Restriction Endonucleases EcoRI Research Articles

Extrachromosomal DNA length and antibiograms of Staphylococcus aureus and Pseudomonas aeruginosa isolated from tears of HIV/AIDS patients after curing with sodium dodecyl sulphate.

Staphylococcus aureus and Pseudomonas aeruginosa strains were isolated from eye swab samples randomly obtained from 100 seropositive HIV/AIDS patients who reported to various anti-retroviral treatment clinics at the University of Benin Teaching Hospital and Central Hospital both based in Benin City, Nigeria. Invitro antibiotic sensitivity patterns of strains before curing were determined by the Kirby-Bauer disc diffusion technique. Resistance plasmid DNA of multidrug resistant strains was cured with 0.1% sodium dodecyl sulphate and cured strains were again subjected to invitro antibiotic sensitivity testing. EcoRI and Hind III restriction endonuclease enzymes were used to make cuts on extracted plasmid DNA whose length sizes were then determined. A total of 36 (36.0%) strains made up of 27 (75.0%) Staphylococcus. aureus and 9 (25.0%) Pseudomonas aeruginosa were isolated of which 7 (19.4%) strains showed multidrug resistance to ciprofloxacin, pefloxacin, ofloxacine, gentamycin, tetracycline, ampicillin, chloramphenicol, nitrofurantoin and erythromycin. All seven multidrug resistant strains before curing, recorded 85.7%, 42.9%, 14.3% and 14.3% sensitivity in that decreasing order to ciprofloxacin, pefloxacin, ofloxacin and gentamycin respectively. There was 0.0% sensitivity each to tetracycline and ampicillin. After curing, there was enhanced sensitivity of 100.0%, 85.7%, 28.6% and 71.4% respectively. There was also 28.6% and 57.1% improved sensitivity to tetracycline and ampicillin after curing. Before curing, there was 76.2% average resistance to all used antibiotics and this reduced to 47.6% after curing Staph. aureus plasmid DNA. In the case of Pseudomonas aeruginosa, there was an average resistance of 76.3% before curing which fell to 42.5% after curing. EcoRI restriction enzyme gave the plasmid DNA length of Staphylococcus aureus strain 04 as 4.0Kb and this size depended upon the distance between recognition sites. Isolation of 36 (36.0%) strains of both isolates from 100 eye swabs shows the danger these organisms portend to all categories of opticians. The cheapness and high sensitivity of gentamycin justifies its use as eye drops for treatment of some eye infections. Curing of plasmid DNA is an indication that if SDS is administered to the organisms in sublethal doses, it can lead to the elimination of plasmid DNA without adverse effect on the genomic DNA of the bacterial strains.

The use of cyprinodont fish, Aphanius fasciatus, as a sentinel organism to detect complex genotoxic mixtures in the coastal lagoon ecosystem

In the present work we aimed to standardise the alkaline comet assay with erythrocytes of the cyprinodont, Mediterranean Killifish, Aphanius fasciatus. The aims of the study were to explore the suitability of this fish to assess biomarkers of genotoxic effects and as a sentinel organism to detect complex genotoxic mixtures in coastal lagoon ecosystems. Following proper optimisation, the application and effectiveness of the comet assay in erythrocytes of A. fasciatus were tested by measuring the tail DNA (%) induced by (a) in vivo exposure of individual fish to X-rays (dose, 3 Gy) and (b) following in vitro challenge of erythrocytes with restriction endonucleases Fok-I and Eco-RI, which selectively induce double-strand breaks with cohesive and blunt termini, respectively. Furthermore, in order to evaluate whether circulating fish blood contained actively proliferating cells that could influence the extent of DNA damage in control (untreated) fish, we measured the number of “comets” positive for 5-bromo-2′-deoxyuridine (BrdU) by the use of anti-BrdU antibody and immuno-histochemical methods. Both treatments (i.e. with X-rays and restriction endonucleases) induced statistically significant increases in tail DNA (%) values compared with the relevant untreated controls, indicating the effectiveness of the comet assay in the erythrocytes of A. fasciatus to detect different types of DNA lesions. Results from anti-BrdU antibody labelling of erythrocytes indicated a very low percentage (5%) of “comets” positive for BrdU. Following optimisation and validation of the assay under laboratory conditions, fish were collected in the Orbetello lagoon (Tuscany, Italy), considered to be a significantly polluted site. The results showed statistically significant increases for tail DNA (%) compared with corresponding values observed in erythrocytes of fish caught in the unpolluted reference site “Saline di Tarquinia”. The effects of physico-chemical parameters of the water (i.e., salinity, pH and oxygen content) did not significantly influence the induction of DNA damage. These results indicate that the comet assay provides a reliable parameter and that A. fasciatus is a promising “sentinel organism” to detect the genotoxic impact of complex mixtures in coastal lagoon ecosystems.

