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This article describes successful insertion the cDNA glucoamylase from Aspergillus awamori into the integrative vector for filamentous fungi pH4Hyg using the CPEC (circular polymerase extension cloning) strategy. The prior step was the PCR amplification of the cDNA glaA gene cloned into a vector based on the plasmid pBluescript II SK(-) and the amplification of linear pH4Hyg vector with promotor and terminator of the glaA gene. The PCR products were purified and used for CPEC reaction. The CPEC product was formed after 20 cycles of the reaction. It is proposed to use the final plasmid pH4HygPTgla in engineering of filamentous fungi strains producing stable forms of glucoamylase with biotechnological interest.

Factors relating to SYBR Green-based quantitative real-time PCR (qPCR) quantification of stubby root nematode Paratrichodorus allius using soil DNA were evaluated in this study. Soils used were loamy sand from potato fields in North Dakota and Idaho. Results showed that the largest nematode individuals (body length >720 µm) produced significant lower Cq values than the smallest individuals (4)(SO4)2 treatment during DNA preparation significantly reduced the inhibitors compared with post-treatment of soil DNA with polyvinylpolypyrrolidone column. The effect of PCR inhibitors on qPCR was suppressed by bovine serum albumin. Quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample when soil grinding and grid sampling strategies were used. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA containing known nematode numbers, produced similar correlations between Cq values and amount of targets. The targets in soil DNA quantified by qPCR using either standard curve correlated well with microscopic observations using both artificially and naturally infested field soils. This is the first study for assessing various factors that may affect qPCR quantification of stubby root nematodes. Results will be useful during the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA.

Background The pOV plasmid isolated from the Pasteurella multocida strain PMOV is a new plasmid, and its molecular characterization is important for determining its gene content and its replicative properties in Pasteurellaceae family bacteria. Methods Antimicrobial resistance mediated by the pOV plasmid was tested in bacteria. Purified pOV plasmid DNA was used to transform E. coli DH5α and Gallibacterium anatis 12656-12, including the pBluescript II KS(−) plasmid DNA as a control for genetic transformation. The pOV plasmid was digested with EcoRI for cloning fragments into the pBluescript II KS(−) vector to obtain constructs and to determine the full DNA sequence of pOV. Results The pOV plasmid is 13.5 kb in size; confers sulfonamide, streptomycin and ampicillin resistance to P. multocida PMOV; and can transform E. coli DH5α and G. anatis 12656-12. The pOV plasmid was digested for the preparation of chimeric constructs and used to transform E. coli DH5α, conferring resistance to streptomycin (plasmid pSEP3), ampicillin (pSEP4) and sulfonamide (pSEP5) on the bacteria; however, similar to pBluescript II KS(−), the chimeric plasmids did not transform G. anatis 12656-12. A 1.4 kb fragment of the streptomycin cassette from pSEP3 was amplified by PCR and used to construct pSEP7, which in turn was used to interrupt a chromosomal DNA locus of G. anatis by double homologous recombination, introducing strA-strB into the G. anatis chromosome. Conclusion The pOV plasmid is a wide-range, low-copy-number plasmid that is able to replicate in some gamma-proteobacteria. Part of this plasmid was integrated into the G. anatis 12656-12 chromosome. This construct may prove to be a useful tool for genetic studies of G. anatis.

The aim of this work is detection of trinitrotoluene (TNT) using aptamer as a new sensing strategy. Two pBluescript plasmids containing RT and ST anti-TNT aptamers were used as templates for aptamer amplification by PCR method. For this purpose, 126 bp ST-aptamer and 118 bp RT-aptamer were amplified using specific primers. TNT bound to bovine serum albumin (TNP-BSA) was used as the antigen, and digoxigenin (DIG)-labeled aptamers were detected by horseradish peroxidase conjugated to anti-DIG monoclonal antibodies. The sensitivity and specificity of ST- and RT-aptamers were determined using optimized PCR and enzyme-linked aptamer-sorbent assay. The sensitivity of this detection after optimization was determined about 1.76 nM of TNT using 1 pM of aptamers. These results indicated favorable functions of both aptamers for TNT detection that can be used in the future as aptasensors for investigation and utilization of the TNT identification.

The synthesis and characterization of two hybrid N-methylated carbazole derivatives containing a thiazolyl or a thienyl ring is reported. The thiazolyl derivative has been also characterised by X-ray diffraction analysis. The study of its reactivity in front of [MCl2(dmso)2] (M = Pd or Pt) or Na2[PdCl4] in methanol has allowed us to isolate and characterize its complexes. However, for the thienyl analogue, the formation of any Pd(II) or Pt(II) complex was not detected, indicating that it is less prone to bind to the M(II) ions than its thiazolyl analogue. Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) calculations have also been carried out in order to rationalize the influence of the nature of the thiazolyl or thienyl group on the electronic delocalization. Molecular mechanics calculations show that the free rotation of the thiazolyl in relation to the carbazole requires a greater energy income than for its thienyl analogue. Studies of the cytotoxic activity of the new compounds on colon (HCT116) and breast (MDA-MB231 and MCF7) cancer cell lines show that the thiazolyl carbazole ligand and its Pt(II) complex are the most active agents of the series and in the MCF7 line their potency is higher than that of cisplatin. In the non-tumoral human skin fibroblast BJ cell line, all the compounds were less toxic than cisplatin. Their potential ability to modify the electrophoretic mobility of pBluescript SK+ plasmid DNA and to act as inhibitors of Topoisomerases I and IIα or cathepsin B has also been investigated.

