Restriction Endonucleases EcoRI Research Articles

Workflow for development of CAPS markers with one type of restriction enzyme to identify citrus cultivars

Given the ease of propagating fruit tree species through cloning, the economic viability of their breeding programs hinges on protecting breeders' rights. This necessitates the development of highly accurate DNA markers for cultivar identification. Here, we present a methodology for the rapid design of cleaved amplified polymorphic sequence (CAPS) markers to discriminate newly bred Japanese citrus cultivars from genetically related cultivars. We first compared the performance of ddRAD-seq and MIG-seq in citrus germplasm. The ddRAD-seq libraries generated using EcoRI and HindIII restriction enzymes yielded the highest number of polymorphisms. Subsequently, ddRAD-seq with EcoRI and HindIII was employed to analyze 29 citrus cultivars and thus identify 331,801 genome-wide polymorphisms. A semi-automated bioinformatics pipeline was then utilized to identify candidate CAPS markers, resulting in the discovery of 14,072 potential markers. Of these candidates, 52 were chosen for validation based on their recognition by the PstI restriction enzyme. This evaluation resulted in the development of 11 highly discriminative CAPS markers. Remarkably, a combination of only six such markers was sufficient to differentiate newly bred cultivars from their genetically related parents. The single restriction enzyme employed for these markers facilitates straightforward multiplexing. Finally, a combination of one multiplex marker testing two loci and four singleplex markers was successfully selected that completely discriminated the cultivars other than the bud sports used in this study. The pipeline established here extends beyond citrus and has the potential to simplify marker development and cultivar protection in various plant species.

Cloning and Optimization of Intracellular Expression of Human Interferon β-1a in Pichia pastoris GS115

Abstract Background: Interferon-beta (IFN-β) is a cytokine with a wide range of biological and pharmaceutical applications, including multiple sclerosis (MS), cancer, some autoimmune disorders, and viral infectious diseases. Thus, many studies have been performed to develop novel strategies for the high-yield production of functional IFN-β in a cost-effective approach. Here, we aimed to improve the intracellular expression of IFN-β-1a in Pichia pastoris. Materials and Methods: The gene of IFN-β-1a was successfully sub-cloned into the pPICZA vector. The recombinant vector was transfected to P. pastoris GS115 cells by electroporation. After screening positive P. pastoris transformants, the expression of IFN-β-1a was evaluated and the cultivation conditions, including temperature, time of incubation, and methanol concentration, were optimized. The protein expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results: The double digestion with EcoRI and XhoI restriction enzymes and sequence analysis confirmed the correct sub-cloning of the IFN-β-1a gene into pPICZA. SDS-PAGE analysis showed that the highest level of IFN-β-1a (25 mg per 1 L of yeast culture) was produced with 2% methanol at 28°C after 72 h incubation. Conclusion: Optimization of cultivation conditions for intracellular expression of IFN-β-1a was successfully performed. This approach can be generally applied to improve the production yield and quality of other recombinant proteins in P. pastoris.

Generation of an Inducible Avp Knockout Mouse Line Using CRISPR/Cas9 and Cre/LoxP