ChemInform Abstract: Inhibition of Restriction Enzymes EcoRI, BamHI and HindIII by Phenethylphenylphthalimides Derived from Thalidomide.

AbstractThe compounds (I) show potent EcoRI‐inhibitory activity (IC50 = 4.2 and 5.3 μm, respectively) and modest BamHI‐ and HindIII‐inhibitory activities.

Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.

Hubungan Polimorfisme Gen Laktoferin dengan Kualitas Susu pada Sapi Perah Friesian-Holstein

Association of Lactoferrin Polymhorphism on Milk Yield and Milk Quality in Holstein-Friesian Dairy CowsABSTRACT. The purposes of this study were to identify the polymorphism of the lactoferrin gene and the association of genetic variants of the lactoferrin gene on milk quality in Holstein-Friesian (HF) cows. The study was conducted on 194 heads of HF lactating cows coming from semi intensive of small dairy farmers in two villages of the KPSBU Lembang, Lembang district. Investigation on variant genotypes of the lactoferrin gene used PCR-RFLP method. Genotyping of the lactoferrin gene with EcoRI restriction enzyme produced two genotypes, i.e. AA (65%) and AB (35%) genotypes. Both the AA and AB genotypes had inconsistency in controlling component of the milk quality. Further, the AB lactoferrin cows had a higher frequency in the classification of low somatic cells count (0.75), while the AA cows were at a higher frequency in the classification of high somatic cells count (0.60). It was concluded that there is no strong relationship between intron 6 lactoferrin gene with milk quality parameters. Further, the AB genotype of the lactoferrin gene had a consistency in producing lower somatic cells count compared to the AA genotype.

Inhibition of influenza A virus replication by RNA interference targeted against the PB1 subunit of the RNA polymerase gene

Influenza (flu) pandemics have posed a great threat to human health in the last century. However, current vaccination strategies and antiviral drugs provide limited protection. RNA interference (RNAi) is an effective means of suppressing influenza virus replication. PB1 is the critical protein subunit of the influenza virus RNA polymerase. The gene encoding this protein, PB1, is highly conserved among different subtypes of IAV and was therefore chosen as the target in this study. The oligonucleotide, PB1-shRNA, contains a 21-bp siRNA corresponding to nucleotides 1,632 to 1,652 of PB1 linear vRNA with BamHI or EcoRI restriction enzyme sites incorporated at the ends. The PB1-shRNA oligonucleotide was directionally cloned into the RNAi-ready pSIREN-shuttle vector. The correct structure of the resulting pSIREN/PB1 plasmid was confirmed by restriction endonuclease digestion. Madin-Darby canine kidney (MDCK) cells were transfected with pSIREN/PB1 and subsequently infected with IAV at an MOI of 0.1 (A/PR/8/34, H1N1). The virus titer in cell culture supernatants was determined 48 hours later, and it was found that virus growth was inhibited by more than 50-fold relative to controls. Furthermore, embryonated eggs and mice were inoculated with liposome-encapsulated pSIREN/PB1 and then challenged with the A/PR/8/34 virus. The results showed at least a 100-fold inhibition in virus replication in egg allantoic fluid and a survival rate of between 50% and 100% in experimental mice. This study demonstrates that PB1-shRNA expressed by the recombinant plasmid pSIREN/PB1 inhibits influenza A virus replication both in vitro and in vivo. These observations provide a foundation for the development of a new and efficient treatment of influenza infections.

