylated DAPK1 in normal L-02 cells, while the conventional assay cannot. Finally, HEADLAMP was applied to the detection of DAPK1 methylation in 20 clinical tissue samples, which revealed hypermethylated DAPK1 in cervical cancer patients. We envisage potential applications of this robust, specific, and sensitive HEADLAMP assay in epigenetic studies and early clinical diagnosis.

A Host-Guest Interaction-Based and Metal-Organic Gel-Based Biosensor with Aggregation-Induced Electrochemiluminescence Enhancement for Methyltransferase Assay.

Metal-organic gels (MOGs) are new soft materials with the characteristics of high colloidal stability, superb luminescence properties, and facile synthesis. Herein, we develop for the first time a host-guest interaction-based and MOG-based biosensor with aggregation-induced electrochemiluminescence (ECL) enhancement for M.SssI methyltransferase (M.SssI MTase) assay. This biosensor employs a MOG as the luminophor and potassium persulfate as the coreactant, and the formation of the Ag-MOG from the aggregation of silver nanoclusters can induce significant ECL enhancement. Two complementary single-stranded DNAs (ssDNAs, i.e., biotinylated DNA-1 and Fc-labeled DNA-2) that contain specific recognition sequence 5'-CCGG-3' can form a double-stranded DNA (dsDNA) probe. In the absence of M.SssI MTase, the dsDNA probe will be digested by restriction endonuclease HpaII, leading to the release of Fc from magnetic beads (MBs). The β-CD can specifically recognize the released Fc through guest-host interaction, resulting in the quenching of an ECL signal. In contrast, the presence of M.SssI MTase enables the formation of fully methylated dsDNA, which cannot be cleaved by HpaII, making Fc remain on the MB surface and consequently generating an improved ECL signal. This biosensor can specifically detect M.SssI MTase with a linear range of 0.05-100 U mL-1 and a limit of detection of 3.5 × 10-3 U mL-1, and it enables accurate detection of M.SssI MTase in human serum. In addition, it can be used for inhibitor screening, with wide applications in drug discovery and disease diagnosis.

Construction of an Enzyme-Free Initiator-Replicated Hybridization Chain Reaction Circuit for Amplified Methyltransferase Evaluation and Inhibitor Assay.

The enzyme-free nucleic acid amplification circuit, for example, hybridization chain reaction (HCR), has paved a broad avenue for evaluating various enzyme-involved biotransformations, including DNA methyltransferases (MTases). The nonenzymatic MTase-sensing platform has supplemented a versatile toolbox for monitoring aberrant methylation in intricate biological samples, yet their amplification efficiency is always constrained by the initiator-depletion paradigm. Herein, the autonomously initiator-replicated HCR (IR-HCR) was developed as a versatile amplification system for detecting MTase with ∼100-fold sensitivity of the conventional HCR system. The initiator I-triggered HCR leads the assembly of a tandem DNAzyme concatemer that cleaves its substrate. This leads to the cyclic replication of a new initiator I for reversely motivating the initial HCR circuit, resulting in a dramatic Förster resonance energy transfer (FRET) readout. Without M.SssI MTase, hairpin HM can be recognized and digested by restriction endonuclease HpaII to release initiator I for stimulating a high FRET signal. While the M.SssI-methylated HM prohibits the HpaII-mediated cleavage of HM, the caged initiator I fails to trigger the IR-HCR circuit. Based on a systematic investigation, the IR-HCR circuit readily achieves selective and sensitive analysis of M.SssI MTase and its inhibitors. As a general MTase-sensing platform, the IR-HCR principle was further applied to analyze another MTase (Dam) by redesigning HM with the Dam recognition sequence. Overall, the versatile homogeneous MTase sensing platform was achieved via an efficient and robust initiator replication amplification circuit and may have enormous potential for early disease diagnosis.

Genetic Polymorphism of Endothelial Nitric Oxide Synthase in Sickle Cell Disease Patients in South-Eastern Nigeria

Background: Sickle cell disease (SCD) is an inherited blood disorder resulting from a point mutation of thymine (T) for adenine (A) at codon 6 of the gene for beta-globin chain of haemoglobin, leading to substitution of Valine for Glutamate at position 6 of the amino acid sequence, thereby shifting the isoelectric point of the protein. The single nucleotide polymorphism (SNP) (T-786C) in the 5’ promoter region which affects the expression of endothelial nitric oxide synthase (ENOS) gene is critical in determining the predisposition of humans to diseases including SCD. This work was therefore aimed at investigating the presence of SNP (T-786C) in the 5’ promoter region of ENOS gene in patients with SCD with respect to controls, visiting the University of Nigeria Teaching Hospital (UNTH) Ituku-Ozalla, Enugu State, south-Eastern Nigeria. Materials and Methods: The study recruited a total of 50 subjects including 20 SCD patients (SS), 15 sickle cell carriers (AS) and 15 healthy non-carriers (AA). DNA was extracted from venous blood samples; PCR amplification was done using specified primers to the region of polymorphism, and finally followed by HpaII restriction enzyme digestion. Results: The results obtained showed only TT genotype across all samples, and so could not establish any relationship between the SNP T-786C ENOS gene region and SCD. There was presence of the same genotype in both test and control groups. Conclusion: This research findings therefore suggest that T-786C ENOS gene polymorphism is not associated with SCD in south-Eastern Nigeria and thus cannot genetically influence the phenotypic outcome of the disease in the clime.