Background: Arginine vasopressin (AVP) is a peptide hormone coded by the Avp gene, synthesized in the hypothalamus and secreted by the posterior pituitary. Dysregulation of AVP secretion contributes to a variety of human diseases. Previous studies of functional roles of AVP have been largely dependent on the use of Brattleboro rats, which manifest a spontaneous mutation in the Avp gene and lack circulating AVP. Despite their utility, Brattleboro rats are diffcult to breed owing largely to the fixed nature of the Avp mutation, resulting in increased neonatal death and behavioral effects in the adults. Consequently, commercial breeders have ceased production, despite a continued need. To fill this need, we have now created a mouse model with a tamoxifen-inducible deletion of AVP. Methods: We employed CRISPR/Cas9 to target the desired regions (first intron and 3' downstream) within the Avp gene. Successful insertion of the two loxP sites was confirmed by PCR using primers flanking the targeted regions. Restriction enzymes BamHI or EcoRI were used to confirm correct targeting. For additional confirmation, PCR products were cloned into the TA-cloning vector and sequenced. Mice harboring the floxed allele were mated to B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J mice that globally express a tamoxifen-inducible Cre recombinase. Results: The resultant homozygous conditional knockout mice ( Cre+ Avpflx/ flx) are viable, fertile, and show no signs of polydipsia or polyuria prior to induction, indicating that the conditional alleles retain their wild-type function. Induction of Cre-mediated recombination by administration of tamoxifen (2-3 mg q.d., i.p. for 5 days) in 8-week-old mice resulted in a decrease in urine osmolality from 2076±138 to 122±6 mOsm/kgH2O on day 31 after induction (p<0.0001). Sanger sequencing demonstrated the expected 1245 bp deletion at the Avp locus. The kidney parenchyma was structurally normal with no evidence of medullary atrophy 6 weeks after induction. Immunoblotting of AQP2 in the inner medulla showed a significant decrease in AQP2 band density in Cre+ Avpflx/ flx mice to 27±14 % of values in Cre− floxed control mice (p<0.05). Conclusion: We developed an inducible AVP knockout mouse line ( Cre+ Avpflx/ flx) that will be shared with the research community and are likely to be useful for further study of regulation of water balance and polycystic kidney disease, as well as neural, vascular, and metabolic roles of vasopressin. The work was funded by the Division of Intramural Research, National Heart, Lung and Blood Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Molecular approach for identification and validation of Eurycoma longifolia, Labisia pumila, and Orthosiphon stamineus in herbal products

BackgroundEurycoma longifolia (E. longifolia), Labisia pumila (L. pumila), and Orthosiphon stamineus (O. stamineus) are popular species known for their therapeutic properties. An increase in local demand for herbal products makes them susceptible to adulteration, which poses a risk to their safety and efficacy. Current identification methods, such as organoleptic, microscopic, and macroscopic analysis, need to be revised to identify plant species in highly processed herbal products due to their limited ability to detect morphological features and provide comprehensive plant taxonomy information.MethodsThis research objective was to develop a simple, reliable, and accurate DNA molecular identification method based on polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) for E. longifolia, L. pumila, and O. stamineus, used to validate the species identification for herbal products. PCR–RFLP was developed for rapid identification using restriction enzymes TaqI, BamH I, HinfI, EcoRI, EcoRV, Mbol, and Mspl.ResultsThe nuclear DNA internal transcribed spacer 2 (ITS2) sequences were identified and compared between plant specimens of E. longifolia, L. pumila, and O. stamineus and 101 samples of commercial herbal products. Plant specimens of E. longifolia, L. pumila, and O. stamineus were successfully identified with high similarity of 100%, 100%, and 99.33%, respectively, based on National Center for Biotechnology Information (NCBI) GenBank. The recovery of DNA sequences from the herbal products was 60.4%, of which 81.97% were identified, and 18.03% showed no sequence through Basic Local Alignment Search Tool (BLAST) identification.ConclusionA reliable approach for identifying and validating plant species in herbal products has been created using restriction enzymes. This simple and accurate PCR–RFLP approach efficiently identifies E. longifolia, L. pumila, and O. stamineus by analysing ITS2 sequences, assuring consumer health and safety.

Molecular Detection and Identification of Candida Species Isolates from Oral by RFLP-PCR

In this study, 10 local isolates from a total of 50 samples of Candida sp. were collected from oral swabs of patients with oral infections in Mosul hospitals. The isolates were diagnosed based on culturing, microscopic and biochemical characteristics, and then molecular methods. The first diagnosis by culturing, microscopic and biochemical tests found the isolates were identified as Candida sp. The ITS region was amplified using universal primers (ITS4-ITS5), The PCR product was size (510-721) bp. Performing RFLP-PCR using MspI, HhaI,and EcoRI, restriction enzyme to detect and identify Candida species, the results showed the presence of the cutting sequence of MspI and HhaI enzymes in the genomic DNA content of local isolate and the absence of the sequences for the EcoRI restriction enzyme. Two Candida species were identified (C. krusei and C. the basis of size and fragment sequences then compared with sequences of standard strains from the gene bank in previous studies. Therefore, it can be observed that there is a genetic variation between the local isolates and that there are different genotypes of rDNA 5.8S have been diagnosed in 10 isolates after the cutting process with three restriction enzymes. We conclude from this study that the RFLP-PCR technique was the best in diagnosing and identifying Candida species compared with traditional methods. and we are d the genetic variation between local isolates.

