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Restriction Sites (restriction + site)
Terms modified by Restriction Sites Selected AbstractsCover Picture: Electrophoresis 4'2010ELECTROPHORESIS, Issue 4 2010Article first published online: 16 FEB 2010 Issue no. 4 is a regular issue with Emphasis on "Bioanalysis". Part I has 12 articles on bioanalysis featuring methodologies for proteomics, proteins, peptides and nucleic acids. Part II has 5 papers on interactive CE, e.g., MEEKC, MEKC and ACE. The remaining 2 articles make up Part III, and are concerned with the regulation of flow in microfluidics and sensitivity enhancement in CEC. Featured articles include: Identification of candidate biomarkers in ovarian cancer serum by depletion of highly abundant proteins and differential in gel electrophoresis. ((10.1002/elps.200900441.R1)) An effective SCAR-PCR method derived from restriction site amplified polymorphism (RSAP) for the identification of female Schistosoma japonicum of zoonotic significance. ((10.1002/elps.200900615.R1)) Rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection based on microchip. ((10.1002/elps.200900538.R1)) [source] Identification of Tetrodotoxin and Fish Species in an Adulterated Dried Mullet Roe Implicated in Food PoisoningJOURNAL OF FOOD SCIENCE, Issue 1 2003Y.W. Hsieh ABSTRACT: There was 1 victim of neurotoxic food poisoning from an adulterated dried mullet roe in Kaohsiung, Taiwan, in March 2001. The victim exhibited typical neurotoxic symptoms. The residue of dried mullet roe retained by the victim was assayed for toxicity and mitochondrial DNA. Its toxicity was 3450 mouse units per gram. The toxin was partially purified and identified as tetrodotoxin and derivative. The sequence of the 376-nucleotide region in the cytochrome b gene of the mitochondrial DNA exhibited the same genotype and the same restriction site for SapI as that of the toxic puffer fish Lagocephalus lunaris. [source] Prenatal diagnosis of 21-hydroxylase deficiency caused by gene conversion and rearrangements: pitfalls and molecular diagnostic solutionsPRENATAL DIAGNOSIS, Issue 13 2002Rong Mao Abstract Objectives The present paper reports the prenatal diagnosis of congenital adrenal hyperplasia (CAH) in two cases of 21-hydroxylase deficiency. DNA diagnostic errors can be caused by the presence of the highly homologous 21-hydroxylase pseudogene, CYP21P, adjacent to the functional gene, CYP21. The present paper details how complex gene conversions and rearrangements between the CYP21 and CYP21P pose unique complications for prenatal diagnosis. Methods Analysis of eight common mutations in the 21-hydroxylase gene as well as deletion of the entire gene is accomplished using polymerase chin reaction (PCR) followed by amplified created restriction site (ACRS) or allele-specific oligohybridization (ASO) and Southern blot followed by hybridization to a CYP21-specific probe. Linkage analysis was performed using microsatellite markers flanking the CYP21 gene. Results The direct mutation detection assay indicated a complicated gene conversion and rearrangement in the probands of both families. Interpretation of these rearrangements made it difficult to determine whether or not the fetuses would be affected with CAH. Linkage studies revealed that each fetus had inherited both parental disease chromosomes and was therefore predicted to be affected with CAH. Conclusion As observed in the two reported cases, direct DNA analysis may provide limited information due to gene conversion or rearrangement between the CYP21 and CYP21P genes. These cases suggest that direct mutation detection should be supported by linkage analysis, whenever possible, to provide more comprehensive information for the family. Copyright © 2002 John Wiley & Sons, Ltd. [source] Molecular analysis of genomic DNA allows rapid, and accurate, prenatal diagnosis of peroxisomal D-bifunctional protein deficiencyPRENATAL DIAGNOSIS, Issue 1 2002B. C. Paton Abstract Prenatal diagnosis was requested for a couple with a previous child affected by the peroxisomal disorder D-bifunctional protein deficiency. Prior analysis of the D-bifunctional protein cDNA sequence from the propositus had shown that it was missing 22,bp. This was subsequently attributed to a point mutation in the intron 5 donor site (IVS5+1G>C) of the D-bifunctional protein gene. Consistent with parental consanguinity, the patient