Production of Restriction Endonuclease Not Ⅰ Utilizing CpG DNA Methylase M.Sss Ⅰ Co-expression Vector / 中国生物工程杂志

Restriction endonucleases are important molecular biology tools for DNA recombination.Because of the cleavage of DNA,their recombinant expression is difficult with low yields and complicated purification processes.In commercial productions,the technology that uses specific methylases to protect host DNA from digestion of the expressed restriction enzymes was cumbersome and practically limited.For solving this problem the expression of restriction enzyme Not Ⅰ was performed by using the DNA methylase M.Sss Ⅰ derived from Spiroplasma sp.MQ1 which specifically kept CpG sequence methylated.The methylated DNA was protected from the cutting of Not Ⅰ whose recognition sequence contained CpG.The gene of methylase M.Sss Ⅰ was introduced into Escherichia coli ER2566 and constitutively expressed,resulting in the CpG methylation pattern of the host DNA.Restriction enzyme Not Ⅰ was successfully expressed in this E.coli strain.Furthermore,by adding a purification tag to one terminus of the enzyme,recombinant Not Ⅰ was prepared as a highly purified and active product through two simple Ni-affinity and anion exchange chromatography steps.This expression system can be applied for the preparation of a series of restriction enzymes with CpG in their recognition sequences.

A putative Type IIS restriction endonuclease GeoICI from Geobacillus sp. – A robust, thermostable alternative to mezophilic prototype BbvI.

Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers’ biofilms, exposed to a broad range of temperatures, from ambient to close to water boiling point, were analysed for the presence of DNA-interacting proteins, including restriction endonucleases (REases). GeoICI, a member of atypical Type IIS REases, is the most thermostable isoschizomer of the prototype BbvI, recognizing/cleaving 5 -GCAGC(N8/12)-3 DNA sequences. As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (λ); (ii) cleavage of custom PCR substrates, (iii) run-off sequencing of GeoICI cleavage products and (iv) shotgun cloning and sequencing of λ DNA fragmented with GeoICI. Geobacillus sp. genomic DNA was PCR-screened for the presence of other specialized REases-MTases and as a result, another putative REase- MTase, GeoICII, related to the Thermus sp. family of bifunctional REases-methyltransferases (MTases) was detected.

Plasmid and Restriction Endonuclease Analyses of Bacteria Associated with Pneumonia in HIV/AIDS Patients in Ekiti State, Nigeria.

Bacterial infections are an important cause of morbidity and mortality in individuals with the human immunodeficiency virus. The emergence of multiple-drug resistant bacteria has been documented by many researches. This study was therefore carried out to determine whether the resistances of bacterial isolates from HIV positive and HIV negative patients are plasmid mediated or chromosomal mediated. The Plasmid, Post Plasmid-curing Sensitivity and Restriction enzymes endonuclease were done using standard methods. The result of plasmid analysis showed that Plasmid-mediated resistance was observed in both populations and the molecular weight of the plasmid DNA was 1000 base pairs. Plasmid mediated resistance was common, and this was observed in all isolates from HIV/AIDS patients with exceptions of P. aeruginosa in which the resistance was chromosomally mediated. Restriction endonuclease analysis from E. coli revealed 3 distinct clusters. The result of restriction enzymes analysis indicate that the pneumonia infection in HIV/AIDS patients is likely to be hospital acquired in the study location. The study also suggests a common source of infection of the patients.

2′-O-methyl nucleotide modified DNA substrates influence the cleavage efficiencies of BamHI and BglII.

