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Gel Electrophoresis of Nucleic Acids : A Practical Approach /
Abstract
2nd Ed Bibliogr. na konci kapitol
... These devices aim to supersede the present methods, which are time consuming and difficult to automate. The separation of DNA molecules, for example, is currently accomplished by gel electrophoresis [2] ; molecules of different size migrate at different speeds through the gel, and can be distinguished from one another after a certain time has elapsed. Using a gel to sieve the DNA (a necessity, since the electrophoretic mobility m 0 in free solution is independent of size) complicates the procedure: A new matrix must be made each time, and recovery of the DNA is awkward, since the gel must either be sliced or blotted [2]. ...
... The separation of DNA molecules, for example, is currently accomplished by gel electrophoresis [2] ; molecules of different size migrate at different speeds through the gel, and can be distinguished from one another after a certain time has elapsed. Using a gel to sieve the DNA (a necessity, since the electrophoretic mobility m 0 in free solution is independent of size) complicates the procedure: A new matrix must be made each time, and recovery of the DNA is awkward, since the gel must either be sliced or blotted [2]. In this Letter, we propose a fresh approach to separating biological macromolecules, based on the technology of microfabricated arrays introduced by Volkmuth and Austin [3] . ...
... An unusual and useful feature is that, since the variance declines as the deflection probability decreases, the resolution is actually higher for larger molecules (smaller D). Let us now evaluate how well this device could perform the task of separating DNA molecules with lengths varying from 100 to 20 000 base pairs—the range for which agarose gel electrophoresis is routinely used [2]. In the buffer solution, these molecules adopt random coil conformations , with size R MM 0 12 b, where M is the number of base pairs, b 100 nm is the Kuhn length [10], and M 0 300 is the number of base pairs per Kuhn length. ...
In a two-dimensional periodic but asymmetric environment, a Brownian particle that is driven in one direction by a potential gradient will also drift in the orthogonal direction at a rate that depends on its diffusion coefficient. On this basis, we propose a new method for separating biological macromolecules according to size. A fine stream of molecules is electrophoresed through a microfabricated sieve, etched from a silicon chip by lithography. The sieve consists of a periodic array of oblong obstacles, which deflect the molecules so that each species follows a different trajectory, oblique to the flow. Advantages promised by the technique include improved efficiency, continuous sorting and ready automation.
... The extraction of the genomic DNA was carried out from foliar tissue using the method of Doyle and Doyle (Stewart and Via, 1993). The amplified products were separated in agarose gels 1.5% and buffer TBE 0.5X (Rickwood and Hames, 1990), applying 100 V during 2 h with a voltage source of Consort E714. The gels were incubated for 40 min in 100 mL of buffer solution TBE 1X, pH 8.3, adding 5 µL of ethidium bromide (10 mg mL −1 ) (Rickwood and Hames, 1990). ...
... The amplified products were separated in agarose gels 1.5% and buffer TBE 0.5X (Rickwood and Hames, 1990), applying 100 V during 2 h with a voltage source of Consort E714. The gels were incubated for 40 min in 100 mL of buffer solution TBE 1X, pH 8.3, adding 5 µL of ethidium bromide (10 mg mL −1 ) (Rickwood and Hames, 1990). The gel documentation system employed was Kodak Digital Science EDAS 120. ...
In order to rind a phenotypic relationship between DNA markers and growth rate of some trees, a study of morphometric and molecular variation was performed in a plantation of Pinus patula Schiede, in Veracruz, Mexico. Growth of nineteen trees 2.5 years of age was measured during two consecutive growing seasons and DNA variation was evaluated in leaf tissue, using the RAPD technique (Random Amplified Polymorphism of DNA). The morphometric study allowed separation of individuals in three groups with different growth rates. The analysis of molecular variation revealed the presence of 38 RAPD's bands by using five primers. Primers OPK-12 and OPK-16 produced a greater percentage of polymorphic bands. A phenotypic association was observed between the OPK-12-9 band and the morphometric group B (trees of average growth), as well as between OPK-16-8 and OPK-16-10 bands and the morphometric group A (fastest growing trees). Further genetic studies should evaluate the potential use of these markers in the selection of P. patula trees with higher increases for the analyzed variables.
... Đối tượng: 10 [13] cho thấy thay đổi thành phần trong cộng đồng Actinobacteria trong quá trình ủ thải hữu cơ theo quy mô của hộ gia đình. Trong thí nghiệm này, chúng tôi chọn phân chuồng ủ từ các hộ gia đình theo phuơng thức truyền thống, nên quá trình tách chiết và tinh sạch cũng như nhân bản đoạn gene 16S rDNA cho kết quả thường không ổn định xem hình 1. ...