An enzyme-responsive electrochemical DNA biosensor achieving various dynamic range by using only-one immobilization probe

Developing a simple and easy-to-operate biosensor with tunable dynamic range would provide enormous opportunities to promote the diagnostic applications. Herein, an enzyme-responsive electrochemical DNA biosensor is developed by using only-one immobilization probe. The immobilization probe was designed with a two-loop hairpin-like structure that contained the mutually independent target recognition and enzyme (EcoRI restriction endonuclease) responsive domains. The target recognition was based on a toehold-mediated strand displacement reaction strategy. The toehold region was initially caged in the loop of the immobilization probe and showed a relatively low binding affinity with target, which was improved via EcoRI cleavage of immobilization probe to liberate the toehold region. The EcoRI cleavage operation for immobilization probe demonstrated the well regulation ability in detection performance. It showed a largely extended dynamic range, a significantly lowered detection limit and better discrimination ability toward the mismatched sequences whether in two buffers (with high or low salt concentrations) or in the serum system. The advantages also includes simplicity in probe design, and facile biosensor fabrication and operation. It thus opens a new avenue for the development of the modulated DNA biosensor and hold a great potential for the diagnostic applications and drug monitoring.

Tối ưu quy trình phân lập hệ gen virus BK từ mẫu máu, nước tiểu của bệnh nhân sau ghép thận

BK polyomavirus (BKV) is a common opportunistic pathogen in the community, infecting humans in their early life stages, often remaining in a latent state. BK virus-associated nephropathy occurs in 2 to 10% of kidney recipients, and up to 40 to 90% of these patients have permanent loss of graft function due to delay in diagnosis. In the present study, we have successfully optimized the component and thermal cycling for the long-PCR assay to amplify the BKV genome, including middle-concentration samples such as plasma (concentration about 105 copy/ml). We have successfully amplified the BKV genome in 41/50 samples, including 04 plasma and 39 urine samples. The optimized amplification protocol of full-length BKV genome includes the following steps: 1) Extraction of DNA-BKV from plasma and urine samples and quantification of BKV-DNA concentrations to ensure that about 105 copies/ml (plasma) and 107 copies/ml (urine); 2) Each full-length genome reaction in the volume of 50 μl containing 1X Phusion HF master mix (Thermo Scientific, USA); 0.5 μM of wBK_a(F+R) primers, 3-5 μl of genomic DNA. Cycling conditions were 98°C for 30 s, followed by 35 cycles of 98°C for 10 s; 58°C for 20 s; 72°C for 2 min, and 72ºC for 6 min and a 4oC hold; 3) All 5.1 kb purified PCR were verified and confirmed by restriction enzymes XbaI, EcoRI, and direct sequencing.

S9.5d Genotyping of Malassezia species from seborrheic dermatitis/dandruff patients