Xanthomonas albilineans is the causal pathogen of leaf scald disease of sugarcane. This devastating disease has been a major problem in most cane growing regions and continues to pose a worldwide economic threat to the sugarcane industry.Chlorosis inducing strains of the pathogen produce several toxic compounds. The major component, albicidin, inhibits DNA replication in sugarcane proplastids, resulting in blocked chloroplast development. Albicidin also inhibits prokaryotic DNA replication and is bactericidal to a range of Gram positive and Gram negative bacteria at concentrations as low as 1 ng ml-1. Albicidin has been partially characterized as a low molecular weight antibiotic that contains 38 carbon atoms with several aromatic rings. Albicidin deficient mutants of X. albilineans fail to cause chlorosis or any other symptom of leaf scald disease in inoculated sugarcane, indicating that albicidin phytotoxins play an import role in disease development. Therefore, resistance to albicidin may provide leaf scald disease resistance in sugarcane. X. albilineans is of interest as a potential source of these resistance genes. This project set out to investigate the genetics and mechanisms of albicidin resistance in X. albilineans.Two strategies were employed to isolate resistance genes from X. albilineans. No albicidin sensitive X. albilineans mutants were generated using transposon insertion mutagenesis; possibly because multiple redundant resistance genes contribute to albicidin resistance in X. albilineans, or because mutations in the resistance gene are lethal, even in Tox- mutants.The second strategy involved the construction of two X. albilineans genomic DNA cosmid libraries, followed by screening for clones conferring albicidin resistance in Escherichia coli. Albicidin resistant clones from the first library in E. coli DH5a proved unstable. One of these clones, E. coli JBDH93, conferred a 350-fold increase in albicidin resistance. However, the resistance gene was not transferrable on the cosmid. The mechanism of resistance did not involve export, binding, enzymatic detoxification or inactivation of the antibiotic, but did show a reduced uptake of the antibiotic. It is known that mutations in the tsx gene can prevent active uptake of the toxin in E. coli. Clone E. coli JBDH93 showed a reduced susceptibility to phage T6, which is characteristic of mutations in the tsx gene. PCR and Southern analysis showed that there had been some rearrangement in the tsx region. However, E. coli JBDH93 showed greater albicidin uptake than previously characterized albicidin resistant tsx mutants, and was resistant to approximately 10-fold higher albicidin concentration than previously characterized tsx mutants. After plating E. coli DH5a cells onto albicidin, no spontaneous mutants were obtained with albicidin resistance comparable to that of E. coli JBDH93. A gene from X. albilineans may be acting in combination with a tsx mutation to increase this clone's resistance to albicidin.The second genomic cosmid library was transformed into PMC 107, a multiple recombination deficient E. coli strain designed to minimise cosmid rearrangements. Several albicidin resistance clones were isolated. One of these clones showed at least a five-fold increased resistance to albicidin. The mechanism of resistance did not involve exclusion, binding, enzymatic detoxification or inactivation of the antibiotic. The 23 kb insert was subcloned and a putative 1487 bp albicidin resistance gene, designated albF was further isolated using PCR methods. In the vector pBlueScript II KS+ with induction of the lac promoter, only clones containing the ORF in the reverse orientation relative to the promoter could be generated, indicating that strong expression of albF is lethal to E. coli cells. Clones with the ORF in the forward orientation could only be maintained with repression of the lac promoter. Under conditions of lac repression, albF increased the minimum inhibitory concentration of albicidin by at least 3000-fold in the E. coli strains PMC107 and DH5a. No resistance to other antibiotics was observed.n n

In December 2012, tomato leaf curl disease (ToLCD) (2) was observed in tomato-growing areas of Gandhinagar District of Gujarat, a state in northwestern India. Incidence of ToLCD was estimated to be between 40 and 70% depending on the cultivars used. Infected plants exhibited symptoms consisting of leaf rolling, leaf curling, and yellowing typical of begomoviruses. Total DNA was isolated from a single affected tomato plant (2). Begomovirus infection in this sample was established by amplification of the expected-size 550-bp DNA fragment from this extract by PCR with degenerate DNA-A primers (3). Rolling circle amplification (RCA) using ϕ29 DNA polymerase was carried out on the total DNA, followed by digestion with Bam HI. An amplicon of ~2.8 kb was gel-eluted and cloned into Bam HI linearized pBluescript II KS(+). Restriction enzyme digestion of plasmid DNA from the resulting clones indicated the presence of one type of molecule. Using PCR and universal betasatellite primers, the expected 1.3-kb fragment was amplified from the DNA extract (1). An amplicon of ~1.3 kb was gel-eluted and cloned into pTZ57RT vector. Sequence analysis revealed that DNA-A (GenBank Accession No. KC952005) is composed of 2,753 nt and showed the highest identity (87.8%) with Tomato leaf curl Kerala virus[India:Kerala:2008] (GenBank Accession No. EU910141). An analysis for recombination showed this begomovirus DNA likely to have originated by recombination between Tomato leaf curl Kerala virus and Tomato leaf curl Karnataka virus. The satellite DNA-β (GenBank Accession No. KC952006) is composed of 1,365 nt and showed the highest identity (75.6%) with Tomato leaf curl betasatellite[India:Ludhiana:2004] (ToLCB-[IN:Lud:04]) (GenBank Accession No. AY765255). On the basis of DNA-A sequence analysis, the ICTV species demarcation criteria of 89% DNA-A sequence identity, and genome organization, the present isolate was considered as a new begomovirus species and named Tomato leaf curl Gandhinagar virus (ToLCGNV). The betasatellite shares less than 78% identity with (ToLCB-[IN:Lud:04]), it is considered a new species of betasatellite and the name, Tomato leaf curl Gandhinagar betasatellite (ToLCGNB) is proposed. Multimeric clones of the begomovirus and betasatellite DNAs were generated in a binary vector and these plasmids transformed into Agrobacterium tumefaciens. Nicotiana benthamiana and tomato plants agroinoculated with the cloned begomovirus DNA developed leaf curl symptoms, whereas plants co-agroinoculated with the cloned begomovirus and betasatellites developed more severe symptoms, including leaf rolling, leaf curling, and yellowing. The symptoms induced by the begomovirus and betasatellite DNAs were indistinguishable from those observed in the field. Thus, ToLCGNV is a new monopartite begomovirus which, in association with a new species of betasatellite, causes ToLCD in Gandhinagar, India. The presence of ToLCGNV needs to be considered, along with the already reported begomoviruses infecting tomatoes in this state, e.g., Tomato leaf curl Gujarat virus (2), in studies aimed to developing tomato cultivars with stable resistance to these tomato-infecting begomoviruses in India. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) C. Reddy et al. Arch Virol. 150:845, 2005. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

The interaction of the recently reported quinazoline derivative (E)-4-(2-(pyridin-2-ylmethylene)hydrazinyl)quinazoline (L) with MnCl2·4H2O resulted in the formation of two manganese(ΙΙ) complexes which were characterized by single-crystal X-ray crystallography, namely [Mn(L)(Cl)2]·H2O, 1·H2O, and [Mn(L)(Cl)(HCOO)(H2O)]·H2O, 2·H2O. The biological profile of complex 2 was further assessed in regard to the binding affinity with calf-thymus DNA, the cleavage ability of pBluescript II KS plasmid DNA, the interaction with bovine serum albumin and the ability to scavenge 1,1-diphenyl-picrylhydrazyl and 2,2΄-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radicals and to reduce H2O2.

Pesticides are among the most extensively chemicals used in the world today and they are also among the most hazardous compounds for the human. This study was designed to use the plasmid relaxation assay to describe the association of markers of DNA damage with pesticide exposure. The DNA damage activity of fluoxastrobin and imazamox were checked on pBluescript M13+ plasmid DNA (3.2 kb) in the absence and presence of Cu(II) ions. It has been found that the fluoxastrobin and imazamox can effectively promote cleavage of plasmid DNA in the absence and presence of Cu(II) ions. DNA cleavage was found to be concentration and dependent. In conclusion, the present study showed that fluoxastrobin and imazamox can induce DNA damage, which warrants for further investigations to correctly evaluate the hazards of exposure to these chemicals.