... Kết quả ở hình 1 ghi nhận được việc tác chiết và thu nhận bộ gene DNA của vi sinh vật trực tiếp từ các mẫu khác nhau trong điều kiện không đồng nhất và lập lại theo phương pháp SDS so sánh cùng các kết quả nghiên cứu trong [17,15,10] và với kết quả này cho phép chúng tôi tiếp tục tinh sạch genomic DNA. Sau khi tinh sạch DNA bằng phương pháp Troughing và kiểm tra trên gel agarose 0,8% ở hình 2 của 10 mẫu phân ủ từ những điều kiện không đồng nhất và so sánh với các kết quả của [9,6,12] kết quả tinh sạch này chúng tôi chọn 10 mẫu (1, 2, 3, 4, 5, 6, 7, 8, 9 và 10) tiếp tục nhân bản đoạn gene 16S rDNA của nhóm vi khuẩn dựa trên 2 cặp mồi 27F và 907R từ bộ gene DNA hỗn hợp trên. ...
In this study, we used DGGE technique to analyze the existence of microbial community of compost. From ten samples extracted, isolated genomic and amplify 16SrDNA region by 27F and 907R primer. And then choice No.6 for analysis by DGGE method with primers (517F-GC, 357R and 517F, 357R) had result nine sequences of V3 region follow as: 6a1, 6a2, 6a3, 6a4, 6d1, 6d2, 6d3, 6e1 and 6e2. The analysis and compared with is database onto GeneBank for sequence similarities follow with Accession number: 6a1, 6a2 similarities 99% with JF987387.1 and JF828759.1, JF 989848.1 similarities 93% with 6a4 and 88% 6a3, 6d1 similarities 82% with AB206012.1, 6d2 similarities 95% JF429206.1, 6d3 similarities 86% with FN781411.1, 6e1 similarities with 99%JF176784.1 and FJ516783.1 similarities 87% with 6e2.
... The extraction of the genomic DNA was carried out from foliar tissue using the method of Doyle and Doyle (Stewart and Via, 1993). The amplified products were separated in agarose gels 1.5% and buffer TBE 0.5X (Rickwood and Hames, 1990), applying 100 V during 2 h with a voltage source of Consort E714. The gels were incubated for 40 min in 100 mL of buffer solution TBE 1X, pH 8.3, adding 5 µL of ethidium bromide (10 mg mL −1 ) (Rickwood and Hames, 1990). ...
... The amplified products were separated in agarose gels 1.5% and buffer TBE 0.5X (Rickwood and Hames, 1990), applying 100 V during 2 h with a voltage source of Consort E714. The gels were incubated for 40 min in 100 mL of buffer solution TBE 1X, pH 8.3, adding 5 µL of ethidium bromide (10 mg mL −1 ) (Rickwood and Hames, 1990). The gel documentation system employed was Kodak Digital Science EDAS 120. ...
... Visualization of both DNAs was performed through denaturalized 0.8% agarose gel electrophoresis. 38 The gel was 0.1% ethidium bromide stained, and the bands were observed in an UV light transilluminator (Gel Doc XR System, Bio-Rad, Hercules, CA, USA). ...
Understanding the origin of life on our planet has generated diverse theories. Currently, the theory is that life has a single origin; however, its starting point has not been defined. As evidenced, it is indispensable to unify the different theories to reach a single theory that would also allow linking the different areas of knowledge to finally understand the mechanism by which life originated on Earth. In this regard, aiming at contributing to the unification of the diverse theories on the origin of life, in this work, the hypothesis based on the condition that silica-carbonates of alkaline earth metals, called biomorphs, are the ones that could unify all the proposed theories on the origin of life is proposed. Aimed at evaluating if this hypothesis is viable, this work assessed whether biomorphs are able to protect the DNA from continuous UV radiation under two conditions that emulate the habitats that could have co-existed in the Precambrian and, after the radiation, evaluated the time during which DNA remained inside the biomorphs. Our results showed that biomorphs can protect the DNA for months after continuous UV exposure. It was also determined that biomorphs protect the DNA from external factors in different habitats, like normal atmospheric conditions and in aqueous environments. The obtained data allowed me to infer that biomorphs may be the gap that unifies the diverse proposed theories on the origin of life in our Planet.
... The obtained DNA was visualized through agarose gel electrophoresis denaturalized at 0.8% using the procedure described by Rickwood and Hames. 41 The gel was stained with 0.1% ethidium bromide, and the bands were observed in a UV transilluminator (Gel Doc XR System, Bio-Rad, Hercules, CA). ...