was shown to be homozygous for this mutation, which is associated with loss of a Hph 1 restriction site in the genomic sequence. Prenatal testing of the fetus using genomic DNA isolated from uncultured amniocytes indicated that both alleles of the D-bifunctional protein had the IVS5+1G>C substitution. The peroxisomal defect was later confirmed biochemically using cultured amniocytes, which were found to have elevated levels of very long chain fatty acids (VLCFA). This is the first report of prenatal diagnosis of D-bifunctional protein deficiency using molecular analysis of genomic DNA. Copyright © 2002 John Wiley & Sons, Ltd. [source] The role of double-strand break-induced allelic homologous recombination in somatic plant cellsTHE PLANT JOURNAL, Issue 3 2002Brigitte Gisler Summary During meiosis, homologous recombination occurs between allelic sequences. To evaluate the biological significance of such a pathway in somatic cells, we used transgenic tobacco plants with a restriction site for the rare cutting endonuclease I- SceI within a negative selectable marker gene. These plants were crossed with two tobacco lines containing, in allelic position, either a deletion or an insertion within the marker gene that rendered both marker gene and restriction site inactive. After the double-strand break induction, we selected for repair events resulting in a loss of marker gene function. This loss was mostly due to deletions. We were also able to detect double strand break-induced allelic recombination in which the break was repaired by a faithful copying process from the homologue carrying the shortened transgene. The estimated frequency indicates that homologous recombination in somatic cells between allelic sites appears to occur at the same order of magnitude as between ectopic sites, and is thus far too infrequent to act as major repair pathway. As somatic changes can be transferred to the germ line, the prevalence of intrachromatid rearrangements over allelic recombination might be an indirect prerequisite for the enhanced genome plasticity postulated for plants. [source] Identification of a novel deletion in the OA1 gene: report of the first Spanish family with X-linked ocular albinismCLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 5 2010Monica Martinez-Garcia PhD Abstract Background:, This study was undertaken to analyse the OA1 gene (GPR143) and its involvement in a Spanish family presenting with nystagmus, a common symptom of X-linked ocular albinism (XLOA). Methods:, DNA samples from the index case and eight relatives were analysed by multiplex ligation-dependent probe amplification (MLPA). Sequence analysis and restriction assay were used to confirm the results. In addition, an analysis of a STR located in intron 1 of the OA1 gene (OA-CA) was performed. Results:, The father of the proband presented with nystagmus, a feature consistent with XLOA. Mutation screening by multiplex ligation-dependent probe amplification and sequence analysis of the exon 2 of the OA1 gene led to the identification of the novel p.Glu129fsX35 (g.5815delA) mutation in two affected males and four carrier females. Three relatives were found to be non-mutated. The deletion detected resulted in a truncated protein 35 codons downstream and generated a new restriction site for the XcmI endonuclease. Additionally, microsatellite analysis showed co-segregation with the disease in the family. Conclusions:, A novel deletion in the OA1 gene was identified in a Spanish family with ocular albinism. The mutation detected is likely a loss-of-function alteration. To the best of our knowledge, we describe the first Spanish family known to present with XLOA due to mutations in the OA1 gene. [source] ,-Globin gene cluster haplotypes and HbF levels are not the only modulators of sickle cell disease in LebanonEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2003A. Inati Abstract: Sickle cell disease (SCD) is an inherited autosomal recessive disorder of the , -globin chain. Despite the fact that all subjects with SCD have the same single base pair mutation, the severity of the clinical and hematological manifestations is extremely variable. This study examined for the first time in Lebanon the correlation between the clinical manifestation of SCD and the , -globin gene haplotypes. The haplotypes of 50 patients diagnosed with SCD were determined using polymerase chain reaction