S9.5 Malassezia: pathogenesis and disease, September 23, 2022, 4:45 PM - 6:15 PM ObjectivesSeborrheic dermatitis/dandruff (SD/D) are common chronic inflammatory skin disorders characterized by recurrent greasy scales with erythema and itchiness. Malassezia lobose and M. restricta are the predominant agents associated with SD/D. The aim of the study was to differentiate Malassezia strains causing SD/D from commensal using a highly discriminatory DNA fingerprinting technique, Fluorescent Amplified Fragment Length Polymorphism (FAFLP).MethodsA total of 154 (M. globosa, n = 85; M. restricta, n = 55; M. arunalokei, n = 14; standard strains, n = 3) isolates from SD/D patients and healthy controls were analyzed using FAFLP. DNA sample was digested with restriction enzymes EcoRI and HindIII, and the fragments were linked to prepared adapters. Pre-selective followed by selective amplification reactions were performed using EcoRI-AC [6-carboxyfluorescein (6-FAM) labeled] and HindIII-T selective primers. The similarity coefficient was determined by Pearson correlation with negative similarities clip to zero. The densitometric curve cluster analysis was performed by unweighted pair group method with arithmetic means using Bionumerics software. The similarity co-efficient of 70% was taken as a cut-off.Results M. restricta and M. arunalokei isolates had a similarity co-efficient of 20%, whereas it was 50% among M. restricta and 60% among M. arunalokei isolates (Fig. 1). Majority of the isolates (n = 38.69%) clustered to form FAFLP type-I, followed by type-II (n = 14.25%), and type-III (n = 3.6%). All three FAFLP types formed tight clustering among themselves (>80% similarity co-efficient) showing less genetic diversity within each FAFLP type. Type-II clustered strains were isolated from the scalp of SD/D patients (P <.05). M. arunalokei isolates were clustered into two FAFLP types (P = .05). All FAFLP type-I isolates (n = 10,71%) were from the scalp of moderate and severe SD/D patients, whereas all type-II isolates (n = 4,29%) were from nasolabial folds of either SD/D patients or healthy controls. All 11 FAFLP types of M. globosa formed very loose structured cluster showing high genetic diversity in intra and inter-FAFLP types (Fig. 2). Majority of the isolates (n = 63,74%) clustered to form FAFLP type-II, followed by type X (n = 5.6%), type-I (n = 4.5%), type-III (n = 3.3.5%), type-IV (n = 3.3.5%), type-V (n = 2.2%), type-VIII (n = 2.2%), and type-VI, VII, IX, XI (n = 1.1% each). No geographic specific isolate clustering was observed in FAFLP.Conclusion M. restricta type-II and M. arunalokei type-I strains were isolated only from the scalp of moderate to severe forms of SD/D. However, no such severity-specific clustering was observed in M. globosa. The results strongly suggest the role of certain genotypes of M. restricta and M. arunalokei in the causation of SD/D.

Enhanced Production, Cloning, and Expression of a Xylanase Gene from Endophytic Fungal Strain Trichoderma harzianum kj831197.1: Unveiling the In Vitro Anti-Fungal Activity against Phytopathogenic Fungi.

Trichoderma sp. is extensively applied as a beneficial fungus for the management of plant diseases, plant growth promotion, induced resistance, and plays an important role in global sustainable agriculture. This study aimed to enhance the production of microbial xylanase in high titer from the endophytic fungus Trichoderma harzianum kj831197.1, and the cloning of xylanase genes in E. coli DH5α using a pUC19 vector. A combination of glucose, 0.1 mM, Tween 80 with lactose, and 2 mM galactose combined with malt extract boostedthe enzyme production. Xylanase production was maximized at a pH of 5.0, temp. of 30 °C, and agitation of 150 rpm in the presence of malt extract and bagasse as the best nitrogen source and waste, respectively, using submerged fermentation. The molecular weight of highly purified xylanase was 32 KDa, identified using SDS-PAGE. The xylanase gene of T. harzianum kj831197.1 was screened in fungal DNA using definite primers specified in the gene bank database. The identified region was excised using restriction enzymes HindIII and EcoRI and cloned into a pUC19 plasmid vector. Optimization of fermentation conditions improved xylanase production about 23.9-fold.The antifungal efficacy of xylanase toward different phytopathogenic fungi was determined. The highest inhibition was against Corynespora cassiicola, Alternaria sp., Fusarium oxysporum, and Botrytis fabae. This study offered an economical, simple, and efficient method using Trichoderma harzianum kj831197.1 for the production of the xylanase enzyme via the submerged fermentation method.