Comparative cytogenetics and heterochromatic patterns in two species of the genus Acanthostracion (Ostraciidae: Tetraodontiformes)

Some groups of fish, such as those belonging to the Order Tetraodontiformes, may differ significantly in the amount and location of heterochromatin in the chromosomes. There is a marked variation in DNA content of more than seven-fold among the families of this Order. However, the karyoevolutionary mechanisms responsible for this variation are essentially unknown. The largest genomic contents are present in species of the family Ostraciidae (2.20–2.60 pg). The present study cytogenetically characterized two species of the family Ostraciidae, Acanthostracion polygonius and A. quadricornis, using conventional staining, C-bandings, Ag-NOR, CMA 3/DAPI, AluI, PstI, EcoRI, TaqI and HinfI restriction enzymes (REs) and double FISH with 18S and 5S rDNA probes. The karyotypes of both species showed 2n = 52 acrocentric chromosomes (FN = 52; chromosome arms) and pronounced conserved structural characteristics. A significant heterochromatic content was observed equilocally distributed in pericentromeric position in all the chromosome pairs. This condition is unusual in relation to the karyotypes of other families of Tetraodontiformes and probability is the cause of the higher DNA content in Ostraciidae. Given the role played by repetitive sequences in the genomic reorganization of this Order, it is suggested that the conspicuous heterochromatic blocks, present in the same chromosomal position and with apparently similar composition, may have arisen or undergo evolutionary changes in concert providing clues about the chromosomal mechanisms which led to extensive variation in genomic content of different Tetraodontiformes families.

Analisis Keragaman Gen Laktoferin Pada Sapi Friesian-Holstein Dengan Metode Pcr-Rflp

Polymorphism analysis of lactoferrin gene on holstein-friesian cow with PCR-RFLP methodABSTRACT. The purposes of this study were to identify the polymorphism of the lactoferrin gene in Holstein-Friesian (HF) cows. The study was conducted on 281 heads of HF lactating cows coming from dairy farmers in Lembang district. Investigation on variant genotypes of the lactoferrin gene used PCR-RFLP method. Genotyping of the lactoferrin gene with EcoRI restriction enzyme produced two genotypes, i.e. AA (65.5%) and AB (34.5%) genotypes.

Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

Elevated homocysteine (Hcys) serum levels represent a risk factor for several chronic pathologies, including cardiovascular disease, atherosclerosis, and chronic renal failure, and affect bone development, quality, and homeostasis. Hcys influences the formation of a stable bone matrix directly through the inhibition of the collagen cross-linking enzyme lysyl oxidase (Lox) and, as we have shown recently, by repressing its mRNA expression. The aim of this study was to investigate the mechanisms involved in this process. Through evaluation of gene arrays, quantitative RT-PCR, immunoblots, and ELISA, we identified a Hcys-dependent stimulation of interleukin 6 (IL-6) and genes involved in IL-6/Janus kinase 2 (JAK2)-dependent signal transduction pathways in pre-osteoblastic MC3T3-E1 cells. Moreover, up-regulation of genes essential for epigenetic DNA methylation (DNA (cytosine-5)-methyltransferases and helicase lymphoid-specific (Hells) was observed. Further investigations demonstrated that Hcys increased via IL-6/JAK2 the expression of Fli1 (Friend leukemia virus integration 1), a transcription factor, which we found essential for IL-6-dependent Dnmt1 stimulation. CpG methylation analysis of CpG-rich Lox proximal promoter revealed an increased CpG methylation status after treatment of the cells with Hcys indicating an epigenetic origin for Hcys-dependent Lox repression. Inhibition of the IL-6/JAK2 pathway or of CpG methylation reversed the repressive effect of Hcys on Lox expression. In conclusion, we demonstrate that Hcys stimulates IL-6 synthesis in osteoblasts, which is known to affect bone metabolism via osteoclasts. Furthermore, IL-6 stimulation results via JAK2, Fli1, and Dnmt1 in down-regulation of Lox expression by epigenetic CpG methylation revealing a new mechanism negatively affecting bone matrix formation.

