Restriction Endonucleases HindIII 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.

Phosphorus-Nitrogen compounds part 74. Syntheses of (p-dimethylamino)benzyl-spiro-(N/N)-cyclotriphosphazenes: Structural characterizations, bioactivity studies, DFT calculations and reactivity parameters

Reactions of hexachlorocyclotriphosphazene (HCCP, N3P3Cl6), with equimolar amounts of N-methyl-N'-(p-dimethylaminobenzyl)-1,2-diaminoethane (1), N-ethyl-N'-(p-dimethyl-aminobenzyl)-1,2-diaminoethane (2), and N-methyl-N'-(p-dimethylaminobenzyl)-1,3-diami-nopropane (3) produced the corresponding tetrachloro-(p-dimethylaminobenzyl)spiro-(N/N)-cyclotriphosphazenes (4, 5, and 6), regioselectively. Tetrapyrrolidino (4a-6a), tetrapiperidino (4b-6b), tetramorpholinophosphazenes (4c and 6c), dichlorodimorpholinophosphazene (5c) and tetra-1,4-dioxa-8-azaspiro[4,5]decanophosphazenes (DASD) (4d-6d) were synthesized from Cl-substitution reactions of 4, 5 and 6 with excess pyrrolidine, piperidine, morpholine, and DASD, respectively. Their structures were elucidated by MS, FTIR, NMR and single crystal X-ray diffraction (for 4, 4b and 6a) techniques. Hirshfeld surface (HS) analyzes of 4, 4b, and 6a were performed to visualize intermolecular interactions in the crystals. In addition, according to the antimicrobial activity test results, compound 6b shows a better inhibitory effect on S. aureus ATCC 25923 G(+) (MİC: 78.1 µM) and C. tropicalis Y-12968 (MİC: 19.54 µM) microorganisms. The interactions of cyclotriphosphazenes with 5′-G/GATCC-3′ and 5′-A/AGCTT-3′ sequences of DNA were evaluated by examining BamHI and HindIII restriction enzyme digestion. The quantum chemical calculations of 4, 4b, and 6a were performed using DFT, and their experimental parameters were compared with the theoretical ones. Natural bond orbitals (NBOs) were determined and HOMO-LUMO energy gaps (Eg) were calculated as 3.55, 5.23, and 5.24 eV for 4, 4b, and 6a, respectively.

Analysis of Genetic Variation of MatK Gene Sequences in Ammothamnus lehmannii NCBI Popset 2440747918 Using In Silico RFLP

Genetic diversity or genetic variation is variation that occurs in an organism due to differences in the sequence of nucleotide bases (adenine, thymine, guanine and cytosine) that form DNA in cells. Variation genetics can be studied in silico using available gene sequences in the NCBI genbank database. This study used the MatK (Maturase-K) gene sequence with the identity number Popset 2440747918 which was downloaded in fasta format from NCBI . Then screening of restriction enzyme candidates was carried out to determine the restriction enzymes prior to in silico RFLP. The restriction enzyme selected from the screening was the restriction enzyme HindIII which has the recognition site A'AGC_T. The results obtained from 79 samples of DNA sequences, 76 samples were cut and 3 samples were not. And found three allele variations with the percentage of the presence of fragments A1 (86.07%), A2 (10.12%) and A3 (3.79%). The percentage values and frequencies of these A1, A2 and A3 alleles indicate a low level of genetic variation.

Construction, Identification, and Expression of Lactococcus lactis-Based Recombinant Vaccine for Echinococcus granulosus

To construct Lactococcus lactis (LL)-based recombinant LL-Eg95 (rLL-Eg95) vaccine for Echinococcus granulosus (Eg) and to examine its expression efficiency. Eg95 gene was obtained by PCR from the template of pCD-Eg95. Then, pMG36e was inserted in the Eg95 gene after double cleaving with restriction endonucleases XbaⅠ and HindⅢ to construct recombinant plasmid pMG36e-Eg95, which was transformed into E.coli BL2 (DE3) competent cells. The recombinant plasmid was extracted and identified by double restriction endonuclease digestion and was then electroporated into LL MG1363 to construct rLL-Eg95 vaccine. Then, the plamid was extracted and identified by PCR. Examination of the recombinant plasmid by double restriction endonuclease digestion showed that the segment was of the expected length. PCR showed that 471 base pairs of Eg95 gene were amplified when the plasmid extracted from roxithromycin-resistant recombinant LL was used as the template. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the relative molecular mass of the Eg95 protein expressed was approximately 16.5×103 and that the amount of the expressed protein was 17% of the total bacterial proteins. Western blot findings suggested that the expressed protein could be recognized by mice serum infected with hydatid cyst. The rLL-Eg95 vaccine was successfully constructed, expressing Eg95 protein that has specific antigenicity.

