EcoRI Restriction Research Articles

The Identification and Characterization of the Resistance Genes of Klebsiella pneumoniae Plasmids Transformed in E. coli BL21(DE3)

The emergence of multidrug-resistant Klebsiella pneumoniae constitutes a serious threat to global public health due to the limited treatment options available. This study aimed to identify some characteristics carried on the plasmids of K. pneumoniae isolated from pathological cases. The plasmid was extracted from K. pneumoniae and transformed into E. coli BL21(DE3) by the heat shock method using 0.1 M CaCl2. The results showed that transformed E. coli BL21(DE3) became resistant to four types of antibiotics which included cefepime (FEP) 10 μg, ceftazidime (CAZ) 30 μg, tetracycline (TE) 10 μg and rifampin (RA) 5 μg. In addition, transforming colonies of E. coli BL21(DE3) were able to producer biofilm as well as production of urease enzyme. The extracted plasmids were digested by 3 types of restriction enzymes (enzyme EcoRI, BamHI and Hind III) to know the restriction sites available in bacterial plasmids. The results showed the presence of some EcoR1 restriction sites by observing the change in the physical shape of the plasmid after being treated with the EcoR1. Meanwhile, no restriction sites were observed for BamH1 and HindIII.

Identification of ß-Catenin Gene as a Colorectal Cancer Controller in Mice <i>(Mus musculus)</i> 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.

Diversity analysis of BmNPV Isolates Infecting Mulberry Silkworm (Bombyx mori) using Restriction Endonuclease Digestion Profiling

Understanding the genetic diversity of BmNPV isolates causing grasserie viral disease of mulberry silkworm Bombyx mori L. is essential for adoption of management strategies including biotechnological tools. The present study was aimed at the use of restriction fragment length polymorphism (RFLP) profiling for studying the molecular diversity analysis of six BmNPV isolates collected from Devanahalli, Kolar, Shidlaghatta, Hosakote, Tumakuru, and Ramanagara areas in Karnataka, India (BmNPV-Ko, BmNPV-Ho, BmNPV-SG, BmNPV-DH, BmNPV-TM, BmNPV-Ram respectively). DNA was extracted from each of these isolates and subjected to digestion with different restriction enzymes EcoR1, BamH1, Sma1, Nco1, and a combination of BamH1+Nco1 and electrophoresed in 0.8% w/v agarose gel to visualize restriction enzyme profile. The analysis revealed that all the six BmNPV isolates had similar Nco1 and Sma1 restriction patterns, although there was variation in low molecular weight fragments. The EcoR1 and BamH1 restriction patterns were nearly the same for all the isolates except for the presence of an approximately 4kb and an additional 1.5kb polymorphic band only in BmNPV-TM and BmNPV-Ram isolates. BamH1+Nco1 digestion of the DNA from each isolate yielded numerous fragments, which was different in BmNPV-Ram isolate. Molecular diversity analysis can helps in understanding the evolution and phylogeny of the virus, enhance the knowledge on its pathogenicity and can help to develop and adopt suitable measures to combat and diagnose the disease to reduce crop loss and increase income generating ability of the farmer.

IN VITRO EFFECT OF NEWLY BACTERIOPHAGES ISOLATES AGAINST ESCHERICHIA COLI SEROGROUPS COLLECTED FROM THE LOCAL HOSPITAL LABORATORY; THEIR SPECIFIC CHARACTERIZATION AND IDENTIFICATION

The aim of this study is to isolate a new bacteriophage with lytic effect against several serogroups of Escherichia coli that were isolated from different samples submitted to the local hospital laboratory. These serogroups are the main causative agents of bloodstream and urinary tract infections (UTIs) among Gram-negative bacteria. Three isolates of E. coli were used as a host to isolate phages from a hospital wastewater treatment plant. Eight phages were isolated and only three of them (EC-BSR1, EC-BSR2, and EC-BSR3) were considered for further characterization and identification. The three phages appear to belong to Myoviridae characterized by icosahedral heads, necks and contractile tails with tail fibers. Phage EC-BSR1 and EC-BSR2 had genome sizes of 67.06 and 68.04 kb, respectively. All three phages were lysed 100% of E. coli serogroups that was tested in vitro and 42.7%, 61.7% and 44.7% in vitro lyses of ECOR collection reference. Phages were resistant to pH from 5 to 9, and phage EC-BSR3 appeared to be more resistant than the other two phages to an acidic and alkaline environment. These phages have the pattern of homologous DNA fragments after being digested with Acc1 and EcoR1 restriction enzymes. It was concluded that these phages are highly coli lytic for the E. coli serogroups isolates.