Here we have developed a novel method of isothermal amplification detection of double-stranded DNA (dsDNA) based on double-stranded fluorescence probe (ds-probe). Target dsDNA repeatedly generated single-stranded DNA (ssDNA) with polymerase and nicking enzyme. The ds-probe as a primer hybridized with ssDNA and extended to its 5′-end. The displaced ssDNA served as a new detection target to initiate above-described reaction. Meanwhile, the extended ds-probe could dynamically dissociate from ssDNA and self-hybridize, converting into a turn-back structure to initiate another amplification reaction. In particular, the ds-probe played a key role in the entire experimental process, which not only was as a primer but also produced the fluorescent signal by an extension and displacement reaction. Our method could detect the pBluescript II KS(+) plasmid with a detection limit of 2.3 amol, and it was also verified to exhibit a high specificity, even one-base mismatch. Overall, it was a true isothermal dsDNA detection strategy with a strongly anti-jamming capacity and one-pot, only requiring one ds-probe, which greatly reduced the cost and the probability of contamination. With its advantages, the approach of dsDNA detection will offer a promising tool in the field of point-of-care testing (POCT).

The present study deals with the synthesis of ZnO nanoflowers (ZnO-1 and ZnO-2) at room temperature using new structurally characterized single molecular precursors (1 and 2). 1, 2 and ZnO-1 were explored for their potential to reduce the viability of the Gram-negative bacteria Escherichia coli.1 and 2 were found to be promising antibacterial agents, while the ZnO nanoflowers demonstrated a relatively non-toxic nature. 1, 2 and ZnO-1 were further evaluated for DNA binding and cleavage behaviour. 1 and 2 showed strong binding affinity towards CT-DNA compared to ZnO-1. In addition, all the three compounds demonstrated oxidative cleavage of pBluescript plasmid DNA in the presence of H2O2.

Plasmids are essential tools for gene transfer and manipulation in many kinds of microorganisms, but they remain mysterious in the Shewanella species. Herein, a novel cryptic plasmid pSXM33 was isolated from marine bacterium Shewanella xiamenensis BC01 (SXM) and followed by sequencing and characterization through bioinformatics approaches. At first, the plasmid DNA was digested to relatively short fragments, sub-cloned into vector pMD19T-Simple and pBluescript SK(II), then transformed into Escherichia coli (E. coli) DH5α for sequencing. A full-length pSXM33 nucleotide sequence revealed 8,068 bp with GC content of 44%, containing 12 putative open reading frames (ORFs) and several unique restriction sites. Based on the annotation of sequences, ORF1 and ORF4 showed the highest similarity to the integrase, while ORF3, ORF7 and ORF8 encoded the replication protein RepB, plasmid stabilization protein and CopG family transcriptional regulator, respectively. The promoter prediction and tandem repeats analyses suggested 15 promoters and multiple tandem repeats. Moreover, pETSXM1 and pETSXM2 were successfully constructed as shuttle vectors for E. coli and Shewanella species, based on the repB from pSXM33 and a kanamycin resistance gene from vector pET28a(+) as a selective marker. These results provide a useful genetic tool for new insight into molecular level study of the Shewanella species.

Antimutagenic and DNA protective effect of an extract VinOserae from Vitis vinifera grapes on oxidative DNA damage was investigated. The extract’s ability to inhibit mutagenicity induced by tert-butyl hydroperoxide (t-BHP) and hydrogen peroxide (H2O2) was determined with Ames test using Salmonella typhimurium His? TA102 strain. Inhibition values of 44.2% and 67.0% were detected for t-BHP and H2O2, respectively. A protective ability of the extract against DNA strand scission induced by hydroxyl radicals was studied with plasmid pBluescript II SK(-). The analysis of DNA strand breaks in plasmid DNA showed a significant inhibition of DNA damage.

Viral vectors based gene therapy is often compromised by adverse immunological reactions raising safety concerns. Hence, improved design and development of non-viral vectors with strong regulatory regions is desired. We describe the design of a non-viral mammalian expression vector in which the primary transgene (a truncated dystrophin gene linked with Duchenne muscular dystrophy (DMD)) named microdystrophin delR4-R23/delCT (MD1) is under the transcriptional control of elements of desmin locus control region (DES-LCR). The designed vector, named as DES-LCR/MD1-EGFP, was constructed by cloning two fragments into the pBluescript backbone. Fragment 1 contains DES-LCR enhancer and DES-LCR promoter region while fragment 2 contains MD1 transgene and reporter EGFP (enhanced green fluorescent protein) gene separated by linker P2A (2A peptide). This vector design provides a framework for strong regulation with non-viral features. This design forms the foundation for application in conditions linked to multisystem diseases.

The nucleotide sequences of plasmids pRC12 (12,342 bp; GC 43.99%) and pRC18 (18,664 bp; GC 34.33%), harbored by the bacteriocin-producer Lactobacillus curvatus CRL 705, were determined and analyzed. Plasmids pRC12 and pRC18 share a region with high DNA identity (> 83% identity between RepA, a Type II toxin-antitoxin system and a tyrosine integrase genes) and are stably maintained in their natural host L. curvatus CRL 705. Both plasmids are low copy number and belong to the theta-type replicating group. While pRC12 is a pUCL287-like plasmid that possesses iterons and the repA and repB genes for replication, pRC18 harbors a 168 amino acid replication protein affiliated to RepB, which was named RepB’. Plasmid pRC18 also possesses a pUCL287-like repA gene but it was disrupted by an 11 kb insertion element that contains RepB’, several transposases/IS elements, and the lactocin Lac705 operon. An Escherichia coli / Lactobacillus shuttle vector, named plasmid p3B1, carrying the pRC18 replicon (i.e. repB’ and replication origin), a chloramphenicol resistance gene and a pBluescript backbone, was constructed and used to define the host range of RepB’. Chloramphenicol-resistant transformants were obtained after electroporation of Lactobacillus plantarum CRL 691, Lactobacillus sakei 23K and a plasmid-cured derivative of L. curvatus CRL 705, but not of L. curvatus DSM 20019 or Lactococcus lactis NZ9000. Depending on the host, transformation efficiency ranged from 102 to 107 per μg of DNA; in the new hosts, the plasmid was relatively stable as 29–53% of recombinants kept it after cell growth for 100 generations in the absence of selective pressure. Plasmid p3B1 could therefore be used for cloning and functional studies in several Lactobacillus species. Fil: Teran, Lucrecia Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Cuozzo, Sergio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: Aristimuño Ficoseco, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Fadda, Silvina G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Chaillou, Stéphane. Institut National de la Recherche Agronomique; Francia Fil: Champomier Vergès, Marie Christine. Institut National de la Recherche Agronomique; Francia Fil: Zagorec, Monique. Institut National de la Recherche Agronomique; Francia Fil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Raya, Raul Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Cervical cancer is the second most common cancer worldwide, after breast cancer. The agent of more than 90% of cervical cancer is Human papillomavirus (HPV). More than 100 various HPV with about 40 types of infective genotypes are identified and among these genotypes, near 15 carcinogenic genotypes are recognized as high risk. Moreover, genotypes 16 and 18 are the main agents of HPV related cancers. Today, HPV vaccines are available, but they could not cover all high-risk genotypes and also, they are really expensive. The aim of this study was to clone and express the L1 protein of human papillomavirus genotype 16 appropriately in the expression host Lactobacillus. The current study is the first step to prepare a live vaccine based on probiotic strain. In this study, the L1 gene of HPV genotype 16 was cloned to Lactobacillus pNZ7021 plasmid and its expression was assessed. So, the DNA of HPV genotype 16 was extracted from a clinical sample and by using specific primers, the L1 gene was amplified and inserted to the pBluescript cloning vector. Then, both amplified L1 gene and pNZ7021 were digested with BanII and HindIII restriction enzymes and ligated. Subsequently, the ligation product was transformed to Lactobacillus cremoris expression host. The recombinant host was cultured and L1 gene expression was evaluated by western blot method. The L1 gene amplifies by PCR successfully. The cloning of the L1 gene in the pBluescript vector was appropriate. The subcloning of the L1 fragment into the expression vector PNZ7021 was correct. Western blot results confirmed of L1 protein. It is suggested that the expression of the L1 protein-coding gene from human papillomavirus genotype 16 in the probiotic strain Lactobacillus cromoris could be considered as an immunogen, and this study needs further investigation.