Production of HPV16-L1 Through BL21/pET32a Expression System

Background: The human papillomavirus (HPV) main capsid protein L1 is naturally capable of self-assembly as virus-like particles (VLPs). There are different recombinant protein expression systems, such as bacteria, yeast, insect, plant, and mammalian cells, for the generation of VLP-based candidate vaccines targeting various pathogens. In this study, we produced the HPV16-L1 protein by BL21/pET32a expression system, and VLP production was confirmed. Methods: The recombinant plasmid pET32/L1 was transformed into Escherichia coli BL21 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/L1 were assessed by restriction endonucleases HindIII and XhoI and nested polymerase chain reaction (PCR). The expression of HPV16-L1 fusion protein in Escherichia coli BL21 was identified by SDS-PAGE and western blotting. Electron microscopy was used to evaluate VLP formation. Results: A codon-optimized L1 gene was expressed in BL21 under the control of the T7/lac promoter. Purification of L1 protein was achieved after Ni NTA chromatography. The 60 kDa protein was detected in the lysates of BL21, recognized as HPV16- L1 protein by western blotting. The VLPs were confirmed using electron microscopy. Conclusion: In this study, we established an efficient recombinant E. coli expression system for the production of HPV 16- L1 protein. The generated L1 protein was correctly self-assembled into VLPs. Therefore, BL21/pET32a as a prokaryotic expression system is a potent tool for HPV16-L1 VLP production.

Novel multi-epitope vaccine against bovine brucellosis: approach from immunoinformatics to expression

Brucellosis is a zoonotic caused by the Brucella which is a well-known infectious disease agent in domestic animals and if transmitted, it can cause infection in humans. Because brucellosis is contagious, its control depends on the eradication of the animal disease in farms. There are two vaccines based on the killed and/or weakened bacteria against B. melitensis and B. abortus, but no recombinant vaccine is available for preventing the disease. The present study was designed to develop a multi-epitope vaccine against of B. melitensis and B. abortus using virB10, Omp31 and Omp16 antigens by the prediction of T lymphocytes, T cell cytotoxicity and IFN-γ epitopes. 50S L7/L12 Ribosomal protein from Mycobacterium tuberculosis was used as a bovine TLR4 and TLR9 agonist. GPGPG, AAY and KK linkers were used as a linker. Brucella construct was well-integrated in the pET-32a Shuttle vector with BamHI and HindIII restriction enzymes. The final construct contained 769 amino acids, that it was soluble protein of about ∼82 kDa after expression in the Escherichia coli SHuffle host. Modeled protein analysis based on the tertiary structure validation, molecular docking studies, molecular dynamics simulations results like RMSD, Gyration and RMSF as well as MM/PBSA analysis showed that this protein has a stable construct and is capable being in interaction with bovine TLR4 and TLR9. Analysis of the data obtained suggests that the proposed vaccine can induce the immune response by stimulating T- and B-cells, and may be used for prevention and remedial purposes, against B. melitensis and B. abortus. Communicated by Ramaswamy H. Sarma

Diversity of partial sequence leptin gene (Exon 3) in crossbred cattle compared to GenBank database

Abstract. Ermawati D, Panjono, Bintara S, Hartatik T. 2022. Diversity of partial sequence leptin gene (Exon 3) in crossbred cattle compared to GenBank database. Biodiversitas 23: 5614-5620. Genetic marker method that is often used to select cattle is Single Polymorphism Nucleotide (SNP). Single Polymorphisms Nucleotide (SNP) has been found in various candidate genes, one of which is the leptin gene. Leptin is a gene that affects reproduction and cattle weight. Therefore, this research aims to identify SNP of the leptin gene (exon 3) in Crossbred cattle by comparison with GenBank data. A sample of Crossbred cattle was taken from Klaten in Central Java, Indonesia. Afterward, DNA sequencing results were analyzed with BioEdit to identify SNP. BioEdit software was used for DNA sequencing alignment part of intron 2, exon 3 and 3'UTR. The MEGA 11.1.1 program was used to analyze and edit the genetic distance and phylogenetic tree based on the SNP leptin gene. The result shows that nine variations of SNPs were found in exon 3. The phylogenetic tree shows that the Crossbred cattle, Bos taurus, Bos indicus, Hereford, Friesian Holstein and Bali, were in the same cluster. HindIII restriction enzyme can be recommended for genotyping Crossbred cattle using PCR RFLP. This research provides information on SNP in the leptin gene so that it can be used for further research on the association of the leptin gene to growth and reproduction.