Cloning, Amplified Expression and Bioinformatics Analysis of a Putative Nucleobase Cation Symporter-1 (NCS-1) Protein From Rhodococcus erythropolis

The Rhodococcus erythropolis gene DYC18_RS18060 (1437 bp) putatively codes for a secondary transporter of the Nucleobase Cation Symporter-1 (NCS-1) protein family (478 amino acids). The DYC18_RS18060 gene was successfully cloned from R. erythropolis genomic DNA with addition of EcoRI and PstI restriction sites at the 5′ and 3′ ends, respectively, using PCR technology. The amplified gene was introduced into IPTG-inducible plasmid pTTQ18 immediately upstream of the sequence coding for a His6-tag. The construct was transformed into Escherichia coli BL21(DE3), then amplified expression of the DYC18_RS18060-His6 protein was achieved with detection by SDS-PAGE and western blotting. Computational methods predicted that DYC18_RS18060 has a molecular weight of 51.1 kDa and isoelectric point of 6.58. The protein was predicted to be hydrophobic in nature (aliphatic index 113.24, grand average of hydropathicity 0.728) and to form twelve transmembrane spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. Whilst database sequence similarity searches and phylogenetic analysis suggested that the substrate of DYC18_RS18060 could be cytosine, this was not certain based on comparisons of residues involved in substrate binding in experimentally characterised NCS-1 proteins. This study has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins.
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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.

Characteristics of Glucose Oxidase Gene (GGOx) from Aspergillus niger IPBCC 08.610

Glucose oxidase is used in various industries for the development of enzymatic fuel cell. Based on prior studies, this compound is sourced from the local isolates of Aspergillus niger IPBCC 08.610, although investigations on the encoding gene have not been conducted. The purpose of this research, therefore, is to identify and characterized the gene responsible for encoding glucose oxidase, in the aspect of sequence, length, and restriction patterns. This experiment involved the amplification of genomic DNA using specific primers for gene recognition, which was followed by the restriction technique with EcoRI and PstI endonucleases. Furthermore, the gene is inserted into vector pGEM®T-Easy and transformed into competent E. coli DH5α cells, in an attempt to perform sequencing. The glucose oxidase gene from A. niger IPBCC 08.610 was confirmed to possess a size of 1848 bp, and a GC content of 57.8%, with a possibility of restriction into two fragments of size 908 bp and 980 bp, using the EcoRI restriction.

Isolation and Genomic Characterization of phiVibrioH1 a Myoviridae Phage for Controlling Pathogenic Vibrio parahaemolyticus from Seafood and Human

Vibrio parahaemolyticus is a zoonotic pathogen causing vibriosis in marine fish and associated with food poisoning outbreaks in humans. This study aimed to isolate and characterize bacteriophage against multidrug resistant (MDR) Vibrio parahaemolyticus from seafood and humans, and also to assess the lytic efficacy of phage on growth. Vibrio Parahaemolyticus was isolated from flesh of 80 white shrimp, 70 blue crabs and 50 mullets, and from 50 hand swabs of humans (fish handlers) on Thiosulphate Citrate bile salts sucrose agar media. The suspected colonies were biochemically identified. Pathogenic V. parahaemolyticus carrying thermostable direct hemolysin (tdh+) gene was molecularly detected . The pathogenic tdh+V. parahaemolyticus (n=25) isolates were explored for antimicrobial vulnerability resistant to 12 antimicrobials employing the disc diffusion method. Fifteen isolates of tdh+V. parahaemolyticus were 100% resistant to five antimicrobials. Bacteriophage was isolated from sewage water using spot test and double over layer agar assay. The phiVibrioH1 was belonged to family Myoviridae according to transmission electron microscopy. This study revealed a polyvalent phage infecting wide host range of MDR V. parahaemolyticus, V. vulnificus, V. fluvialis, V. alginolyticus, Pseudomonas aeruginosa, E. coli O26 and Proteus vulgaris. The genome of phage was sensitive to digestion with BamH1 and ECoR1 restriction enzymes indicating double stranded DNA. From 24 to 42 hours post-treatment of pathogenic V. parahaemolyticus with the PhiVibrioH1 phage showed complete lysis of bacterial cells. This study confirmed that the phiVibrioH1 is a lytic phage and has a high potential to control pathogenic V. parahaemolyticus strains recovered from seafood and humans.