Occurrence and identification of a phytoplasma associated with Pinus brutia witches’ broom disease in Isfahan, Iran

Between 2016 and 2017, symptoms of witches’ broom, proliferation, drying of twigs and ball-like structures formation were observed in Pinus brutia in urban green areas and parks in Isfahan (Iran). All samples from affected P. brutia trees resulted positive in nested-PCR using primer pairs targeting the phytoplasma 16S rRNA gene. Amplicons were obtained only from all the symptomatic pine tree samples and RFLP analyses of R16F2n/R16R2 amplicons with KpnI, AluI, HaeIII, HhaI, HpaII, MseI, RsaI,ThaI, BfaI and TaqI restriction enzymes showed the presence of phytoplasmas enclosed in the 16SrVI group. Four amplicons were sequenced and showed 100% identity to each other. One of these sequences was deposited in the GenBank database (accession number MK158098) and the virtual RFLP pattern was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group VI, subgroup A confirming that the phytoplasma detected in pine is a member of 16SrVI-A subgroup. This is the first report of a 16SrVI-A phytoplasma strain associated with pine tree witches’ broom (P. brutia) disease.

Occurrence of ‘Candidatus Phytoplasma omanense’-related strains and other phytoplasmas in Sophora alopecuroides plants showing dwarfing and yellowing

Sophora alopecuroides dwarfing and yellowing symptoms were observed at the edge of several Damaneh and Fereydun Shahr (Isfahan province, Iran) fields. Polymerase chain reaction using primers P1/P7 and nested PCR using the same primers followed by R16mF2/R16mR2 and R16F2n/R16R2 primer pairs amplified the 16S ribosomal RNA-encoding gene in all the symptomatic plants tested, but not in the asymptomatic ones. RFLP analysis of R16F2n/R16R2 amplicons with AluI, HaeIII, HhaI, HpaI, HpaII, RsaI, MseI and TaqI restriction enzymes showed profiles referable to 16SrII, 16SrVI, 16SrIX, 16SrXII and 16SrXXIX phytoplasma ribosomal groups indicating also the presence of mixed infection in several samples. The identity percentage and the phylogeny obtained after direct sequencing of selected 16S rRNA-encoding gene sequences (R16F2n/R2 amplicons) from symptomatic S. alopecuroides confirmed the presence of phytoplasma strains related to ‘Candidatus Phytoplasma omanense’ (group 16SrXXIX), ‘Ca. P. australasia’ (group 16SrII), and ‘Ca. P. trifolii’ (16SrVI). Moreover, in a few samples/amplicons, 16SrXII-A, 16SrIX-H, 16SrVI-F and 16SrXXIX-A phytoplasma subgroups were identified by real and virtual RFLP analyses. The other phytoplasmas detected were classified in subgroups 16SrII-D and 16SrVI-A. This is the first report of the presence of 16SrII and 16SrXXIX phytoplasma groups and of mixed infection of phytoplasma strains belonging to groups 16SrII, 16SrVI, 16SrIX, 16SrXII and 16SrXXIX in S. alopecuroides worldwide.

Identification of phytoplasma strains associated with witches’ broom and yellowing in Ziziphus jujube nurseries in Iran

Phytoplasma symptoms, including proliferation, witches’ broom, leaf rolling and yellowing, were observed in jujube (Ziziphus jujube) nurseries in the East of Iran. Total nucleic acid was extracted from symptomatic and symptomless plants, and was tested for phytoplasma presence using nested PCR. Amplicons of about 1.8 kb (primer pair P1/P7) and 1.25 kb (R16F2n/R16R2) were obtained from all symptomatic plants but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons using KpnI, HaeIII, RsaI, AluI, HpaII, HhaI, TaqI, MseI, BfaI and ThaI restriction enzymes showed two RFLP patterns referable to 16SrI and 16SrVI phytoplasma groups. The consensus sequences of Z. jujube yellowing and witches’ broom of six samples correspond to ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma trifolii’-related strains. Two R16F2n/R16R2 16S rDNA sequences representative of each RFLP profile, one each from witches’ broom (accession number MK379605) and yellowing (MK379604) host symptoms, were submitted to the GenBank. Phylogenetic analysis confirmed that the phytoplasma strains associated with jujube yellowing clustered within the 16SrI phytoplasma clade, and those associated with witches’ broom clustered within the 16SrVI clade. Restriction analysis confirmed that virtual RFLP patterns of the jujube yellowing and witches’ broom phytoplasma strains were identical to the reference pattern of 16SrI-B and 16SrVI-A. This is the first report of these phytoplasma strains associations with witches’ broom and yellowing in jujube plants.