Quantum biology. π–π entanglement signatures in protein-DNA interactions

The biological functions of DNA are carried out by individual proteins that interact with specific sequences along the DNA in order to prime the molecular processes required by the cellular metabolism. Protein-DNA interactions include DNA replication, gene expression and its regulation, DNA repair, DNA restriction and modification by endonucleases, generally classified as enzymatic functions, or transcription factors functions. To find specific binding target sequences and achieve their aims, in less than one second proteins operate in symbiosis with a crowded cellular environment, identifying extremely small cognate sequences along the DNA chain, which range from 15–20 bps for repressors to 4–6 bps for restriction enzymes. In a previous work, we proposed that the extraordinary ability of proteins to identify consensus sequences on DNA in a short time appears to be dependent on specific quantum signatures such as the entanglement of π–π electrons between DNA nucleotides and protein amino acids, where the couple of π electrons function as a radical pair, one π electron is located on a specific site of sequence to be identified and the other one performs a quantum walk to identify possible sites of consensus sequence. In this paper, we use the restriction endonucleases enzymes, EcoRV and EcoRI as a case study. These enzymes are able to recognize 3′-GATACT-5′ or 3′-GAATCT-5′ sequences, respectively. We exploit the analogy of a coin operator with a Bloch sphere to demonstrate that the entanglement between π–π electrons generated at the contacts on specific GA dimers between proteins and DNA relies on the spin of the electrons that form an initial singlet state. The latter is a maximally entangled state so that the identification of specific nucleotides is associated with the formation of singlet states. On the other hand, during the identification of subsequent GA dimers, the spin–orbit interaction on walking π electron induces triplet transitions so that singlet–triplet transitions should manifest an experimentally measurable effect. We propose that the possible experimental evidence of entanglement between π–π electrons may be due to the phosphorescence signal correspondence to triplet decay processes.

Association of Lactoferrin (LTF) Gene Variation to Milk Yield in Indonesian Friesian Holstein

Abstract Lactoferrin (LTF) is a gene that play role in dairy cattle’s health and immunity. Since animal health is an integral factor supporting milk production in dairy cattle, the purpose of the current study was to find an association between LTF gene variation and the amount of milk yield in Indonesian Friesian Holstein (IFH). Fifty cows were sampled in this study. The PCR – RFLP reaction using EcoRI restriction enzyme was employed to genotype the LTF in individual cow samples. As the result, AB and AA genotype was found, and it has frequencies 0.58 and 0.42 respectively. The AB genotype shows a higher amount of milk per lactation when compared to the AA genotype (3741 ± 1520.29 kg vs. 3338 ± 1317.57 kg), but not significantly different (p &gt; 0.05). In this studied dairy cattle population, the variation of the LTF gene didn’t associate with milk yield.

Identification of ß-Catenin Gene as a Colorectal Cancer Controller in Mice &lt;i&gt;(Mus musculus)&lt;/i&gt; Induced by Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS) Using PCR RFLP Method with EcoR1 and Hinif1

Colorectal cancer is cancer attacking the colon to the rectum. The pathophysiology of colorectal cancer occurs due to several causes, such as changes in normal colonic epithelial cells histopathologically through molecular processes. Another cause is that the adenomatous polyps become colorectal cancer due to the carcinogenesis process. Most colorectal cancers originate from adenocarcinomas. Colon cancer is characterized by the uncontrolled growth of cells in the epithelial lining of the large intestine. The type of this study was laboratory experimental research. The population was 2 mice that had been induced by Azoxymethane (AOM) and Dextran sulfate sodium (DSS) and 1 mouse did not get any treatment for 2 months. From the result, the results of RFLP on PCR products from 3 samples that had been used showed that only one sample showing the presence of the β-catenin gene by marking the formation of a 227bp Deoxy Nucleic Acid (DNA) band and testing the EcoR1 restriction enzyme did not show any cutting of DNA fragments with no DNA bands. The size of 81bp and 146bp for Hinf1 restriction showed a mutation in P3 with the formation of bands of 89bp and 138bp in the large intestine that had been induced by azoxymethane and dextran sodium sulfate. Through this study, it can be seen that the occurrence of mutations in the-catenin gene as a marker of colorectal cancer can be identified using the Hinif1 enzyme with the RFLP PCR method.