Molecular Characterization of Fusarium oxysporum Causing Chickpea Wilt by Internal Transcribed Spacer (ITS) Marker

Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris is one of the serious disease causes tremendous loss to crop throughout the world and has assumed serious proportions in the recent years. Wilt affected chickpea plant roots samples were collected from forty eight different regions of Maharashtra. The collected samples were characterized by designed ITS primers. The Fusarium oxysporum f. sp. ciceris (Foc) showed about 302 bp amplicon when subjected to PCR with these primers. Further the amplified product was digested with five restriction enzymes viz. AluI, EcoRI, HaeIII, MboI and MspI and restriction fragment length polymorphism (RFLP) pattern were analysed. A dendrogram derived from ITS-RFLP analysis of the rDNA region divided the Foc isolates into four clusters. The maximum genetic distance 0.75 exhibited by five Foc isolates viz. Foc-30, Foc-36, Foc-24, Foc-25 and Foc-48 belonging to Shahartakli, Shelur, Nashik, Satara, Kolhapur regions respectively and found to be more diverse among the other Foc isolates. Whereas isolate no. Foc-20, Foc-21 and Foc-8 from Ambajogai, Karjat and Rahata shown minimum genetic distance (0.13). This showed the genetic variability among the Foc isolates collected from different regions of Maharashtra.

Molecular diversity of hpd gene in clinical isolates of Haemophilus influenzae

Infections due to Haemophilus influenzae result in tremendous global morbidity. The conjugated vaccines against H. influenzae type b (Hib) have dramatically reduced the incidence of invasive Hib disease in the routine immunization of infants. The several proteins used as vaccine candidates for this pathogen, but they don't produce efficient immune in animal models against all strains of H. influenzae. This study aimed to determine the diversity of hpd gene nucleotide sequences of Iranian native clinical isolates of H. influenzae as a native vaccine candidate compared to standard strains.Twenty isolates of H. influenzae recovered from different clinical specimens of patients admitted to Milad and Imam Khomeini hospitals, Tehran, Iran. Then, isolates detected and identified as H. influenzae using biochemical tests, and further confirmation through omp6 gene PCR. The hpd gene was amplified by PCR using gene-specific primers, and the amplicons digested with EcoR1. For four isolates, the Amplicon of hpd gene sequenced, and the sequences aligned with sequences harbored in GenBank. Subsequently, sequences were submitted to the EMBL site (http://www.ebi.ac.uk/embl/).EcoR1 restriction enzyme pattern was the same among the 19 clinical isolates, and only one isolate was different. That different one with 3 out of 19 isolates were sequenced. The results showed that the nucleotide sequences and the deduced amino acid sequences for protein D in clinical isolates were highly conserved with similarities >95%.In conclusion, regarding high similarity up to 99% in clinical isolates, protein D can be a novel vaccine candidate against all types of H. influenza from Iran. This finding should be proved with more isolates, and also, evaluate the immunological features of protein D in animal models.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