Insights on Copper Coordination and Reactivity of Endonuclease EcoRI by ESR Spectroscopy and Modeling

Restriction endonuclease EcoRI cleaves a specific six base pair sequence of DNA in the presence of some divalent metal ions such as Mg2+. Cu2+, on the other hand, does not catalyze the cleavage of DNA by EcoRI. To understand the functional difference between Cu2+ and Mg2+, electron spin resonance (ESR) spectroscopy was used to determine the local environment of Cu2+ in EocRI. Two Cu2+ components are observed from the continuous wave-ESR spectrum. Electron spin echo envelope modulation (ESEEM) spectra under different external magnetic fields have contributions from both of the two Cu2+ components (except at a low magnetic field). Both components contain peaks below 2 MHz and broad peaks at ∼4 MHz. A peak at ∼14 MHz corresponding to proton ESEEM is also observed in each spectrum. To quantitatively analyze the spectra, we developed a theoretical simulation package to simulate these spectra. Based on the simulation, both of the two Cu2+ components have a 14N of histidine imidazole coordinated to Cu2+. The peaks below 2 MHz and at ∼ 4 MHz are assigned to the nuclear quadrupole interaction and the double quantum transitions of the remote 14N of histidine imidazole, respectively. We discovered H114 of EcoRI coordinates to the Cu2+, based on distance measurements using double electron electron resonance spectroscopy. And molecular dynamics simulation confirms the coordination of H114 to Cu2+. These results help us to get an understanding of the different coordination environments between Cu2+ and Mg2+ in EcoRI-DNA, and thus to explain the functional difference between them. This work supported by NSF.

Exploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping.

We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing the cofactor magnesium, we determine the binding location of the enzyme by the cleavage process of dsDNA as in optical restriction mapping, however here the DNA need not be immobilized on a surface. We note that no special labeling of the enzyme is required, which makes it simpler than our previous scheme using stagnation point flows for sequence detection. Our accuracy in determining the location of the recognition site is comparable to or better than other single molecule techniques due to the fidelity with which we can control the linearization of the DNA molecules. In addition, since the cleavage process can be followed in real time, information about the cleavage kinetics, and subtle differences in binding and cleavage frequencies among the recognition sites, may also be obtained. Data for the five recognition sites for the type II restriction endonuclease EcoRI on λ-DNA are presented as a model system. While the roles of the varying fluid velocity and tension along the chain backbone on the measured kinetics remain to be determined, we believe this new method holds promise for a broad range of studies of DNA-protein interactions, including the kinetics of other DNA cleavage processes, the dissociation of a restriction enzyme from the cleaved substrate, and other macromolecular cleavage processes.

Inhibition of Restriction Enzymes EcoRI, BamHI and HindIII by Phenethylphenylphthalimides Derived from Thalidomide

We discovered inhibitors of the restriction enzymes EcoRI, BamHI and HindIII by screening our library of compounds with a phenethylphenylphthalimide skeleton, based on α-glucosidase inhibitors and liver X receptor antagonists derived from thalidomide. Structural development afforded the potent restriction enzyme inhibitors 25 and 26.

Molecular Characterization of Phytoplasmas Associated with Potato Purple Top Disease in Iran

Potato plants with symptoms suggestive of potato purple top disease (PPTD) occurred in the central, western and north-western regions of Iran. Polymerase chain reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7 followed by primer pairs R16F2n/R16R2 and fU5/rU3 for phytoplasma detection. Using primer pairs R16F2n/R16R2 and fU5/rU3 in nested PCR, the expected fragments were amplified from 53% of symptomatic potatoes. Restriction fragment length polymorphism (RFLP) analysis using AluI, CfoI, EcoRI, KpnI, HindIII, MseI, RsaI and TaqI restriction enzymes confirmed that different phytoplasma isolates caused PPTD in several Iranian potato-growing areas. Sequences analysis of partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma solani’, ‘Ca. Phytoplasma astris’ and ‘Ca. Phytoplasma trifolii’ are prevalent in potato plants showing PPTD symptoms in the production areas of central, western and north-western regions of Iran, although ‘Ca. Phytoplasma solani’ is more prevalent than other phytoplasmas. This is the first report of phytoplasmas related to ‘Ca. Phytoplasma astris’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ causing PPTD in Iran.