Polymorphism Study of POU1F1 Gene in Mandya and NARI-Suwarna Sheep

Background: The mutations in POU1F1 gene result in deficiency of GH, PRL and TSH-â expression and thus have an indirect effect on development and reproduction. The present study was conducted to determine and compare the polymorphism of POU1F1 gene in Mandya and NARI-Suwarna Sheep. Methods: Blood samples were collected from 50 each of Mandya and NARI-Suwarna sheep and genomic DNA was isolated following Miller’s high salt method. PCR-RFLP analysis was performed for exon 3 and exon 4 regions of POU1F1 gene, whereas, PCR-SSCP analysis was performed for exon 6 region of POU1F1 gene. Result: The PCR products of sizes 365 bp, 508 bp and 501 bp for exon 3, exon 4 and exon 6 regions of POU1F1 gene, respectively were successfully amplified. PCR-RFLP analysis of exon 3 and exon 4 regions of POU1F1 gene with AluI and EcoRI restriction enzymes, respectively revealed monomorphism in both Mandya and NARI-Suwarna sheep. The PCR-SSCP analysis of exon 6 region of POU1F1 gene revealed two patterns, P1 and P2 with frequencies of 98 and 2 per cent, respectively in Mandya sheep and 90 and 10 per cent, respectively in NARI-Suwarna sheep. The alignment of P1 and P2 pattern sequences revealed two SNPs (109 G greater than C and 112 T greater than A) in Mandya sheep, whereas 3 SNPs (218 T greater than G, 225 G greater than A and 264 T greater than A) were revealed in NARI-Suwarna sheep. Polymorphism was successfully established at exon 6 region of POU1F1 gene.

Genetic polymorphism of ALDH2 in Indonesia’s Minang ethnic

Background: In some people, acetaldehyde, a toxic product from ethanol oxidation, cannot be oxidized to acetate. The excess of acetaldehyde could cause facial flushing, dizziness, and hypertension when they consume ethanol. This ethanol sensitivity is caused by a deficiency of ALDH2.&#x0D; Objective: This study aims to analyze and count the polymorphism frequency of the ALDH2 gene in Indonesia’s Minang ethnic.&#x0D; Methods: DNA samples were taken randomly from hair bulbous of 60 subjects (male and female, 3rd generation). A nested polymerase chain reaction was conducted to amplify the ALDH2 in the samples. Afterward, restriction fragment length polymorphism (RFLP) was conducted to the amplicons using the EcoRI restriction enzyme. The measured parameters were the distribution of the wildtype, atypical homozygote, and heterozygote.&#x0D; Results: Results showed that out of 60 subjects, 53.33% have an atypical homozygote gene (subjects prone to hypersensitive to alcohol), 28.33% have a heterozygote gene, and 18.33% have a wildtype gene. The frequency of the atypical alleles in Minang ethnic is 0.675.&#x0D; Conclusion: The atypical ALDH2 allele was much higher than the normal ALDH2 allele, in which most participants have atypical homozygote ALDH2, suggesting the samples are sensitive to alcohol.

Investigation and rapid detection of fungal pathogens causing mulberry sclerotial disease in south‐west China

AbstractMulberry sclerotial disease is a destructive disease that afflicts mulberry fruits throughout the world. In recent years, the impact of mulberry sclerotial disease has increased with the development of the mulberry fruit industry. Ciboria carunculoides, C. shiraiana, and Scleromitrula shiraiana are all pathogens causing mulberry sclerotial disease, but the dominant pathogen may be different in different mulberry planting areas. In this study, we investigated the causal agents of mulberry sclerotial disease in Chongqing and Sichuan, the main mulberry planting areas in south‐west China. The results showed that C. carunculoides was the dominant pathogen in the investigated areas, and the disease incidence rate on some widely cultivated mulberry varieties exceeded 90%. Based on the differences in internal transcriber spacer sequences of different pathogens, we developed a method using cleaved amplified polymorphic sequences (CAPS) from digestion with the three restriction enzymes HindIII, EcoRI, and MluI for rapid detection of pathogens on mulberry fruits. Use of this method confirmed that the diseased fruits of some mulberry varieties were infected by a mixture of C. carunculoides and S. shiraiana. Long‐fruit mulberry, which was considered to be resistant to sclerotial disease in the past, was also infected by C. carunculoides. Diseased fruit infected by C. shiraiana or by S. shiraiana alone were not detected. Our results suggest that C. carunculoides is probably the dominant pathogen causing mulberry sclerotial disease in south‐west China. The CAPS method for rapid detection of pathogens could assist diagnosis, epidemiology, and disease monitoring.