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.

Inverse PCR-based detection reveal novel mobile genetic elements and their associated genes in the human oral metagenome

The human oral cavity is one of the hotspots harboring multiple mobile genetic elements (MGEs), which are segments of DNA that can move either within bacterial genomes or between bacterial cells that can facilitate the spreading of genetic materials, including antimicrobial resistance genes. It is, therefore, important to investigate genes associated with the MGEs as they have a high probability of dissemination within the bacterial population under selective pressure from human activities. As one-third of oral bacteria are not yet culturable in the laboratory condition, therefore, in this work, it is aimed to detect and identify the genetic contexts of MGEs in the oral cavity through an inverse PCR (IPCR)-based approach on the oral metagenomic. The human oral metagenome was extracted from saliva samples collected from healthy individuals in Tromsø, Norway. The extracted DNA was partially digested with the HindIII restriction enzyme and self-circularized by ligation. DNA primers targeting each MGE were designed to amplify outwards from the MGEs and used for the IPCR on the circularized DNA products. The IPCR amplicons were cloned into a pCR-XL-2-TOP vector, screened, and sequenced. Out of 40 IPCR amplicons, we confirmed and verified the genetic contexts of 11 samples amplified with primers targeting integron gene cassettes (GCs), IS431 composite transposons, and Tn916 conjugative transposons (tet(M) and xis-int). Novel integron GCs, MGEs, and variants of Tn916 conjugative transposons were identified, which is the first report using the IPCR technique to detect the genetic contexts of MGEs in the oral metagenomic DNA.

Phosphorus-nitrogen compounds. Part 62. Preparation of tetraaminobis(4-fluorobenzyl)spiro(N/N)cyclotriphosphazenes: Chemical, structural characterizations, antimicrobial, antioxidant and DNA-binding activity studies

Substitution reaction of hexachlorocyclotriphosphazatriene (trimer; N3P3Cl6; HCCP) with N,N'-bis(4-fluorobenzyl)ethane-1,2-diamine (1) gave the starting compound tetrachlorobis(4-fluorobenzyl)monospiro(N/N)cyclotriphosphazene (2). Tetraaminobis(4-fluorobenzyl)spiro(N/N) cyclotriphosphazenes (2a-2c) were synthesized from the substitution reactions of spiro (2) with excess piperidine, ethylpiperazine and phenylpiperazine, respectively, in boiling THF. Elemental analysis, ESI-MS, FTIR, 1H, 13C and 31P NMR data verified the structures of new compounds; and the solid-state crystal structures of 2a and 2b were elucidated using the X-ray crystallography method. Hirshfeld surface (HS) analyses were performed to figure out the intermolecular interactions in these crystals as well. Moreover, the minimum inhibitory, bactericidal and fungicidal concentrations (MIC, MBC and MFC) were determined using the microdilution method, which was used to detect the compounds that have the antimicrobial effects on microorganisms exhibited the effect at which concentration. It was concluded that MIC, MBC and MFC values ranged from 312.5 and > 2500 µM. Furthermore, DNA cleavage and unwinding properties of 2a, 2b and 2c with pBR322 plasmid DNA were examined by agarose gel electrophoresis. Also, compound-DNA incubation and BamHI and HindIII restriction enzyme digestions were realized to provide more insight into changes in DNA conformation. Finally, the antioxidant activity was determined that 2c had the highest antioxidant capacity with a 38.38% radical scavenging activity value.

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.

Genetic variations of renin-angiotensin and fibrinolytic systems and susceptibility to coronary artery disease: a population genetics perspective.