The origin of life on Earth is associated with the Precambrian era, in which the existence of a large diversity of microbial fossils has been demonstrated. Notwithstanding, despite existing evidence of the emergence of life many unsolved questions remain. The first question could be as follows: Which was the inorganic structure that allowed isolation and conservation of the first biomolecules in the existing reduced conditions of the primigenial era? Minerals have been postulated as the ones in charge of protecting theses biomolecules against the external environment. There are calcium, barium, or strontium silica-carbonates, called biomorphs, which we propose as being one of the first inorganic structures in which biomolecules were protected from the external medium. Biomorphs are structures with different biological morphologies that are not formed by cells, but by nanocrystals; some of their morphologies resemble the microfossils found in Precambrian cherts. Even though biomorphs are unknown structures in the geological registry, their similarity with some biological forms, including some Apex fossils, could suggest them as the first "inorganic scaffold" where the first biomolecules became concentrated, conserved, aligned, and duplicated to give rise to the pioneering cell. However, it has not been documented whether biomorphs could have been the primary structures that conserved biomolecules in the Precambrian era. To attain a better understanding on whether biomorphs could have been the inorganic scaffold that existed in the primigenial Earth, the aim of this contribution is to synthesize calcium, barium, and strontium biomorphs in the presence of genomic DNA from organisms of the five kingdoms in conditions emulating the atmosphere of the Precambrian era and that CO2 concentration in conditions emulating current atmospheric conditions. Our results showed, for the first time, the formation of the kerogen signal, which is a marker of biogenicity in fossils, in the biomorphs grown in the presence of DNA. We also found the DNA to be internalized into the structure of biomorphs.
... La cuantificación del DNA se realizó asumiendo que una unidad de absorbancia a 260 nm es igual a 50 µg/µL de DNA de doble cadena (Rickwood y Hames, 1990). La calidad del DNA fue inferida mediante el cálculo de la relación A260/A280. ...
... Zurich. This gel electrophoresis model has developed as the standard technique widely applied to separate, identify and purify DNA fragments (Sambrook & Russel, 2001;Rickwood & Hames, 1982). The DNA separation on the gel is based on the negative charge of DNA molecules due to the negatively-charged oxygen of the phosphate group of their backbone. ...
Marine sponges are a rich source of bioactive natural products with potent anticancer activities. Currently, the limited availability of most of these substances prohibits further drug development. Highly complex consortia of bacterial symbionts associated with sponges have been frequently proposed to be the true producers of many secondary metabolites . However, the majority of these complex microbial assemblages are not amenable to cultivation, thereby hampering efforts to prove the symbiont hypothesis as well as to access their biosynthetic potential. Using metagenomic-based approaches, Piel and colleagues have previously provided the first genetic evidence for the bacterial origin of invertebrate-derived natural products by cloning the entire gene cluster for onnamide/theopederin from the Japanese sponge Theonella swinhoei. In this work, we investigated further natural product biosynthetic pathways from uncultivated symbiotic bacteria using Japanese T. swinhoei as a symbiotic assemblage model. The reasons for selecting this sponge are the wide variety of pharmaceutically important secondary metabolites isolated from this sponge as well as the high complexity of the associated bacteria, which might play an important role in metabolite biosynthesis. Metagenome mining strategies that we applied and developed in this work have led to the cloning of two new biosynthetic pathways from this complex symbiosis model. Our bioinformatic analysis predicted that one pathway is responsible for the biosynthesis of misakinolide A, and another one for keramamide H. Interestingly, we found that the first pathway contains additional components that match structures of swinholide A and hurghadolide A, potent actin polymerization inhibitors isolated from other sponges. Both biosynthetic pathways were encoded on two different gene clusters that exhibited typical bacterial gene features, strongly indicating that the producer of misakinolide A and keramamide H is a symbiotic bacterium. Since the screening system used to clone the gene clusters was based on the filamentous fraction dominated by “Candidatus Entotheonella sp.”, a heterothropic delta-proteobacterium associated with T. swinhoei, we assumed that misakinolide A and keramamide H are produced by “Entotheonella sp.” To confirm the taxonomic status of the bacterial producer, further analysis either by single cell studies or its combination with complete genome is currently underway. Subsequent genome sequencing of another member of this as-yet uncultivable candidate genus from a different chemotype of T. swinhoei led to the identification of genes for the biosynthesis of orbiculamide-like structure, which is structurally related to keramamides. Therefore, the results in this work provide not only convincing proof for the microbial origin of marine natural products but also specific taxonomic information as well as the potential to sustainable supply of pharmacologically potential compounds. The Full-text is available in the following link: http://hss.ulb.uni-bonn.de/2013/3082/3082.htm
... Larger fragments migrate more slowly, at a rate approximately proportional to its charge to mass ratio, toward the positive electrode. Complete complexation and neutralization of RNA is indicated by complete retention of the RNA in the wells of the gel [25]. The gel retardation assay has been used here to find out if there are cosolutes binding a fragment of RNA (in a RNA-cosolute complex) by watching how fast the RNA fragment moves through an electric field and seeing whether it moves more slowly when a particular cosolute is also present. ...