CLONING OF SHRNA SEQUENCE SPECIFIC TO ELASTASE OF MACROPHAGES INTO EXPRESSION VECTOR PGPV-17019250

BACKGROUND: Macrophage elastase/MMP12 is one of the most important matrix metalloproteinase that cells secrete into the extracellular space. Secreted and activated MMP12 is capable to digest the proteins of extracellular matrix, such as fibronectin, laminin and elastin, facilitating the migration of immune cells to the inflamed tissue.&#x0D; AIM: The aim of our study was to create a vector that would express a specific small interfering RNA (shRNA) to suppress the expression of human macrophage elastase.&#x0D; METHODS: The online tool "siRNA Wizard" was used to design cDNA encoding shRNA specific to human macrophage elastase. The restriction endonucleases BamH1 and EcoRI, as well as a commercial T4 DNA ligase we used to clone the named cDNA into the expression vector pGPV-17019250. The presence of the cDNA in the vector was confirmed by PCR and DNA-sequencing with vector-specific primers.&#x0D; RESULTS: In this study, we obtained the sequence of cDNA, encoding shRNA specific to macrophage elastase. We also cloned the named cDNA into the expression vector pGPV-17019250 and confirmed its exact location in the vector.&#x0D; CONCLUSION: The generated expression vector pGPV-17019250-EM designated to suppress human macrophage elastase in cultured cells.

Multiplex PCR - RFLP Assay for Identification of Cryptococcus neoformans and Cryptococcus gattii Isolated from Birds’ Droppings and Eucalyptus Trees

Cryptococcus neoformans/ Cryptococcus gattii (C. neoformans/ C. gattii) species complex are encapsulated basidiomycetous yeasts, causing cryptococcosis which is a life-threatening fungal disease of the pulmonary and central nervous system of humans and animals. This study aimed to investigate the recovery rate of C. neoformans and C. gattii from bird droppings and Eucalyptus trees in Egypt as well as the performance of the phenotypic and molecular identification methods for Cryptococcus species identification. Overall, 27 Cryptococcus isolates (13.5%) were isolated from 200 examined samples including 70 pigeon droppings, 50 captive birds’ droppings, and 80 Eucalyptus trees samples. The recoveredisolates were phenotypically identified based on macro- and micro-morphological characters, urease test, and differentiation using cryptococcus differential agar media. Multiplex polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis using EcoRIrestriction enzyme for confirmation of species identification. The molecular methods identified 20 C. neoformansfrom pigeon’s droppings (12/70, 17.14%) and captive birds’ droppings samples (8/50, 16%), as well as 7 C. gattii from Eucalyptus trees (7/80, 8.75%). Molecular identification results did not correspond with those of the phenotypic identification methods in three isolates (11.11%), as phenotypic methods identified only 4 C. gattii isolates and molecular methods identified 7 isolates. In conclusion, multiplex PCR and RFLP analysis of multiplex PCR products are rapid, sensitive, species-specific, and more reliable methods for identification of Cryptococcus species and may be used as a complementary to phenotypic methods to avoid false-negative and false-positive results.

EcoRII Restriction Endonuclease Forms Specific Contacts to the Bases of Its Target Sequence Flipped from DNA in a Transition Complex with Photoactivatable Substrates