Genetic predisposition is an important risk factor in coronary artery disease (CAD).This study was conducted to determine the polymorphism frequencies of the plasminogen activator inhibitor-1(PAI-1) gene 4G/5G, angiotensin-converting enzyme (ACE) gene I/D, and angiotensin II type 1 receptor (AT1) gene A1166C genotypes and to examine the role of these polymorphisms in CAD. Genomic DNAs obtained from 260 subjects (130 CAD patients and 130 control) were used in the study. ACE I/D and PAI-1 4G/5G polymorphism genotypes were determined using polymerase chain reaction (PCR) and electrophoresis. AT-1 A1166C polymorphism was determined using the PCR, restriction fragment length polymorphism (RFLP) and electrophoresis. The products amplified from AT1 gene by PCR were cut with HindIII restriction endonuclease and then analyzed by 2% agarose gel electrophoresis. The results were statistically analyzed with the chi-square test, Mann-Whitney U test, and independent two-sample t-test. Allele frequencies showed statistically significant differences between the patient and control groups. There was no statistically significant difference in ACEI/D genotype frequencies between the twogroups. Likewise, no statistically significant difference was found in the AT1 A1166C genotype frequencies; however, a statistically significant difference was found in allele frequencies. The PAI-1 4G/5G genotype frequency was significantly higher in the patient group. While there is a relationship between of PAI-1 gene 4G/5G polymorphism and CAD, ACE gene I/D and AT1 gene A1166C polymorphisms are not related. PAI-1 gene homozygous genotypes may be considered as a prognostic marker for CAD patients.

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.

Identification of Main Oyster Species and Comparison of Their Genetic Diversity in Zhejiang Coast, South of Yangtze River Estuary

Oysters are an important aquaculture species distributed worldwide, including in Zhejiang Province, located on the east coast of China. Because of the high diversity and complicated introduction history of oysters and their seedlings, there has been much disagreement regarding the origin of each species, and the dominant and indigenous species remain unclear. We sampled 16 batches of oysters from seven sites in three aquaculture bays and found two main oyster species, Crassostrea sikamea and Crassostrea angulata. The former occupied the higher intertidal zone and comprised more than 70% of the cultured oysters. Based on the cytochrome oxidase C subunit I (COI) and mitochondrial noncoding region (MNR), C. sikamea showed higher genetic diversity than C. angulata. The analysis of molecular variance among COI sequences of these species from the Xiangshan Bay populations were comparable to those of other populations and showed that most of the molecular variance was within groups, which was consistent with the low pairwise fixation index FST values. The neutrality test revealed that C. sikamea experienced population expansion events, whereas for C. angulata, the significant Fu’s Fs and non-significant Tajima’s D test results may indicate a possible population expansion event, implying that C. sikamea is likely an indigenous species. The method established based on internal transcribed spacer 1 digestion by the HindIII restriction enzyme is useful for identifying C. sikamea and C. angulata in the local region. The specific primers on the MNR sequence show potential for distinguishing C. sikamea from four other important Crassostrea oysters. These results highlight the abundance of C. sikamea on the Zhejiang coast and lay the foundation for protecting and utilizing the local oyster germplasm resources and for the sustainable development of the oyster industry.

Association of Factor V Leiden G1691A and Prothrombin gene G20210A mutations with adverse pregnancy outcomes.

To determine the association of Factor V Leiden / prothrombin gene mutation in Pakistani women with adverse pregnancy outcomes. The prospective study was conducted at the Aga Khan University Hospital, Karachi, from January 1 to December 31, 2016, and comprised females > 40 years having history of two or more foetal losses with no apparent aetiology. Restriction fragment length polymorphism- Polymerase chain reaction was performed using MnlI and HindIII restriction enzymes for factor V Leiden G1691A and prothrombin gene mutation G20210A. Females with two or more consecutive normal pregnancies were enrolled as the control group. Data was analysed using SPSS 19. Of the 172 participants with a mean age of 29.3±5.9 years (range: 19-38 years). 86(50%) each were healthy controls and those with recurrent pregnancy loss. There were 238 livebirths among the controls compared to 13 in the other group. Factor V Leiden G1691A was identified in 2(2.3%) women, and prothrombin gene mutation G20210A in 1(1.2%) woman in the patient group, while no mutation was identified in the control group. The prevalence of Factor V Leiden / prothrombin gene mutation in women with recurrent pregnancy loss was found to be very low.