English

This study characterized Cucumber mosaic virus (CMV) infecting pepper in Southwestern Nigeria with the aim of providing up-to-date information on the taxonomic subgroup(s) of the parading strains in the study area. Fifty pepper leaf samples with one or more symptoms of mottling, mosaic, necrosis, leaf distortion and stunting were collected from eight commercial farms across Southwestern Nigeria and screened for CMV using antigen coated plate enzyme linked immunosorbent assay (ACP-ELISA). From the seropositive samples, four representative samples were selected, labelled PSWN1-4 before subjected to reverse transcription polymerase chain reaction (RT-PCR) and nucleic acid sequencing using a pair of primers specific for the amplification of RNA 3 genomic fragment of CMV. These were followed by multiple nucleotide sequence alignment and phylogenetic estimations of the isolates alongside selected CMV strains that were reported in previous studies using the molecular evolutionary genetics analysis (MEGA) software. The result of the ACP-ELISA test revealed that 14 (28%) of the samples collected were positive for CMV infection. The resulting nucleotide sequences from RT-PCR revealed 98% nucleotide homologies with several corresponding sequences of CMV strains from the Genbank. Further analysis of the PSWN nucleotide sequences through multiple nucleotide sequence alignment revealed the absence of the EcoR1 restriction site found only within the examined genomic portion of RNA 3 of subgroup II strains. These features defined the detected isolates as subgroup I strains. Phylogenetic analysis and estimations of genetic distances, conclusively distinguished the pepper-infecting CMV isolates from Southwestern Nigeria as members of subgroups IA and IB, which are the most virulent subgroups. Key words: Cucumber mosaic virus (CMV), pepper, Southwestern Nigeria.

Designing and construction of a DNA vaccine encoding ag85a and tb10.4 fusion genes of Mycobacterium tuberculosis

Background and Aim : Novel TB vaccines that aim to boost and/or replace Bacillus Calmette-Guerin (BCG) are currently in development. DNA vaccines can stimulate both humoral and cell-mediated immunity in different animal models of TB and is thought to be a promising strategy in the development of new vaccines against TB. The aim of this study was to design and construct a DNA vaccine encoding ag85a and tb10.4 fusion genes of Mycobacterium tuberculosis . Materials and Methods: Tb10.4 fragment was amplified by PCR and the products were digested with restriction enzymes. Next, it was cloned into the pcDNA3.1 + plasmid. The ag85a gene and pcDNA3.1 + / tb10.4 plasmid were digested by EcoRI and BamH1 restriction enzymes. Eukaryotic cells were transfected with pcDNA3.1 + / tb10.4 - ag85a plasmid for confirming expression of tb10.4 - ag85a in these cells. Results: Using electrophoresis of PCR products, fragments 297 bp for tb10.4 and 1017 bp for ag85a were observed. Eukaryotic cells transfection with pcDNA3.1 + / tb10.4 - ag85a vector was confirmed with cDNA synthesis and existence of tb10.4-ag85a was confirmed with RT-PCR. Conclusion: In this study, we constructed a DNA vaccine encoding tb10.4-ag85a fusion fragment. It can be used for development of more new DNA vaccines in future studies .

Polymorphisms of TLR4 Gene and Its Association With Genetic Resistance to Salmonella Enteritidis Infection in Fayoumi Breed and Hy-line Strain in Egypt

The aim of this study is to screen for genetic resistance to different doses of salmonella infection and its possible association with polymorphisms in exon 1 and 2 of TLR4 in Fayoumi breed and Hy-line strain of chickens. Two experiments was done, first one for measuring the variation of TLR4 -exon 1 and exon 2 of different genotypes of chicken under study and resistance to salmonella enteritidis. This study include 100 birds of each breeds, were infected orally with LD50 (108 cfu) salmonella enteritidis at age of two weeks and was divided into two groups (susceptible and resistance). TLR4 gene was genotyped with PCR-RFLP, The genotyping results on exon 1 (596 bp) by Taq1 restriction enzyme showed that the BB genotypes is more resistance to infection in the Fayoumi breed and also the AB genotypes is more resistance to infection in the Hy-line strain. Also The genotyping result on exon 2 (793 bp) by EcoR1 restriction enzyme showed only one genotype BB in Fayoumi breed and The frequency of AB genotypes is more resistant to infection than BB genotypes in Hy-line strain. The second experiment to evaluate the variation in immune response to the different genotypes of TLR4 -exon 1 and exon 2 this study include 100 birds of each breeds were infected with (5× 107 cfu) salmonella enteritidis and according to Elisa results the chickens devided into two groups (high and low immune response). The genotyping result on exon 1 (596 bp) by Taq1 restriction enzyme showed that the AB genotypes produce high immune response in both Fayoumi breed and Hy-line strain so can be used as a genetic marker for increase the immune response in chicks under study. Also The genotyping result on exon 2 (793 bp) by EcoR1 restriction enzyme showed only one genotype BB genotypes in Fayoumi breed and Hy-line strain.