Induction of endonucleolytic DNA cleavage is an essential event that links the initiating stimuli to the final effects of cells. The cleavage efficiency and thus the final yield could be affected by many factors, including structures of DNA substrates, composite structures of enzymes–substrates or enzymes–nucleic analogs and so on. However, it is not clear whether a nucleotide derivative-substituted in DNA substrates can influence the efficiency of enzymatic cleavage. To investigate the effect of sugar pucker conformation on DNA–protein interactions, we used 2′-O-methyl modified nucleotides (OMeN) to modify DNA substrates of isocaudemers BamHI and BglII in this study, and used FRET assay as an efficient method for analysis of enzyme cleavage. Experimental results demonstrated that OMeN-substituted recognition sequences influenced the cleavage rates significantly in a position-dependent manner. OMeN substitutions can reduce the cleavage as expected. Surprisingly, OMeN substitutions can also enhance the cleavage rates. The kinetics parameters of Vmax and Km have been obtained by fitting the Michaelis-Menten kinetic equation. These 2'- OMe nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property could enrich our understanding about the endonuclease cleavage mechanism and enhance our ability to regulate the enzymatic cleavage efficiency for applications in synthetic biology.

Rotavirus A genotype G1P[8]: a novel method to distinguish wild-type strains from the Rotarix® vaccine strain

Rotaviruses are important enteric pathogens for humans and animals. Group A rotaviruses (RV-A) are the most common agents of severe gastroenteritis in infants and young children and vaccination is the most effective method to reduce RV-A-associated diseases. G1P[8], the most prevalent RV-A genotype worldwide, is included in the RV-A vaccine Rotarix®. The discrimination between wild-type G1P[8] and vaccine G1P[8] strains is an important topic in the study of RV-A epidemiology to manage outbreaks and to define control measures for vaccinated children. In this study, we developed a novel method to segregate the wild-type and vaccine strains using restriction endonucleases. The dsRNA from the Rotarix® vaccine was sequenced and the NSP3 gene was selected as the target gene. The vaccine strain has a restriction pattern that is different than that of wild-type RV-A G1P[8] isolates after digestion with the restriction endonuclease BspHI. This pattern could be used as a marker for the differentiation of wild-type G1P[8] strains from the vaccine strain.

Hidden heterochromatin: characterization in the Rodentia species Cricetus cricetus, Peromyscus eremicus (Cricetidae) and Praomys tullbergi (Muridae)

The use of in situ restriction endonuclease (RE) (which cleaves DNA at specific sequences) digestion has proven to be a useful technique in improving the dissection of constitutive heterochromatin (CH), and in the understanding of the CH evolution in different genomes. In the present work we describe in detail the CH of the three Rodentia species, Cricetus cricetus, Peromyscus eremicus (family Cricetidae) and Praomys tullbergi (family Muridae) using a panel of seven REs followed by C-banding. Comparison of the amount, distribution and molecular nature of C-positive heterochromatin revealed molecular heterogeneity in the heterochromatin of the three species. The large number of subclasses of CH identified in Praomys tullbergi chromosomes indicated that the karyotype of this species is the more derived when compared with the other two genomes analyzed, probably originated by a great number of complex chromosomal rearrangements. The high level of sequence heterogeneity identified in the CH of the three genomes suggests the coexistence of different satellite DNA families, or variants of these families in these genomes.

BpuAmI: a novel SacI neoschizomer from Bacillus pumilus discovered in an isolate from Amazon Basin, recognizing 5'-GAG FONT FACE=Symbol>¯ /FONT>CTC-3'

A strain of Bacillus pumilus was isolated and identified from water samples collected from a small affluent of the Amazon River. Type II restriction endonuclease activity was detected in these bacteria. The enzyme was purified and the molecular weight of the native protein estimated by gel filtration and SDS-PAGE. The optimum pH, temperature and salt requirements were determined. Quality control assays showed the complete absence of "nonspecific nucleases." Restriction cleavage analysis and DNA sequencing of restriction fragments allowed the unequivocal demonstration of 5´GAG FONT FACE=Symbol>¯ /FONT>CTC3´ as the recognition sequence. This enzyme was named BpuAmI and is apparently a neoschizomer of the prototype restriction endonuclease SacI. This is the first report of an isoschizomer and/or neoschizomer of the prototype SacI identified in the genus Bacillus.