Presence of low concentrations (1-2%) of ethanol during irradiation exhibited significant protection against DNA damage caused by very high doses (2-12 kGy) of 60 Co-gamma-rays in vitro. Radiation-induced DNA damage was substantially reduced in different types of DNA molecules (chromosomal DNA from Anabaena 7120 or Deinococcus radiodurans or bacteriophage Lambda, and plasmid pBluescript DNA) when irradiated in the presence of ethanol, thus indicating the generic nature of ethanol protection. The radioprotection appeared to be a consequence of the well known ability of ethanol to scavenge hydroxyl radicals. Addition of ethanol during 6 kGy irradiation also reduced DNA damage in vivo and improved post-irradiation growth recovery of Anabaena 7120 cultures. To our knowledge, this is the first instance of ability of very low ethanol concentrations to protect DNA from damage triggered by extremely high doses of 60 Co-gamma rays.

An advanced shuttle-vector for efficient nuclear transformation and genetic engineering of Haematococcus pluvialis has been developed and tested by inserting linked trans-genes. The phytoene desaturase ( pds ) gene mutated in the codon for the amino acid at position 504 (from leucine to arginine), yields an enzyme resistant to the herbicide norflurazon and is used as an endogenous selection marker for transformation of H. pluvialis . A novel cloning vector allowing insertion of additional genes, both at the 5′ and the 3′ end of the mutated pds , has been designed by inserting the selection marker into the cloning vector pBluescript II SK(−) to give pBS– pds featuring the genomic mutated pds including 2000 bp of its promoter. In a second version pBS– pds short was created, by shortening the promoter sequence to 1000 bp. Unique restriction sites 5′ and 3′ of the selection marker have been reserved for insertion and simultaneous transformation with two transgenes. Transformation efficiency was significantly better than previously reported, achieving transformation frequencies of 2 × 10 − 5 both with long and short promoters, as well as with linear constructs. An expression cassette for the ble derived from vector pGenD- ble was inserted into pBS- pds either 5′ or 3′ of the pds , and successfully transformed into H. pluvialis , resulting in engineered strains weakly expressing the ble mRNA driven by the Chlamydomonas reinhardtii PsaD promoter. Both circular plasmids, as well as linear DNA fragments only consisting of the ble cassette fused to the pds selection marker were used successfully to engineer H. pluvialis . The plasmid constructs presented here, as well as the use of an endogenous dominant selection marker, represent a blueprint for the future successful production of safe, genetically modified microalgae, for the possible production of high value products or biofuels.

In this study, the complete genome of the virulent strain LH of goose parvovirus (GPV) was sequenced and cloned into the pBluescript II (SK) plasmid vector. Sequence alignments of the inverted terminal repeats (ITR) of GPV strains revealed a common 14-nt-pair deletion in the stem of the palindromic structure in the LH strain and three other strains isolated after 1982 when compared to three GPV strains isolated earlier than that time. Transfection of 11-day-old embryonated goose eggs with the plasmid pLH, which contains the entire genome of strain LH, resulted in successful rescue of the infectious virus. Death of embryos after transfection via the chorioallantoic membrane infiltration route occurred earlier than when transfection was done via the allantoic cavity inoculation route. The rescued virus exhibited virulence similar to that of its parental virus, as evaluated by the mortality rate in goslings. Generation of the pathogenic infectious clone provides us with a powerful tool to elucidate the molecular pathogenesis of GPV in the future.

Our previous studies have demonstrated that Fam20C promotes differentiation and mineralization of odontoblasts, ameloblasts, osteoblasts and osteocytes during tooth and bone development. Ablation of the Fam20C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice. However, control and regulation of the expression of Fam20C are still unknown. In this study, we generated a transgenic reporter model which expresses green fluorescence protein (GFP) driven by the Fam20C promoter. Recombineering was used to insert a 16 kb fragment of the mouse Fam20C gene (containing the 15 kb promoter and 1.1 kb of exon 1) into a pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence. GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice. Fluorescence was evident in the bone and teeth as early as E17.5. The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth. The expression of GFP was significantly reduced in teeth, alveolar bone and muscle by 8 weeks of age. We also observed colocalization of the GFP signal with the Fam20C antibody in postnatal 1- and 7-day-old animals. Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20C gene expression and the biological function of this gene during odontogenesis and osteogenesis.

The anisotropic shape of DNA molecules allows them to form lyotropic liquid crystals (LCs) at high concentrations. This liquid crystalline arrangement is also found in vivo (e.g., in bacteriophage capsids, bacteria or human sperm nuclei). However, the role of DNA liquid crystalline organization in living organisms still remains an open question. Here we show that in vitro, the DNA spatial structure is significantly changed in mesophases compared to non-organized DNA molecules. DNA LCs were prepared from pBluescript SK (pBSK) plasmid DNA and investigated by photochemical analysis of structural transitions (PhAST). We reveal significant differences in the probability of UV-induced pyrimidine dimer photoproduct formation at multiple loci on the DNA indicative of changes in major groove architecture.