Characterisation of Klebsiella pneumoniae Xylanase and Increment of Its Activity in Heterologous Expression System

A xylanase DNA sequence with a total length of 642 bp was previously isolated from a xylanolytic Klebsiellapneumoniae. Xylanase gene primers were designed with the addition of BamH1 and EcoR1 restriction enzymesites in order get a full xylanase gene that is in-frame with pSTAG expression vector. The isolated xylanasegene was amplified using the designed primers through PCR, then cloned and expressed in E. coli BL21 (DE3).In-silico characterization showed that the recombinant xylanase has a molecular weight of 23.9 kDa and a pI of9.32. The signal peptide cleavage site for the recombinant xylanase was predicted to be between residues 61and 62. The activity of the crude recombinant xylanase was 2.015 U/mL, which was higher than the crudenative xylanase activity, with maximum at 0.642 U/mL. Staining of the birchwood xylan agar plate with Congored showed a clearing zone around E. coli BL21 (DE3) colonies with recombinant pSTAG plasmid evenwithout being induced with IPTG. This implied leaky expression of the E. coli BL21 (DE3) secretion system,which recognized the signal sequence of the recombinant xylanase, and proceeded to cleave and secreted outthe mature protein into the culture medium. MALDI-TOF analysis of a 20 kDa protein present in the culturemedium confirmed that the recombinant xylanase had been secreted into the culture medium.

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

Generation of Thy1 Constructs for Pronuclear Injection.

With easy access to core facilities or commercial providers of pronuclear injections, generating simple Thy1-XFP transgenic mice (where XFP stands for any fluorescent protein) is now a possibility even for small laboratories. The generation of new Thy1 transgenic lines generally consists of five steps: (1) engineering and characterization of the desired fluorescent reporter protein, (2) cloning of the reporter protein into the Thy1 vector, (3) linearization and purification of the new Thy1 construct, (4) pronuclear injection to generate founders, and (5) screening of founder progeny to establish transgenic lines. Here, we provide a protocol for Steps 2 and 3. The sequence for a desired fluorescent reporter protein is cloned into the XhoI restriction site of the Thy1 vector. This usually involves blunt-end cloning because the traditional Thy1 vector does not carry an intact multiple cloning site. Following successful cloning, the DNA is prepared for pronuclear injection by linearizing it using EcoRI and PvuI restriction enzymes. The purified linearized DNA must then be sent to a facility specializing in pronuclear injection to generate transgenic founder mice.

Interactions of DNA with H2TMPyP4+and Ru(II)TMPyP4+: Probable Lead Compounds for African Sleeping Sickness

Interactions of DNA with free base porphyrin, tetrakis(1-methylpyridium-4-yl)porphyrin (H2TMPyP4+) and its metallo-derivative of ruthenium(II) have been investigated by UV-Vis, fluorescence and circular dichroism (CD) spectroscopy at 0.1 M NaCl, 7.5 pH and 25 °C. The results suggest that Ru(II)TMPyP4+ interacts with DNA via outside binding in self-stacking manner as indicated by UV-Vis data, a small red shift (?? =3 nm) and a minimal hypochromicity (10%) upon addition of DNA. CD spectra showed the presence of a new peak in the Soret region on addition of Ru(II)TMPyP4+ to DNA solution. On the other hand, the interaction of free base porphyrin, H2TMPyP4+ with DNA revealed a significant hypochromicity (30%) and a large red shift (??=20 nm)in the UV-Vis results which conforms intercalation of free base porphyrin with DNA. In this case, the CD results showed only a negative peak developed in the Soret region during titration with DNA. Fluorescence spectroscopy revealed that initially aggregated porphyrin species were de-aggregated after addition of DNA. This phenomenon has been verified with the fluorescence experiments for the porphyrins in the presence of different concentrations of NaCl and ethanol. Ru(II)TMPyP4+ showed enhanced DNA cleavage in the presence of EcoR1 restriction enzyme, where the enzyme did not cleave DNA. Metallo-porphyrins having the ability to cleave DNA could be used as chemotherapeutic agents for the treatment of African sleeping sickness (Trypanosomiasis). DOI: http://dx.doi.org/10.3329/bpj.v17i1.22321 Bangladesh Pharmaceutical Journal 17(1): 79-85, 2014