SEQUENCING OF THE U6 PROMOTERS IN CASTOR BEANS AND VECTOR CONSTRUCTION FOR CRISPR/Cas9 GENOMIC EDITING ON THEIR BASIS

Castor bean is an important crop in many countries. It is mainly used to obtain the castor oil, which is widely applied in various industries. The rich protein cakes and meals are remains after the oil is pressed. They are promising for use as protein additives in the fodder production. However, it is limited due to the presence of toxins. These are ricin protein and ricinine alkaloid. One of such methods can be genomic editing using the CRISPR/Cas9 system. The technology consists in cutting the target region of the intact DNA by the Cas9 enzyme with the assistance of a short guide RNA fragment. The delivery of the Cas9 and guide RNA genes into the cell of the edited plant is often carried out by plasmid vectors. For efficient synthesis of the guide RNA in such vectors, the promoters of small nuclear RNA genes are usually used. In dicotyledonous plants editing, the promoter of the Arabidopsis U6 gene is most often used. In this work, the amplification, sequencing and analysis of the castor bean U6 promoters were carried out at the first time. The obtained sequences were analyzed and used in the construction of CRISPR/Cas9 vectors for the ricin gene editing. Our aim was to study castor bean U6 promoters with help by bioinformatics and molecular genetic approaches. Zanzibar Green and Gibzonskaya varieties of castor bean plants were used in this work. DNA was isolated from young leaves. The preparation of sequences, alignments and homology level calculation were carried out by GenDoc program (http://www.nrbsc.org/gfx/genedoc/index.html). Primers for amplification of castor bean U6 promoters were designed by Primer3 program (http://bioinfo.ut.ee/primer3-0.4.0/). PCR was performed using a C-100 PCR machine (Bio-Rad Laboratories, Inc., USA). PCR products were separated in 1.5 % agarose gel at 6 B/cm in the Sub-Cell GT electrophoresis camera (Bio-Rad Laboratories, Inc., USA). The amplicons were purified with the GeneJET PCR Purification Kit (Thermo Fisher Scientific, Inc., USA). The purified amplicons were cloned into the pAL2-T vector (Evrogen, Russia) according to protocol of manufacturer. In the CRISPR/Cas9 vector construction, the pRGE31, HindIII and SbfI restriction enzymes (Sibenzyme, Russia) and nucleotides were used. Purification of the digested products was carried out with the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Inc., USA). Bioinformatic search were conducted in the GenBank base, and 12 scaffolds with the castor bean U6 gene were found. Six promoters with intact USE and TATA-box elements of regulation were used in the primer design for amplification with cv. Zanzibar Green and cv. Gibzonskaya DNA matrices. One fragment PCR products were cloned and sequenced. The analysis of the obtained amplicon sequences reveled that promoter regions of two studied varieties were similar. The level of promoter identity from different amplicons ranged within 51-77 %. In comparison of these promoters with ones from other plants, the level of homology was 42-64 %. The promoter sequences with intact USE and TATA-box motifs were used in construction of the CRISPR/Cas9 vectors which can be used for efficient editing of the castor bean genes involved in the ricin and ricinine synthesis.

Expression, Purification, and Bioactivity of a Soluble Recombinant Ovine Interferon-tau in Escherichia Coli.

IntroductionOvine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein.Material and MethodsFirst, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography.ResultsThe protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays.ConclusionPurified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

The rs78378222 prevalence and the copy loss of the protective allele A in the tumor tissue of diffuse large B-cell lymphoma.