amplification of fragments containing nine polymorphic restriction sites around and within the ,,G,,A,,,,,,,, -globin gene complex. Most reported haplotypes were found in our population with the Benin haplotype as the most prevalent one. When the patients were divided according to their HbF levels into three groups (Group A: HbF < 5%, Group B: HbF between 5 and 15%, and Group C: HbF > 15%), surprisingly, the highest levels of HbF were associated with the most severe clinical cases. Our findings suggest that fetal hemoglobin levels are important but not the only parameters that affect the severity of the disease. In addition, the high levels of HbF in patients with CAR haplotypes did not seem to ameliorate the severity of symptoms, suggesting that genetic factors other than haplotypes are the major determinants of increased HbF levels in Lebanon. [source] Genetic variations among Mycoplasma bovis strains isolated from Danish cattleFEMS MICROBIOLOGY LETTERS, Issue 1 2000Lughano J.M. Kusiluka Abstract The genetic heterogeneity of Mycoplasma bovis strains isolated in Denmark over a 17-year period was investigated. Forty-two field strains isolated from different geographic locations and specimens, including strains from 21 herds involved in two outbreaks of M. bovis -induced mastitis, and the type strain of M. bovis (PG45T) were assayed for variations in the BglII and MfeI restriction sites in the chromosomal DNA by using the amplified fragment length polymorphism (AFLP) fingerprinting technique. The obtained genomic fingerprints consisted of 62,68 AFLP fragments in the size range of 50,500 bp. Among the analyzed strains, 18 different AFLP profiles were detected. The similarity between individual fingerprints, calculated by Dice similarity coefficient, ranged from 0.9 to 1.0. Twenty-five strains, including 23 which were isolated during two outbreaks of M. bovis -induced mastitis which occurred 2 years apart, showed indistinguishable AFLP patterns. More genetic diversity was observed among the recent strains. The similarity of the genotypes of the field strains to that of the M. bovis type strain (PG45T) was 97.7%. The results of this study have demonstrated a remarkable genomic homogeneity of Danish strains of M. bovis that were probably epidemiologically related and which have remained stable for a considerable length of time. Furthermore, this study has demonstrated that AFLP can be used for genomic fingerprinting and discrimination of M. bovis strains. [source] cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the Indianmeal moth, Plodia interpunctellaINSECT MOLECULAR BIOLOGY, Issue 1 2000Y. C. Zhu Abstract Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two trypsin-like proteinases, which is found in both the Bt-susceptible and -resistant strains of the Indianmeal moth. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF064525 for the RC688 strain and AF064526 for HD198). [source] The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLPJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2004C. Whitby Abstract Aims:, To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis. Methods and Results:, Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site. Conclusions:, Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1,16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences. Significance and Impact of the Study:, This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment. [source] Mitochondrial DNA sequence variation within and between tuna Thunnus species and its application to species identificationJOURNAL OF FISH BIOLOGY, Issue 6 2001H. Takeyama Restriction analysis detected two types of bigeye tuna (, and ,); the , type was in the majority in the Atlantic but nearly absent in the Indo-Pacific. The , type shared a larger number of restriction sites with other species than the conspecific , type, but bigeye-specific nucleotide substitutions with a novel diagnostic restriction profile were found. Although the nucleotide sequence difference between Atlantic and Pacific sub-species of the northern bluefin tuna was nearly the largest among species, individuals possessing the Atlantic type of mtDNA were found at very low frequency in the Pacific and vice versa. Previous RFLP markers were found to be diagnostic for the other five species (albacore, blackfin, longtail, southern bluefin and yellowfin tunas). Genetic information is