PRESENCE OF SHARKA DISEASE IN THE NORTH-HUNGARIAN COUNTIES

In the concerned region, such as Hungary Plum pox virus (PPV) causes the most serious yield loss in the stone fruit orchards. A survey of sharka disease was conducted in the north-Hungarian border region from 2010 to 2012. The main goals of the survey were to isolate the virus, to map the spreading and the actual status, to identify the PPV strains and inform the orchard owners and manufacturers about the results of the study. This survey was a part of the Hungary-Slovakia Cross-Border Co-operation Programme 2007-2012. Nearly 250 leaf samples were taken from 51 different settlements of the northern counties from stone fruit orchards, home gardens and natural environment, showing the symptoms of PPV. The surveyed species were plum, apricot, peach, sweet and sour cherry, and blackthorn. To identify the strains the following molecular methods were used: conventional RT-PCR with strain-specific primers, RT-PCR followed by RFLP analysis using EcoRI, DdeI and EcoRV restriction enzymes in the 3’P3-6K1-5’CI genomic region and RT-PCR followed by sequence analysis in the formerly mentioned region. The results show that the dominant strains were PPV-Rec in the central and PPV-D in the eastern region, but PPV-M was also identified. The apricot and peach orchards were less infected than plum orchards in the northern counties of Hungary. A suspected triple-mixed infection (M, D and Rec) is under further studies. After the survey a map was prepared, the dominant isolates, the infection rate, the host plants and the type of the sample collection places were marked. From the results a bilingual booklet was also published with the great help of the Slovak partners, for producers and orchard owners including all important knowledge about the virus and the disease prevention.

FIRST REPORT OF PEPINO MOSAIC VIRUS ON TOMATO IN APULIA, ITALY

In October 2013, unusual chlorotic patches were observed on the middle leaves of a few tomato plants cv. Lotty grown in a greenhouse located in the countryside of Fasano (Apulia, southern Italy). Younger leaves and fruits were symptomless and no aggravation of symptoms was detected as time went by. Electron microscope observations of leaf dips revealed the presence of filamentous virus particles ca. 520 nm in length. Mechanical inoculations with leaf extracts from symptomatic tomatoes elicited a mosaic reaction in Nicotiana benthamiana, but not in tomato plants cv. UC82, which, however, were systemically infected, as shown by RT-PCR. A RT-PCR product of the expected size (ca. 700 bp) was obtained using the degenerate broad-spectrum potexvirus primers Potex5/Potex1RC (van der Vlugt and Berendsen, 2002). The amplicon was custom sequenced (BMR Genomics, Italy) and the sequence deposited in GenBank under the accession No. KM923762. BLAST alignment showed that the 700 bp amplicon shared 97-99% homology with the RNA-dependent RNA polymerase (RdRp) gene of several isolates of Pepino mosaic virus (PepMV) genotype CH2, 82% homology with the LP and EU genotypes, and 79-80% with the US genotype. A RT-PCR-restriction fragment length polymorphism (RFLP) analysis of the RdRp amplicon with EcoRI and BglII restriction endonucleases confirmed that our isolate, designated PUG1, belongs to the CH2 genotype (Hanssen, 2010). Our observations and assays are consistent with the presence of PepMV in the tomato plants tested, and the fact that PUG1 is a mild isolate of the virus. PepMV has previously been recorded from tomato in three Italian regions, i.e. Sardinia, Sicily and Campania, but this the first report from Apulia.