BackgroundRare single nucleotide polymorphisms (SNPs) are likely to be a crucial genetic factor for human diseases, including cancer. rs78378222 is rare SNP in 3′-untranslated region (UTR) of TP53 gene leading to disturbance of 3′-end mRNA processing. The frequency of rs78378222 varies in several studied populations. The meta-analysis of 34 genome-wide association studies indicated that rs78378222 was significantly associated with an increased risk of cancer overall. Bioinformatic analysis indicates that somatic loss of the protective A allele of rs78378222 occurs in the tumor tissue of some malignant. The goal of the current study is to document the rs78378222 prevalence and evaluate the copy loss status of the protective allele A in the tumor tissue of patients with diffuse large B-cell lymphoma (DLBCL).MethodsTotal DNA was isolated from FFPE-samples and peripheral blood of patients with DLBCL and comparable in age and sex controls. rs78378222 genotyping was performed by the PCR-RFLP method using restriction endonuclease HindIII. Direct Sanger’s sequencing was used to confirm the presence of C allele of the rs78378222. The search for TP53 gene mutations was carried out by Sanger’s direct sequencing method, according to the IARC protocol.ResultsThe result of genotyping of 136 DNA samples from DLBCL tumor tissue suggested that frequency of the rs78378222 was 11/136 (8.1%). Rare allele C frequency was 11/272 (4.2%). A total of 5/11 DLBCL rs78378222 heterozygous samples had the heterozygosity loss in the TP53 gene. Only one of these cases was combined with TP53 gene mutations which have proven oncogenic potential—p.Arg196Gln, other four cases have not mutations in the coding regions of gene.ConclusionsAt the stages of DLBCL initiation or progression a loss of the protective allele A of rs78378222 occurs. Further efforts are needed to study possible molecular mechanisms underlying somatic alterations in DLBCL in this region of the TP53 3′-UTR as well as functional studies to illustrate how the presents of rs78378222 may affect tumor progression of lymphoma.

Genetic variations of Renin-angiontensin and Fibrinolytic systems and susceptibility to coronary artery disease: a population genetics perspective

Genetic predisposition is an important risk factor in coronary artery disease (CAD).This study was conducted to determine the polymorphism frequencies of the plasminogen activator inhibitor-1(PAI-1) gene 4G/5G, angiotensin-converting enzyme (ACE) gene I/D, and angiotensin II type 1 receptor (AT1) gene A1166C genotypes and to examine the role of these polymorphisms in CAD. Genomic DNAs obtained from 260 subjects (130 CAD patients and 130 control) were used in the study. ACE I/D and PAI-1 4G/5G polymorphism genotypes were determined using polymerase chain reaction (PCR) and electrophoresis. AT-1 A1166C polymorphism was determined using the PCR, restriction fragment length polymorphism (RFLP) and electrophoresis. The products amplified from AT1 gene by PCR were cut with HindIII restriction endonuclease and then analyzed by 2% agarose gel electrophoresis. The results were statistically analyzed with the chi-square test, Mann-Whitney U test, and independent two-sample t-test. Allele frequencies showed statistically significant differences between the patient and control groups. There was no statistically significant difference in ACEI/D genotype frequencies between the twogroups. Likewise, no statistically significant difference was found in the AT1 A1166C genotype frequencies; however, a statistically significant difference was found in allele frequencies. The PAI-1 4G/5G genotype frequency was significantly higher in the patient group. While there is a relationship between of PAI-1 gene 4G/5G polymorphism and CAD, ACE gene I/D and AT1 gene A1166C polymorphisms are not related. PAI-1 gene homozygous genotypes may be considered as a prognostic marker for CAD patients.

Molecular cloning and characterizing of Bacillus subtilis cellulase collected from central-northern Iran forests

In the large-scale commercial bioconversion of complex lignocellulosic materials to biofuels, a cost-effective purification and production of cellulase is a huge concern. In this regard, genetically engineered strains have shown great merits for producing active cellulase. As an attempt to improve cellulase production; herein, bacterial strains with cellulolytic activity were collected from forest soils located across Mazandaran Province, in central-northern Iran. To verify cellulolytic activity, carboxymethyl cellulose (CMC) in aka cellulose gum medium was used. Afterward, in order to characterize the bacteria with most cellulolytic activity, 16S rDNA of bacterial colonies were amplified and then phylogenetic analysis was conducted. Cellulase gene was isolated from the most active bacterial strain, Bacillus subtilis B2 and then it was cloned into the bacterial expression vector pET-26b using HindIII and BamHI restriction endonuclease sites. The generated plasmid containing cellulase and expression promoter system was transformed into the BL21 (DE3) competent Escherichia coli. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed to verify presence of the protein. The sequencing of cloned gene revealed 77% identity with endoglucanase of Bacillus subtilis subsp. Moreover, the molecular weight of cloned cellulase was 48 k Da as revealed by SDS-PAGE. Significance statementHerein, a newly isolated bacterial strain with high potential of cellulase production is introduced that benefits commercial bioconversion of complex lignocellulosic materials to biofuels.