Nine aph genes, including aph(2″)-Ib , aph(2″)-Ic , aph(2″)-Ig , aph(2″)-If , aph(2″)-If1 , aph(2″)-If3 , aph(2″)-Ih , aac(6′)-Ie – aph(2″)-Ia , and aac(6′)-Ie – aph(2″)-If2 , were previously identified in Campylobacter . To measure the contribution of these alleles to aminoglycoside resistance, we cloned nine genes into the pBluescript and expressed them in Escherichia coli DH5α. The nine aph expressed in E. coli showed various levels of resistance to gentamicin, kanamycin, and tobramycin. Three genes, aac(6″)-Ie – aph(2″)-Ia , aph2″-If1 , and aph2″-Ig , showed increased MICs to amikacin, and five aph genes were transferrable.

For members of the family Parvoviridae, rescue of infectious virus from recombinant plasmid is usually done in cultured cells. In this study, the whole genome of the pathogenic Muscovy duck parvovirus (MDPV) strain YY was cloned into the pBluescript II (SK) vector, generating recombinant plasmid pYY. With the aid of a transfection reagent, pYY plasmid was inoculated into 11-day-old embryonated Muscovy duck eggs via the chorioallantoic membrane route, resulting in the successful rescue of infectious virus and death of the embryos. The rescued virus exhibited pathogenicity in Muscovy ducklings similar to that of its parental strain, as evaluated based on the mortality rate. The results demonstrate that plasmid transfection in embryonated Muscovy duck eggs is a convenient and efficacious method for rescue of infectious MDPV in comparison to transfection of primary cells, which is somewhat time-consuming and laborious.

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, KmR), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×101 to 1.0×105CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila.

The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes.

REMI (restriction enzyme-mediated integration) technique was employed to construct Trichoderma atroviride strain T23 mutants with degrading capability of neonicotinoid insecticide, imidacloprid. The plasmid pBluescript II KS-hph used for integration in REMI mutants was confirmed by PCR and Southern hybridization. Among 153 transformants, 57% of them have showed higher neonicotinoid insecticide, imidacloprid, degradation ability than the wild strain T23 (p

We constructed a phagemid consisting of the whole genome of the Neisseria gonorrhoeae bacteriophage NgoΦ6 cloned into a pBluescript plasmid derivative lacking the f1 origin of replication (named pBS::Φ6). Escherichia coli cells harboring pBS::Φ6 were able to produce a biologically active phagemid, NgoΦ6fm, capable of infecting, integrating its DNA into the chromosome of, and producing progeny phagemids in, a variety of taxonomically distant Gram-negative bacteria, including E. coli , Haemophilus influenzae , Neisseria sicca , Pseudomonas sp., and Paracoccus methylutens . A derivative of pBS::Φ6 lacking the phage orf7 gene, a positional homolog of filamentous phage proteins that mediate the interaction between the phage and the bacterial pilus, was capable of producing phagemid particles that were able to infect E. coli , Haemophilus influenzae , N. sicca , Pseudomonas sp., and Paracoccus methylutens , indicating that NgoΦ6 infects cells of these species using a mechanism that does not involve the Orf7 gene product and that NgoΦ6 initiates infection through a novel process in these species. We further demonstrate that the establishment of the lysogenic state does not require an active phage integrase. Since phagemid particles were capable of infecting diverse hosts, this indicates that NgoΦ6 is the first broad-host-range filamentous bacteriophage described.

The synthesis and characterization of the Pd(II) complex of the formula [Pd(L)2] 1 with the Schiff base 4-chloro-2-(N-ethyliminomethyl)-phenol (HL) as derived in situ via the condensation reaction of 5-chloro-salicylaldehyde and ethylamine was undertaken. The structure of 1 was verified by single-crystal X-ray crystallography. The ability of 1 to interact with calf-thymus (CT) DNA was studied by UV–vis and viscosity experiments, and its ability to displace ethidium bromide (EB) from the DNA-EB conjugate was revealed by fluorescence spectroscopy. It was found that intercalation is the most possible mode of interaction with CT DNA. Additionally, DNA electrophoretic mobility experiments showed that 1 interacts with the plasmid pBluescript SK(+) (pDNA) as proved by the formation of unusual mobility DNA bands and degradation of relaxed pDNA at concentration of 5 mM. The interaction of 1 with human (HSA) and bovine serum albumin (BSA) was monitored revealing its reversible binding to albumins. The complex showed noteworthy antimicrobial activity against one (Bacillus subtilis) of the five tested bacteria. In order to explain the described in vitro activity of the compound, we adopted molecular docking studies on the crystal structure of HSA, BSA, CT DNA and DNA-gyrase. Furthermore, in silico predictive tools have been employed to study the properties of the complex. The in silico studies are adopted on a multitude of proteins involved in cancer growth, as well as prediction of drug-induced changes of gene expression profile, protein- and mRNA-based prediction results, prediction of sites of metabolism, cytotoxicity for cancer cell lines, etc.

The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the 'switching mechanism at the 5'-end of the RNA transcript' (SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu mu L-1 and a storage capacity of 1.05x10(6) cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes (E-value 80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.

The gene encoding extracellular thermostable carboxylesterase (estA) from Geobacillus thermoleovorans YN was fused to six histidines and expressed under dif- ferent promoters. Three different clones carrying this gene were constructed in host cell Escherichia coli BL21 (DE2) using three expression systems pET-17b, pBluescript II KS(?) and pCYTEX-P1 (pET-estA T7 promoter; pBlue- estA T7 promoter and pBlue-estA T7 promoter and Lambda pL promoter). The efficiency of expression of the three constructs was evaluated, where the highest esterase expression (589 lmol/ml) using p-nitrophenyl laurate as substrate (pNP-laurate: C18H27NO4) was measured under the control of T7 promoter in expression vector pET-17b after 4 h of the induction. A 1.5-fold increase in enzyme activity was measured with specific activity 1,043 lmol/ mg protein on growing the clone expressed the target gene under T7 promoter (pET-estAT7) in 2-L working volume BIOFLO III bioreactor under optimal conditions. Recom- binant expression of the tested thermostable esterase was monitored by sodium dodecyl sulfate polyacrylamid gel electrophoresis, zymogram and activity measurements with a-naphthyl acetate (C12H10O2) and Fast Red. The SDS- PAGE analysis of E. coli BL21(DE3)/pET-estA lysates indicated the presence of a protein band (29 kDa) related to the targeted expressed gene.

The bacterial respiratory gene, nuoA, was previously used as a reporter gene in an amperometric, whole cell biosensor for tetracycline (Tet) detection. While the nuoA-based bioassay responded sensitively to Tet, the signal declined at high Tet concentrations, probably partly due to transgene over-expression. Also, at zero concentration of Tet, the assay registered a relatively high background signal when compared to the nuoA knockout Escherichia coli strain without the biosensor transgene construct. This was probably due to incomplete repression of nuoA expression. In order to reduce gene over-expression, the sensor cells were incubated with Tet at a relatively low temperature (15 °C). Also, a low-copy number plasmid pBR322 was used to carry the transgene, instead of the high-copy number plasmid pBluescript in order to reduce over-expression and to reduce background expression. Both assays improved the biosensor response. By using a low-copy number plasmid and tetracycline resistance, the sensor was less inhibited at higher Tet concentrations; but, this did not significantly increase the linear range of the sensor. The low temperature nuoA assay could detect Tet at a range of 0.001-1 μg ml(-1). In contrast, the low-copy number nuoA assay was able to detect Tet at a range of 0.0001-1 μg ml(-1). The detection limit of Tet determined by the low-copy number nuoA assay was 0.00023 μg ml(-1), which is one order of magnitude more sensitive than in the previous nuoA assay.