provided to discriminate all Thunnus species regardless of their origin and to identify the ocean of capture in the northern bluefin and bigeye tunas. [source] GENETIC DIVERGENCE CORRELATES WITH MORPHOLOGICAL AND ECOLOGICAL SUBDIVISION IN THE DEEP-WATER ELK KELP, PELAGOPHYCUS PORRA (PHAEOPHYCEAE)JOURNAL OF PHYCOLOGY, Issue 5 2000Kathy Ann Miller Pelagophycus porra (Leman) Setchell has a narrow distribution confined to deep water from the Channel Islands off the southern California coast to central Baja California, Mexico. Distinct morphotypes are consistently correlated with distinctive habitats, that is, windward exposures characterized by strong water motion and rocky substrates, and sheltered areas with soft substrates found on the lee sides of the islands. We tested the hypothesis that morphologically and ecologically distinct forms reflect genetically distinct stands. Individuals representing populations from three islands and the mainland were compared using RFLP analyses of the nuclear rDNA internal transcribed spacers (ITS1 and ITS2), chloroplast trnL (UAA) intron sequences, and random amplified polymorphic DNA (RAPDs). No variation was found in a survey of 20 restriction sites of ITS1 (ca. 320 base pair [bp]) and ITS2 (ca. 360 bp) among individuals from six populations. Likewise, comparisons of trnL intron (241 bp) sequences among nine individuals from seven populations were identical with the exception of a CATAGT insert in two adjacent stands. A RAPD analysis of 24 individuals from nine populations (4 windward and 5 leeward) using 16 primers generated 166 bands. Thirty-eight percent of the bands did not vary, 16% were unique to a given individual, and 46% were variable. Neighbor joining analysis produced a well-resolved tree with moderately high bootstrap support in which windward and leeward populations were easily distinguished. The lack of divergence in both the fast evolving nuclear rDNA-ITS and the chloroplast trnL intron does not support the morphotypes as different species. However, the compartmentalized differentiation shown in the RAPD data clearly points to isolation. This, and previous ecological studies that demonstrate habitat specificity suggest that leeward stands probably comprise a species in statu nascendi. [source] Identification and differentiation of Heterotardigrada and Eutardigrada species by riboprintingJOURNAL OF ZOOLOGICAL SYSTEMATICS AND EVOLUTIONARY RESEARCH, Issue 3 2007R. O. Schill Abstract In the last decades, the number of known tardigrade species has considerably increased to more than 960 species with new ones being discovered every year. However, the study of tardigrade species presents a general problem which is frequently encountered during the work with invertebrates: small size and remarkable degrees of phenotypic plasticity may sometimes not permit a definite identification of the species. In this investigation we have used riboprinting, a tool to study rDNA sequence variation, in order to distinguish tardigrade species from each other. The method combines a restriction site variation approach of ribotyping with amplified DNAs. In eight investigated species of heterotardigrades and eutardigrades we have amplified the genes for the small subunit ribosomal RNA (SSU; 18S) and subsequently sequenced the genes. Virtual riboprints were used for identification of restriction sites from ten already published 18S rDNA sequences and seven new 18S rDNA sequences. On the basis of the obtained sequences, diagnostic restriction fragment patterns can be predicted with only 11 restriction enzymes. The virtual digestion confirmed the obtained restriction fragment patterns and restriction sites of all amplified and digested tardigrade DNAs. We show that the variation in positions and number of restriction sites obtained by standard restriction fragment analysis on agarose gels can be used successfully for taxonomic identification at different taxonomic levels. The simple restriction fragment analysis provides a fast and convenient method of molecular barcoding for species identification in tardigrades. Zusammenfassung Im Laufe der letzten Jahrzehnte wurden viele neue Tardigradenarten beschrieben. Zur Zeit sind mehr als 960 Arten bekannt und jedes Jahr kommen neue Arten hinzu. Die Arbeit mit Tardigraden stellt jedoch oftmals ein Problem dar, das generell auch bei anderen Organismen von großer Bedeutung ist: die