Tranformasi Fragmen DNA Kromosom Xanthomonas Campestris Ke Dalam Escherichia Coli

Research on DNA transformation of Xanthomonas campestris into Escherichia coli DH5αα using plasmid vector Escherichia coli (pUC19). was carried out. DNA chromosome was isolated using CTAB method, alkali lysis method was used to isolate DNA plasmid. Both of DNA plasmid and chromosome were digested using restriction enzyme EcoRI. Competent cell was prepared with CaCl2 and heat shock method for transformation procedure. The result revealed transformation obtain 5 white colonies, with transformation frequency was 1,22 x 10-8 colony/competent cell. Electrophoresis analysis showed the DNA fragment (insert) in range 0.5 – 7,5 kb. Further research should be carried out to prepare the genomic library to obtain better result of transformant. Keywords: Tranformation, restriction enzyme, plasmid and chromosome

ChemInform Abstract: 3-Deaza-2′-deoxyadenosine: Synthesis via 4-(Methylthio)-1H-imidazo(4,5-c)pyridine 2′-Deoxyribonucleosides and Properties of Oligonucleotides.

The synthesis of 4-(methylthio)-1H-imidazo[4,5-c]pyridine 2′-deoxy-β-D-ribonucleosides 2 and 9 and the conversion of the N1-isomer 2 into the 2′,3′-didehydro-2′,3′-dideoxyribonucleoside 3a or (via7) 3-deaza-2′-deoxyadenosine (1) is described. Phosphonate building blocks of 1 were employed in solid-phase synthesis of self-complementary base-modified oligonucleotides. Their properties were studied with regard to duplex stability and hydrolysis by the restriction enzyme Eco RI.

Toward specific detection of Dengue virus serotypes using a novel modular biosensor

Arthropod-borne diseases affect a significant portion of the world's population. Dengue fever, a viral disease carried by the Aedes aegypti mosquito, is one of the most wide-spread, with many fatalities evident each year. To date, Dengue viral diagnostic technologies have been too complex, time-consuming and expensive to be widely deployed, particularly in developing countries where the disease is most prevalent. Here we demonstrate a modular biosensor that is able to rapidly identify sequences associated with the Dengue virus genome. The biosensor consists of an oligonucleotide linker module, an aptamer/restriction endonuclease signal transducer and a fluorescent signalling molecule. The linker molecule has a simple stem/loop conformation and comprises a target-complementary moiety within the loop and a trigger moiety within the stem. When bound to the target nucleic acid, the trigger strand of the denatured stem can bind to the aptamer within the signal transducer. Disruption of the aptamer releases the restriction endonuclease EcoRI from aptamer-mediated inhibition. Active EcoRI is able to rapidly cleave multiple signalling molecules to generate a detectable signal. The biosensor was able to detect sequences derived from each of the four Dengue virus serotypes with a great degree of specificity. Along with sequences specific to each serotype, a pan-Dengue sequence, common to all serotypes, was also detected.

Adh1 first intron polymorphisms in Argentinean populations of Ceratitis capitata

AbstractDNA size polymorphisms were utilized in a study of 24 natural populations of Ceratitis capitata Wiedemann (Diptera: Tephritidae) from Argentina. The first intron of alcohol dehydrogenase 1 gene (Adh1) was amplified using exon priming intron crossing‐polymerase chain reaction. Three size variants were detected among the 307 samples analyzed. To better differentiate the size variants, further digestion of PCR products with the EcoRI restriction enzyme was carried out. Complete nucleotide sequences of the three‐allele variants were obtained and single changes, insertions, deletions, and EcoRI recognition sites were located. Population allele frequencies were analyzed and a global mean heterozygosity (He) of 0.33 was obtained. In most populations, observed allelic frequencies conformed to Hardy–Weinberg expectations. Significant differences between provinces and sampling sites within these provinces, and among some populations were found. The average number of insects exchanged among populations (Nm) was estimated and high values were observed between Argentina and populations from two African countries (Morocco and Kenya), Australia, and Hawaii (Kauai). Pest introduction sources and dispersion patterns in Argentina are discussed based on these results as well as on available bibliographical data.