Uma linhagem de Bacillus pumilus foi isolada e identificada de amostras de águas coletadas em um pequeno Igarapé do Rio Amazonas. Foi detectada atividade de restrição do tipo II nesta bactéria. A enzima foi purificada e o peso molecular da proteína nativa foi estimado por gel filtração e por eletroforese em gel de poliacrilamida. Foram determinados, o pH e temperatura ótimos e as necessidades de sais. Os ensaios do controle de qualidade mostraram uma ausência completa de "nucleases não específicas". As analises das clivagens e o seqüenciamento do DNA dos fragmentos de restrição permitiram uma demonstração inequívoca de que 5´GAG FONT FACE=Symbol>¯ /FONT>CTC 3´ é a seqüência de reconhecimento da enzima. Esta enzima foi denominada de BpuAmI e aparentemente é um neoesquisômero da enzima protótipo SacI. Este é o primeiro relato de um isoesquisômero e/ou neoesquisômero da enzima protótipo SacI identificada no gênero Bacillus.

A Novel Method for Quickly Cloning Genes With Multiple DNA Fragments Using Isoschizomer-heterotail Restriction Endonuclease (IHRE) / 生物化学与生物物理进展

A novel method for quickly cloning genes with multiple DNA fragments--one step cloning technique using isoschizomer-heterotail restriction endonuclease (IHRE) is described. Up to six DNA segments are ligated by using only one restriction endonuclease in this method. Comparing with routine method,it is simple, fast, economical and generates products with higher purity and achievement. Light chain of human enterokinase, DNA multi-epitope vaccine to HSV2 have been designed and successfully constructed via this method.

A Case of Nocardia asteroides type I Induced Pneumonia / 대한진단검사의학회지

Nocardia species are opportunistic pathogens that are known to affect mostly the immunocompromised patients. Recently, we experienced a young systemic lupus erythromatosus female patient having infected with Nocardia species, which we were able to isolate from her lung abscess. The patient is twenty-nine years old female who was diagnosed as having systemic lupus erythromatosus two years ago and is currently engaged with ongoing treatment. During her admission, new symptoms of fever and dyspnea along with a lesion in the apical lobe of her left lung found by simple chest X-rays were observed. Under lung biopsy examination, there were seen neutrophilic exudates that were gram-positive, AFB-negative, and modified AFB-positive. By culturing the biopsy material, we found gram-positive, AFB-negative, and modified AFB-positive branching hyphaes that were morphologically matched for Nocardia species. We have analyzed the Nocardia DNA by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and found the organism to be Nocardia asteroides type I. Treatment of patient was done using sulfamethoxazole/trimethoprim and ceftriazone, and her clinical conditions as well as her radiological findings improved.

Characterization of Type 2 Restriction Endonucleases (Hpy51) from Helicobacter pylori Strain 51

This study describes the purification and characterization of type II restriction endonuclease of Helicobacter pylori in order to understand the DNA restriction and modification of H. pylori. H. pylori cell extract was subjected to polyethyleneimine treatment, salt precipitation, heparine-sepharose column chromatography, and fast protein liquid chromatography (FPLC) using Resource Q column and Mono Q column to purify the type II restriction endonuclease. Hpy51-I was characterized to recognize the sequneces 5`-GT(G/C)AC-3`, yielding 5-base 5` protruding ends. The restriction sequence was identical to that of Tsp 45 I. The enzyme exhibited its maximal activity in the presence of 10-20 mM LaCl, but was inhibited completely in the presence of more than 80 mM NaCl. The enzyme showed its maximal activity in the presence of 1-10 mM MgC1(2). The optimal pH and temperature for enzyme activity was pH 9.0 and 37 degrees C, respectively. MnC1(2) could not substitute for MgC1(2) in reaction mixture. And addition of j3-mercaptoethanol and bovine serum albumin in reaction mixture led to loss of enzyme activity of Hpy51-I. The whole cell extract of H. pylori strain 51 was confirmed to carry the enzyme activity for methylation of Hpy51-I-recognised sequence. Hpy51-I digested genomic DNAs of enteric bacteria to less than I kb while it could not cut the genomic DNAs of H. pylori isolates. In this study, the type II restriction enzyme (Hpy51-I) of H. pylori was identified and characterized its biochemical properties, demonstrating that Hpy51-I might be one of the barriers for preventing the introduction of foreign DNAs into H. pylori.