The photoactivatable modified oligonucleotides were used to investigate direct contacts formed by the type IIE EcoRII restriction endonuclease and the T/A bases of its recognition site (5'-CCT/AGG). EcoRII dimer consists of a central catalytic core, made of two C-terminal endonuclease-like domains (EcoRII-C) from different subunits, and two N-terminal effector DNA binding domains (EcoRII-N). According to co-crystal structure of isolated EcoRII-C with DNA catalytic dimer EcoRII-C flips nucleotides of the central T/A pair into the enzyme binding pockets. Нere, photocross-linking technique was used to investigate the direct contacts formed by extrahelical T/A bases in the protein pockets of full-length EcoRII within the pre-reactive EcoRII–DNA complex obtained in the presence of Ca2+ in solution. Photoreactive zero-length agent 5-iodo-2'-deoxyuridine (IdU) was introduced as single substituent into the central T/A position of EcoRII recognition site or into the flanking nucleotide sequences of 14-mer DNA substrate. The substitution of only dT or dA residues in EcoRII recognition site resulted in formation of photocross-links upon irradiation only in the presence of Ca2+. Proteolytic digestion of the enzyme-oligonucleotide conjugates followed by MALDI-MS analysis have allowed to identify the 224VEYD227 EcoRII region involved in the formation of the cross-links. This region belongs to the central part of H-10 α-helix. Y226 residue was suggested to form cross-link with T or A bases of EcoRII site replaced by IdU within the pre-reactive complex. The flipped base pair protein pockets of EcoRII seem to accommodate equally well both A and T bases of the DNA substrate. Altogether, IdU-containing photoactivatable DNA substrates have allowed to trap the flipped bases in complex with full-length EcoRII before DNA cleavage in the solution and to identify direct enzyme–DNA contacts important for high specificity of EcoRII for the Т/A nucleotides providing a highly specific cleavage reaction.

In vitro expression of the recombinant fusion protein of Newcastle disease virus from local Indonesian isolates by using a cell-free protein expression system

The aim of this work was the in vitro expression of the recombinant fusion (F) protein of Newcastle disease virus (NDV). The pBT7-N-His-Fusion-NDV expression plasmid which carries the recombinant F protein encoding gene from local Indonesian isolates, was prepared and transformed into E. coli BL21 (DE3). To detect bacterial colonies carrying the recombinant plasmid, a restriction endonuclease analysis was performed using the EcoRI restriction endonuclease. These results showed that the pBT-N-His-Fusion-NDV plasmid was successfully isolated with a size of 4.601 bp, and three recombinant plasmids carrying the gene coding for the recombinant F protein of NDV were obtained. Selected recombinant plasmids were then in vitro by using a cell-free protein expression system followed by visualization of the recombinant F protein on a 12% SDS-PAGE gel both by Coomassie Brilliant Blue staining and Western blotting. Recombinant F protein was successfully in vitro expressed by using a cell-free protein expression system as indicated by a specific single protein band with a molecular mass of 25.6 kDa.

Pseudomonas karstica sp. nov. and Pseudomonas spelaei sp. nov., isolated from calcite moonmilk deposits from caves.

A taxonomic study of two fluorescent Pseudomonas strains (HJ/4T and SJ/9/1T) isolated from calcite moonmilk samples obtained from two caves in the Moravian Karst in the Czech Republic was carried out. Results of initial 16S rRNA gene sequence analysis assigned both strains into the genus Pseudomonas and showed Pseudomonas yamanorum 8H1T as their closest neighbour with 99.8 and 99.7 % 16S rRNA gene similarities to strains HJ/4T and SJ/9/1T, respectively. Subsequent sequence analysis of rpoD, rpoB and gyrB housekeeping genes confirmed the highest similarity of both isolates to P. yamanorum 8H1T, but phylogeny and sequences similarities implied that they are representatives of two novel species within the genus Pseudomonas. Further study comprising whole-genome sequencing followed by average nucleotide identity and digital DNA-DNA hybridization calculations, repetitive sequence-based PCR fingerprinting with the REP and ERIC primers, automated ribotyping with the EcoRI restriction endonuclease, cellular fatty acid analysis, quinone and polar lipid characterization, and extensive biotyping confirmed clear separation of both analysed strains from the remaining Pseudomonas species and showed that they represent two novel species within the genus Pseudomonas for which the names Pseudomonas karstica sp. nov. (type strain HJ/4T=CCM 7891T=LMG 27930T) and Pseudomonas spelaei sp. nov. (type strain SJ/9/1T=CCM 7893T=LMG 27931T) are suggested.