geringe Größe und die außergewöhnliche phenotypische Plastizität machen in vielen Fällen eine genaue Artidentifikation schwierig. In der vorliegenden Untersuchung verwenden wir das Riboprinting, eine Technik, rDNA Sequenzunterschiede zu erfassen, um damit verschiedene Tardigradenarten voneinander zu differenzieren. Diese Methode vereint den Ansatz der Restriktionsschnittstellenanalyse des Riboprinting mit amplifizierten DNAs. Von acht untersuchten Heterotardigraden und Eutardigraden wurden die Gene für die kleine ribosomale RNA Untereinheit (SSU; 18S) amplifiziert und sequenziert. Virtuelle Riboprints wurden zur Identifikation von zehn bereits publizierten 18S rDNA Sequenzen und sieben neuen 18S DNA Sequenzen erstellt. Auf der Basis der vorliegenden Sequenzen können die diagnostischen Restriktionsfragmentmuster mit insgesamt elf Restriktionsenzyme vorhergesagt werden. Der virtuelle Verdau bestätigt die Restriktionsfragmentmuster und Restriktionsschnittstellen aller amplifizierten und verdauten Tardigraden DNAs. Wir zeigen, dass die unterschiedlichen Variationen in den Positionen und Anzahl der Restriktionsschnittstellen erfolgreich zur taxonomischen Identifikation auf verschiedenen taxonomischen Ebenen verwendet werden können. Die einfache Restriktionsfragmentanalyse stellt eine schnelle und geeignete Methode für das molekulare Barcoding zur Artidentifikation bei Tardigraden dar. [source] Rapid amplification and cloning of Tn5 flanking fragments by inverse PCRLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2000G. Huang A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies. [source] Creep Resistant Polymer Nanocomposites Reinforced with Multiwalled Carbon NanotubesMACROMOLECULAR RAPID COMMUNICATIONS, Issue 8 2007Jinglei Yang Abstract Poly(propylene) (PP) nanocomposites filled with shorter- and longer-aspect-ratio multiwalled carbon nanotubes (MWNTs) were compounded using a twin-screw extruder and an injection moulding machine. It is shown that with only 1 vol.-% of MWNTs, creep resistance of PP can be significantly improved with reduced creep deformation and creep rate at a long-term loading period. Additionally, the creep lifetime of the nanocomposites has been considerably extended by 1,000% compared to that of a neat PP. Three possible mechanisms of load transfer were considered that could contribute to the observed enhancement of creep resistance, which are: (1) fairly good interfacial strength between MWNTs and polymer matrix, (2) increasing immobility of amorphous regions due to nanotubes acting as restriction sites, and (3) high aspect ratio of MWNTs. DSC results showing crystallinity changes in the specimens before and after creep deformation present evidence to confirm these mechanisms. Our results should lead to improved grades of creep resistant polymer nanocomposites for engineering applications. [source] Molecular characterization of sickle cell anemia in the Northern Brazilian state of ParáAMERICAN JOURNAL OF HUMAN BIOLOGY, Issue 5 2010Greice De Lemos Cardoso To assess ,+-thalassemia deletion alleles, ,-thalassemia mutations and haplotypes linked to the HBB*S cluster in a sample of 130 unrelated sickle cell anemia (SCA) patients (55% female) from Belém, Pará State, for their possible effects on the patients' survival. -,3.7, -,42, -,20.5, and ,MED ,+-thalassemia deletion alleles were investigated using multiplex gap-PCR method. Characterization of ,-thalassemia mutations was made by direct genomic sequencing of the ,-globin gene amplified through polymerase chain reaction (PCR). Haplotypes were determined by analysis of six polymorphic restriction sites [(1) XmnI-5,,G, (2) HindIII-,G, (3) HindIII-,A, (4) HincII-,,, (5) HincII-3,,,, and (6) HinfI-5,,] followed by restriction digestion and agarose gel electrophoresis. Twenty-one patients (16%) presented -,3.7 thalassemia. Sixteen of those (76%) were heterozygous (-,3.7/,,) and 5 (24%) were homozygous (-,3.7/-,3.7). -,4.2, -,20.5 and ,MED deletions were not found. Nine cases of sickle cell-, thalassemia were found and four different ,-thal mutations were identified: ,+ ,88 (C>T), 3.8%; ,+ codon 24 (T > A), 1.5%; ,+ IVSI-110 (G > A), 0.7% and , (IVSI-1 (G > A), 0.7%. No differences according to age were observed in -,3.7 deletion, ,-thalassemia and HHB*S haplotypes distribution. Our results suggest that although ,- and ,-thalassemia and ,S haplotypes may have modulating effect on