The heteroduplex mobility assay (HMA) is widely used to characterize strain variants of human viruses. To determine whether it can detect small sequence differences in homologous templates, we constructed a series of deletion constructs (1–10 bp deletions) in the multiple cloning site (MCS) of pBluescript II. After PCR amplification of the MCS using a mixture of wild-type and one of the deletion constructs, the resulting PCR amplicons were electrophoresed using 15% polyacrylamide gels. Two types of heteroduplexes exhibited retarded electrophoretic migration compared with individual homoduplexes. Therefore, we applied this HMA to detect transcription activator-like effector nucleases (TALEN)-induced insertion and/or deletion (indel) mutations at an endogenous locus. We found that TALEN in vivo activity was easily estimated by the degree of multiple HMA profiles derived from TALEN-injected F0 embryos. Furthermore, TALEN-injected F0 founder fish produced several unique HMA profiles in F1 embryos. Sequence analysis confirmed that the different HMA profiles contained distinct indel mutations. Thus, HMA is a rapid and sensitive analytical method for the detection of the TALEN-mediated genome modifications.

Radish mosaic virus (RaMV) is a crucifer-infecting comovirus that has been detected worldwide. Here, we report the successful construction of a full-length infectious cDNA clone of RaMV. The full-length cDNA clones corresponding to RNA1 and RNA2 of a Japanese isolate of RaMV were cloned into the pBlueScript plasmid or the binary vector pCAMBIA1301 downstream of the cauliflower mosaic virus 35S promoter. Mechanical inoculation or agroinoculation of Nicotiana benthamiana with these vectors resulted in systemic RaMV infections causing symptoms similar to those caused by the wild-type parental virus. The presence of progeny virus was verified by western blot analysis and electron microscopy.

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis, and in more severe cases, a serious clinical complication called hemolytic uremic syndrome (HUS). Shiga toxin (Stx)is one of the factors that cause HUS. Serotypes of Stx produced by EHEC include Stx1 and Stx2. Although some genetically mutated toxoids of Stx have been developed, large-scale preparation of Stx that is practical for vaccine development has not been reported. Therefore, overexpression methods for Stx2 and mutant Stx2 (mStx2) in E. coli were developed. The expression plasmid pBSK-Stx2(His) was constructed by inserting the full-length Stx2 gene, in which a six-histidine tag gene was fused at the end of the B subunit into the lacZα fragment gene of the pBluescript II SK(+) vector. An E. coli MV1184 strain transformed with pBSK-Stx2(His) overexpressed histidine-tagged Stx2 (Stx2-His) in cells cultured in CAYE broth in the presence of lincomycin. Stx2-His was purified using TALON metal affinity resin followed by hydroxyapatite chromatography. From 1 L of culture, 68.8 mg of Stx2-His and 61.1 mg of mStx2-His, which was generated by site-directed mutagenesis, were obtained. Stx2-His had a cytotoxic effect on HeLa cells and was lethal to mice. However, the toxicity of mStx2-His was approximately 1000-fold lower than that of Stx2-His. Mice immunized with mStx2-His produced specific antibodies that neutralized the toxicity of Stx2 in HeLa cells. Moreover, these mice survived challenge with high doses of Stx2-His. Therefore, the lincomycin-inducible overexpression method is suitable for large-scale preparation of Stx2 vaccine antigens.

Background Muscovy duck parvovirus (MDPV) is the etiological agent of Muscovy duckling parvoviral disease, which is characterized by diarrhea, locomotive dysfunction, stunting, and death in young ducklings, and causes substantial economic losses in the Muscovy duck industry worldwide. FZ91-30 is an attenuated vaccine strain that is safe and immunogenic to ducklings, but the genomic information and molecular mechanism underlining the attenuation are not understood. Methods The FZ91-30 strain was propagated in 11-day-old embryonated goose eggs, and viral particles were purified from the pooled allantoic fluid by differential centrifugation and ultracentrifugation. Single-stranded genomic DNA was extracted and annealed to form double-stranded DNA. The dsDNA digested with NcoI resulted two sub-genomic fragments, which were then cloned into the modified plasmid pBluescript II SK, respectively, generating plasmid pBSKNL and pBSKNR. The sub-genomic plasmid clones were sequenced and further combined to construct the plasmid pFZ that contained the entire genome of strain FZ91-30. The complete genome sequences of strain FM and YY and partial genome sequences of other strains were retrieved from GenBank for sequence comparison. The plasmid pFZ containing the entire genome of FZ91-30 was transfected in 11-day-old embryonated goose eggs via the chorioallantoic membranes route to rescue infectious virus. A genetic marker was introduced into the rescued virus to discriminate from its parental virus. Results The genome of FZ91-30 consists of 5,131 nucleotides and has 98.9 % similarity to the FM strain. The inverted terminal repeats (ITR) are 456 nucleotides in length, 14 nucleotides longer than that of Goose parvovirus (GPV). The exterior 415 nucleotides of the ITR form a hairpin structure, and the interior 41 nucleotides constitute the D sequence, a reverse complement of the D′ sequence at the 3′ ITR. Amino acid sequence alignment of the VP1 proteins between FZ91-30 and five pathogenic MDPV strains revealed that FZ91-30 had five mutations; two in the unique region of the VP1 protein (VP1u) and three in VP3. Sequence alignment of the Rep1 proteins revealed two amino acid alterations for FZ91-30, both of which were conserved for two pathogenic strains YY and P. Transfection of the plasmid pFZ in 11-day-old embryonated goose eggs resulted in generation of infectious virus with similar biological properties as compared with the parental strain. Conclusions The amino acid mutations identified in the VP1 and Rep1 protein may contribute to the attenuation of FZ91-30 in Muscovy ducklings. Plasmid transfection in embryonated goose eggs was suitable for rescue of infectious MDPV.

To study DNA transport in isolated mitochondria from potato tubers (Solanum tuberosum), we developed an approach permitting the analysis of internalized DNA by quantitative PCR. It was shown that mitochondrial transport of recombinant plasmid pBluescript IIKS (+) of the size of 3050 bp and DNA fragment of 89 bp amplified from human LINE1 repeat did take place, apparently through different membrane translocation mechanisms. Regardless of the length of the polynucleotide chain, DNA transport into mitochondria was a reversible process.