Construction of recombinant retroviral vector containing human tissue factor pathway inhibitor and its expression in endothelial progenitor ceils

Objective To construct the recombinant retroviral vector capable of expressing human tissue factor pathway inhibitor and GFP in rat endothelial progenitor cells (EPCs). Methods Full length TFPI cDNA obtained from pIRES-TFPI by PCR amplification was digested with EcoRI and Xhol restriction enzymes and subsequently inserted into pMSCV-IRES-GFP expression vector to create the recombinant bicistronic retroviral vector pMSCV- TFPI- IRES -GFP encoding both TFPI and GFP . The recombinant plasmid was identified with restrictive endonuclease digestion and DNA sequencing. The recombinant plasmid was transfected into 293T and the supernatant containing packaged recombinant retroviral particles was collected and used to infect the EPCs isolated from rat bone marrow. TFPI mRNA was measured by RT-PCR and the amount of TFPI protein secreted from the transfected cells was determined by ELISA. GFP expression in the infected cells was analyzed by fluorescent microscopy and fluorescence activated cell sorting (FACS). Results Restriction endonuclease mapping and DNA sequencing confirmed the in-frame insertion of TFPI cDNA into the constructed vectorpMSCV-TFPI-IRES-GFP. Both RT-PCR and ELISA analysis demonstrated increased TFPI expression in the EPCs infected with pMSCV-TFPI-IRES-GFP. FACS analysis demonstrated that the transduction efficiency of EPCs with pMSCV-TFPI-IRES-GFP in vitro was over 90%. Conclusion pMSCV-TFPI-IRES-GFP could be effectively expressed in cultured EPCs and may provide a useful tool for further study on the application of TFPI in the prevention of restenosis. Key words: Endothelial progenitor cells; Retroviral vector; Tissue factor pathway inhibitor

Induced expression of EcoRI endonuclease as an active maltose-binding fusion protein in Escherichia coli

Among the numerous bacterial Type II restriction enzymes, EcoRI endonuclease is the most extensively studied and is widely used in recombinant DNA technology. Its heterologous overexpression as recombinant protein has already been studied. However, very limited information concerning its fused product is available thus far. In the present study, the EcoRI restriction endonuclease gene was cloned and expressed as a part of maltose-binding fusion protein under the control of strong inducible tac promoter in TB1 strain of Escherichia coli cells. Transformed cells containing pMALc2X-EcoRI recombinant plasmid were unable to grow under experimental conditions. However, fused EcoRI protein was purified (with the yield of 0.01 mg/l of bacterial culture) by affinity chromatography from E. coli cells induced at the late exponential phase of growth. Restriction quality test revealed that the purified product could restrict a control plasmid DNA in vitro.

Ubiquitination of PTEN (Phosphatase and Tensin Homolog) Inhibits Phosphatase Activity and Is Enhanced by Membrane Targeting and Hyperosmotic Stress

The PTEN (phosphatase and tensin homolog) tumor suppressor is a phosphatase that inhibits phosphoinositide 3-kinase-dependent signaling by metabolizing the phosphoinositide lipid phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) at the plasma membrane. PTEN can be mono- or polyubiquitinated, and this appears to control its nuclear localization and stability, respectively. Although PTEN phosphorylation at a cluster of C-terminal serine and threonine residues has been shown to stabilize the protein and inhibit polyubiquitination and plasma membrane localization, details of the regulation of ubiquitination are unclear. Here, we show that plasma membrane targeting of PTEN greatly enhances PTEN ubiquitination and that phosphorylation of PTEN in vitro does not affect subsequent ubiquitination. These data suggest that C-terminal phosphorylation indirectly regulates ubiquitination by controlling membrane localization. We also show that either mono- or polyubiquitination in vitro greatly reduces PTEN phosphatase activity. Finally, we show that hyperosmotic stress increases both PTEN ubiquitination and cellular PtdInsP(3) levels well before a reduction in PTEN protein levels is observed. Both PTEN ubiquitination and elevated PtdInsP(3) levels were reduced within 10 min after removal of the hyperosmotic stress. Our data indicate that ubiquitination may represent a regulated mechanism of direct reversible control over the PTEN enzyme.