clinical expression and hematological parameters of SCA, these genetic variables probably have little influence on the subjects' survival. Am. J. Hum. Biol. 22:573,577, 2010. © 2010 Wiley-Liss, Inc. [source] VIRS: A visual tool for identifying restriction sites in multiple DNA sequencesBIOTECHNOLOGY PROGRESS, Issue 5 2009Xiang Chen Abstract VIRS (A visual tool for identifying restriction sites in multiple DNA sequences) is an interactive web-based program designed for restriction endonuclease cut sites prediction and visualization. It can afford to analyze multiple DNA sequences simultaneously and produce visual restriction maps with several useful options intended for users' customization. These options also perform in-depth analysis of the restriction maps, such as providing virtual electrophoretic result for digested fragments. Different from other analytical tools, VIRS not only displays visual outputs but also provides the detailed properties of restriction endonucleases that are commercially available. All the information of these enzymes is stored in our internal database, which is updated monthly from the manufacturers' web pages. It is freely available online at http://bis.zju.edu.cn/virs/index.html. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] A Polylinker Approach to Reductive Loop Swaps in Modular Polyketide SynthasesCHEMBIOCHEM, Issue 16 2008Laurenz Kellenberger Dr. Abstract Multiple versions of the DEBS 1-TE gene, which encodes a truncated bimodular polyketide synthase (PKS) derived from the erythromycin-producing PKS, were created by replacing the DNA encoding the ketoreductase (KR) domain in the second extension module by either of two synthetic oligonucleotide linkers. This made available a total of nine unique restriction sites for engineering. The DNA for donor "reductive loops," which are sets of contiguous domains comprising either KR or KR and dehydratase (DH), or KR, DH and enoylreductase (ER) domains, was cloned from selected modules of five natural PKS multienzymes and spliced into module 2 of DEBS 1-TE using alternative polylinker sites. The resulting hybrid PKSs were tested for triketide production in vivo. Most of the hybrid multienzymes were active, vindicating the treatment of the reductive loop as a single structural unit, but yields were dependent on the restriction sites used. Further, different donor reductive loops worked optimally with different splice sites. For those reductive loops comprising DH, ER and KR domains, premature TE-catalysed release of partially reduced intermediates was sometimes seen, which provided further insight into the overall stereochemistry of reduction in those modules. Analysis of loops containing KR only, which should generate stereocentres at both C-2 and C-3, revealed that the 3-hydroxy configuration (but not the 2-methyl configuration) could be altered by appropriate choice of a donor loop. The successful swapping of reductive loops provides an interesting parallel to a recently suggested pathway for the natural evolution of modular PKSs by recombination. [source] Molecular determinants of hyperosmotically activated NKCC1-mediated K+/K+ exchangeTHE JOURNAL OF PHYSIOLOGY, Issue 18 2010Kenneth B. Gagnon Na+,K+,2Cl, cotransport (NKCC) mediates the movement of two Cl, ions for one Na+ and one K+ ion. Under isosmotic conditions or with activation of the kinases SPAK/WNK4, the NKCC1-mediated Cl, uptake in Xenopus laevis oocytes, as measured using 36Cl, is twice the value of K+ uptake, as determined using 86Rb. Under hyperosmotic conditions, there is a significant activation of the bumetanide-sensitive K+ uptake with only a minimal increase in bumetanide-sensitive Cl, uptake. This suggests that when stimulated by hypertonicity, the cotransporter mediates K+/K+ and Cl,/Cl, exchange. Although significant stimulation of K+/K+ exchange was observed with NKCC1, a significantly smaller hyperosmotic stimulatory effect was observed with NKCC2. In order to identify the molecular determinant(s) of this NKCC1-specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2 nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the first intracellular loop between membrane-spanning domains one and two and the second extracellular loop between membrane-spanning domains three and four of NKCC1 are necessary components of the hyperosmotic stimulation of K+/K+ exchange. [source] | |||||||||||||||||||