One of the most important consequences of oxidative stress is the induction of oxidation in DNA molecules. Permanent modification of genetic material resulting from “oxidative damage” incidents represents the first step involved in mutagenesis, carcinogenesis, and aging. Therefore, there is an urgent need for monitoring DNA oxidative damage in a quantitative way. For this purpose, the present work evaluates the use of gel electrophoresis (GE) on-line coupled to inductively coupled plasma-mass spectrometry (ICP-MS) to address induced oxidative events in plasmid DNA pBlueScript SK (2961 bp). Oxidative stress is induced in the samples by addition of Fe2+ and H2O2 through the Fenton reaction. After optimization of the set-up for large DNA fragments, the GE separation of the different oxidation products (as induced by the Fenton reaction) were followed by 31P+ monitoring with ICP-MS and compared with conventional slab gels. The main advantage with the proposed set-up is that the quantification of each resulting oxidation product could be performed using just an inorganic phosphate as internal standard.

Reaction of [RuII(tpy)(pic)(H2O)]+ (1) with KHSO5 resulting in the formation of [RuIV(tpy)(pic)(O)]+ (2) was studied kinetically as a function of [KHSO5], temperature (15–35°C), and pressure (10–30 MPa) at a fixed pH of 5.1 using spectrophotometric techniques. A suggested mechanism that is in agreement with the observed rate and activation parameters is presented. Complex 1 was found to induce DNA (pBluescript) cleavage in the presence of KHSO5, which proceeds via oxygen transfer from 2.

Several biotechnological processes rely on the utilization of high-copy-number plasmids for heterologous gene expression, and understanding the interactions between plasmid DNA and bacterial hosts is highly relevant for bioprocess optimization. We assessed metabolic modifications and physiological changes exerted by expression of a plasmid-encoded alcohol-acetaldehyde dehydrogenase from Leuconostoc mesenteroides (adhE Lm ) in Escherichia coli redox mutants. Plasmid pET Lm , a pBluescript II KS(–)-derivative carrying adhE Lm , was introduced in E. coli CT1061 [arcA creC(Con)]. This recombinant was able to attain a higher ethanol concentration in glycerol cultures compared to the parental strain. pBluescript II KS(–) was rapidly lost in 72-h bioreactor cultures (7.8 ± 1.2% of plasmid-bearing cells), while pET Lm was present in 92.4 ± 7.2% of the cells. In E. coli CT1061 carrying pBluescript II KS(–) the plasmid copy number steadily diminished in bioreactor cultures to reach 334 ± 45 copies per chromosome at 72 h, while pET Lm was stably maintained, reaching 498 ± 18 copies per chromosome at the end of the cultivation. Plasmid pETΩ Lm , bearing a defective copy of adhE Lm interrupted by cat, reached 293 ± 62 copies per chromosome, implying a functional role of adhE Lm on plasmid maintenance. The intracellular NADH/NAD+ content suggest that regeneration of oxidized co-factors by the heterologous bioreaction might play a relevant role in plasmid maintenance.

Light Upon eXtension real-time PCR (LUX real-time PCR) assay was developed for the detection of porcine circovirus type 2 (PCV2). The primers flanking a 114 bp fragment were selected from ORF1. The optimized assay could detect 20 viral copies of pBluescript SK+ plasmid containing inserted PCV2 DNA. The dynamic range of quantitative analysis covered a 7-order interval ranging from 20 to 2 × 108 genome equivalents per assay with the best results in the range from 2 × 102 to 2 × 107 viral copies. The LUX real-time PCR assay had a high specificity since it detected PCV2 but not PCV1, CSFV, PRRSV or negative samples. There was good agreement between the LUX real-time PCR and the conventional PCR when lymph nodes from PCV2 infected animals were tested. A comparison of the LUX real-time PCR with the TaqMan PCR and SYBR Green PCR indicated that the amount of viral copies determined using linear calibration curve differed from assay to assay but not more than an order. LUX real-time PCR, similar to the TaqMan PCR, was more specific for generation of fluorogenic signal than SYBR Green PCR.

A full-length cDNA clone of duck hepatitis virus type 1 (DHV-1) isolated from duck was assembled in the plasmid vector pBluescript II SK+. RNA synthesized in vitro by means of a Sp6 promoter inserted in front of the cDNA produced infectious particles after transfection of BHK-21 cells, as shown by the appearance of cytopathic effect. The rescued virus was also found to be highly pathogenic to young ducks by intradermal injection and resulted in a fatal disease indistinguishable from that of wild-type virus. The availability of the first cDNA clone of DHV-1 will allow examination of the molecular mechanisms behind DHV-1 virulence and attenuation, which could in turn lead to the production of second-generation, genetically engineered DHV-1 vaccines.

Summary A small-insert bovine genomic library was constructed in pBluescript II SK(+) and enriched for microsatellites by selective rescue of single-stranded pBluescript DNA carrying (CA)n/(TG) n tandem repeats. Approximately 50% of the clones in the enriched library contained (CA) nrepeats or CA-rich sequences. Sequencing of clones selected for (CA) n repeats resulted in the identification and characterization of 45 (CA) npolymorphic microsatellites. Genotyping in 9 large paternal half-sib families indicated that 40 of these microsatellite markers exhibit autosomal Mendelian inheritance. The numbers of alleles range from 2 to 18, with an average of 6-3 per locus. The polymorphic microsatellite markers we have identified and characterized will contribute to the construction of a high-resolution linkage map of bovine genome.

RuIII-edta (edta, ethylenediaminetetraacetate) induced cleavage of pBluescript SK+ plasmid DNA in the presence of air with primary oxidant, PO (PO = H2O2, KHSO5) or reductant (L-ascorbic acid) has been studied at pH 7.2. The studies revealed that the RuIII-edta complex induces DNA cleavage in different ways. A mechanism suggesting the involvement of [RuV(edta)O]− in the oxidative cleavage of DNA is proposed for H2O2 and KHSO5. Generation of active oxygen radical species ( /OH•) is proposed for cleavage of DNA with RuIII-edta/ascorbate system. Results are discussed in reference to the data reported for the reaction of Ru-edta with DNA constituents, H2O2, KHSO5, and L-ascorbic acid.

The levansucrase gene (lsrA) from Rahnella aquatilis was strongly expressed in a constitutive manner in Escherichia coli when cloned into a pBluescript KS-based pRL1CP plasmid vector. The native promoter upstream of lsrA and the lacZ promoter cooperatively enhanced the expression of lsrA to a level that was comparable to that of the T7 promoter, which is used in commercial pET expression vector system. A putative rho-independent transcription termination signal downstream of lsrA was crucial for gene expression. This plasmid vector also proved to be applicable for efficient expression of other foreign genes in E. coli.

Methylation of CpGs is generally thought to repress transcription without significant influence from the sequence surrounding the methylated dinucleotides. Using the mouse Igf2/H19 imprinting control region (ICR), Igf2r differentially methylated region 2 (DMR2) and bacterial sequences, we addressed how methylation-dependent repression (MDR) from a distance varies with CpG number, density and surrounding sequence. In stably transfected F9 cells, the methylated ICR repressed expression from a CpG-free reporter plasmid more than 1000-fold compared with its unmethylated control. A segment of pBluescript, with a CpG number equal to the ICR's but with a higher density, repressed expression only 70-fold when methylated. A bacteriophage lambda fragment and the Igf2r DMR2 showed minimal MDR activity, despite having CpG numbers and densities similar to or greater than the ICR. By rearranging or deleting CpGs, we identified CpGs associated with three CTCF sites in the ICR that are necessary and sufficient for sequence-specific MDR. In contrast to F9 cells, the methylated ICR and pBS fragments exhibited only 3-fold reporter repression in Hela cells and none in Cos7. Our results show that the strength of MDR from a distance can vary a 1000-fold between different cell types and depends on the sequence surrounding the methylated CpGs, but does not necessarily increase with CpG number or density.

To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK(+). The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

UDP-GlcNAc:α-6–D-mannoside [GlcNAc to Manα1–6] β-1,2-N-acetylglucosaminyltransferase II (GlcNAc-T II, EC 2.4.1.143) is a Golgi enzyme catalyzing an essential step in the conversion of oligo-mannbse to complex N-glycans. A 1.2-kb probe from a rat liver cDNA encoding GlcNAc-T II was used to screen a human genomic DNA library in λEMBL3. Southern analysis of restriction endonuclease digests of positive phage clones identified two hybridizing fragments (3.0 and 3.5 kb) which were sub-cloned into pBlueScript. The inserts of the resulting plasmids (pHG30 and pHG36) are over-lapping clones containing 5.5 kb of genomic DNA. The pHG30 insert (3.0 kb) contains a 1341-bp open reading frame encoding a 447-amino-acid protein, 250 bp of G+C-rich 5′-upstream sequence and 1.4kb of 3′-downstream sequence. The pHG36 insert (3.5 kb) contains 2.75 kb of 5′-upstream sequence and 750 bp of the 5′-end of the open reading frame. The protein sequence showed the domain structure typical of all previously cloned glycosyltransferases, i.e. a short 9-residue putative cytoplasmic N-terminal domain, a 20-residue hydrophobic non-cleavable putative signal-anchor domain and a 418-residue C-terminal catalytic domain. Northern analysis of human tissues showed a major message at 3 kb and minor signals at 2 and 4.5 kb. There is no sequence similarity to any previously cloned glycosyltransferases including human UDP-GlcNAc:α-3-d-mannoside [GlcNAc to Manα;1–3] β-1,2-N-acetylglucosaminyltransferase I (GlcNAc-T I) which has 445 amino acids with a 418-residue C-terminal catalytic domain. The human GlcNAc-T I and II genes (MGAT1 and MGAT2) map to chromosome bands 5q35 and 14q21, respectively, by fluorescence in situ hybridization. The entire coding regions of human GlcNAc-T I and II are each on a single exon. There is 92% identity between the amino acid sequences of the catalytic domains of human and rat GlcNAc-T II. Southern analysis of restriction enzyme digests of human genomic DNA indicates that there is only a single copy of the MGAT2 gene. The full-length coding region of GlcNAc-T II has been expressed in the baculovirus/Sf9 insect cell system, the recombinant enzyme has been purified to near homogeneity with a specific activity of about 20 μmol · min-1· mg-1· and the product synthesized by the recombinant enzyme has been identified by high-resolution 1H-NMR spectroscopy and mass spectrometry.

A full-length cDNA encoding ribosome-inactivating/antiviral protein from the leaves of Bougainvillea xbuttiana was recently isolated. The coding region of cDNA was cloned and expressed in Escherichia coli, and the protein product was designated as BBAP1 (Bougainvillea xbuttiana antiviral protein 1). BBAP1 showed ribonuclease activity against Torula yeast RNA. It also exhibited depurination activity against supercoiled pBlueScript SK+ plasmid DNA in a concentration dependent manner, and was found to convert nicked circular DNA into linear form only at higher concentration. On bioassay, BBAP1 exhibited antiviral activity against sunnhemp rosette virus infecting Cyamopsis tetragonoloba leaves in which 95% inhibition of local lesion formation was observed.

In this work the first protease gene encoding a novel zinc-metalloprotease from the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 has been cloned, sequenced and reported to the GenBank. We have generated a library containing about 10,000 transformants whose screening yielded one clone harboring plasmid pBluescript with 3.6 kb inserted fragment (pBlueSVP2) with positive caseinolytic activity. Nucleotide sequence analysis of the selected clone revealed a single open reading frame (ORF) of 1833 bp encoding 611 amino acids. The deduced amino acid sequence includes a zinc-metalloprotease HEXXH-E consensus motif which is highly conserved in the M4 family of proteases. The primary amino acid sequence alignment search in the database revealed a moderate homology between the deduced amino acid sequence and the known zinc-metalloproteases including vibriolysin from Vibrio vulnificus and Pseudomonas aeruginosa elastase. The full length of SVP2 gene was subcloned into pQE-80L (pQEVP1) and transformed into Escherichia coli BL21 (DE3) for recombinant overexpression of the protease. Following induction by IPTG, active enzyme was found within cells and in the extracellular medium, where it slowly accumulated to high levels. Mass spectrometric fingerprinting of trypsin digested rSVP2 analysis identified the processed mature protease which starts at Ala-200 of a SVP2 full length protein. Although this result suggested a mature protein of 412 amino acids (44.8 kDa), electrospray-ionisation mass spectrometry revealed that the molecular mass of purified rSVP2 was only 34.2 kDa, which indicates a further cleavage site at the C-terminal.

The genetics of streptomycin production is well characterized in Streptomyces griseus. More than 25 clustered genes encode proteins involved in biosynthesis, regulation and transport functions. StrR, the pathway specific transcriptional activator or regulator that located in this cluster, then induces transcription of other streptomycin production genes by binding multiple sites in the gene cluster. We aim to put strR gene in to different multicopy and integrated expression vector specifically designed for Streptomyces. To start with, the isolated strR gene was ligated into pBluescript (pBs) vector and transformed into different strains of Escherichia coli. The correct structure of the recombinant plasmid, isolated from transformed E. coli, was confirmed using gel electrophoresis, PCR and double digested with restriction enzymes BamHI and EcoRI. Finally the plasmid map, named pFDstrR. This unique vector has a much expanded Multiple Cloning Site (MCS), which makes it suitable for different purposes of gene cloning and also site directed mutagenesis or gene targeting. This gene will be lifted up and transfer into different varieties of Streptomyces specific vectors in order to make different transgenic or genetically manipulated Streptomyces.