The design of synthetic carriers for nucleic acid delivery has become a research field of increasing interest. Studies on the delivery of DNA have brought up a variety of gene delivery vehicles. Recent studies in our group have demonstrated the preparation of new gelatin-based nanoparticles for the sustainable intracellular DNA delivery. Furthermore, the more recently emerged strategy by the intracellular delivery of RNA takes benefit from existing expertise in DNA transfer. In this work, the preparation and physicochemical characterization of new nucleic acid-based particles for the sustainable RNA delivery have been demonstrated. Gelatin (either high or low gel strength) and protamine sulfate have been selected to form particles by interaction of oppositely charged compounds. Particles in the absence of RNA (binary system) and in the presence of RNA (ternary system) have been prepared. The physicochemical characterization (particle size, polydispersity index, degree of RNA entrapment and RNA binding efficiency) has been evaluated as a function of the nature of the RNA derivative (acid form, diethylaminoethanol salt or core form) from torula yeast. The pH-dependent response of nanoparticles co-incubated in buffers at defined pHs that mimic late endo-lysosomal environment has demonstrated that the nanoparticles tend to destabilize and RNA can be successfully released as a consequence of changes in the intracellular pHs. Among the different systems, gelatin B (RNA)-PS nanoparticles using RNA acid form from torula yeast has proved to be the best system, from an effective and economic point of view.
... The last years witnessed a blooming of nanotechnology and the use of nanoparticles (NPs) and nanomaterials (NMs) for scientific purpose and commercial applications is continu-method, allowing multiple runs in parallel on the same gel [6]. The most commonly used GE methods include PAGE [7], commonly used to separate protein molecules to a high degree of purity, and agarose GE (AGE) mainly used for separating charged biopolymers, such as DNA and RNA [8]. Although PAGE has been used for characterization of NPs, such as CdTe Quantum dots (QDs) bioconjugates [9] and CdTe QDs stabilized with mercaptosuccinic acid [10], the small pore size of polyacrylamide gels (usually of less than 10 nm) limits its application for other NPs. ...
Agarose gel electrophoresis (AGE) has been used extensively for characterization of pure nanomaterials or mixtures of pure nanomaterials. We have evaluated the use of AGE for characterization of Ag nanoparticles (NPs) in an industrial product (described as strong antiseptic). Influence of different stabilizing agents (PEG, SDS and Sodium dodecylbenzenesulfonate), buffers (TBE and Tris Glycine) and functionalizing agents (Mercaptosuccinic acid (TMA) and proteins) have been investigated for the characterization of AgNPs in the industrial product using different sizes-AgNPs standards. The use of 1% SDS, 0.1% TMA and Tris Glycine in gel, electrophoresis buffer and loading buffer led to the different sizes-AgNPs commercial standards moved according to their size/charge ratio (obtaining a linear relationship between apparent mobility and mean diameter). After using SDS and TMA, the behaviour of the AgNPs in the industrial product (containing a casein matrix) was completely different, being not possible their size characterization. However we demonstrated that AGE with LA-ICP-MS detection is an alternative method to confirm the protein corona formation between the industrial product and two proteins (Bovine Serum Albumin and transferrin) maintaining NPs-protein binding (what is not possible using SDS-PAGE). This article is protected by copyright. All rights reserved.
... The sample was divided into three aliquots and the subsequent unwinding reaction was carried out as described above. Samples were separated on 3% composite acrylamide-agarose gels (19) and gels were dried and subjected to autoradiography. ...
DbpA is a putative Escherichia coli ATP dependent RNA helicase belonging to the family of DEAD box proteins. It hydrolyzes ATP in the presence of 23S ribosomal
RNA and 93 bases in the peptidyl transferase center of 23S rRNA are sufficient to trigger 100% of the ATPase activity of DbpA.
In the present study we characterized the ATPase and RNA unwinding activities of DbpA in more detail. We report that—in contrast
to eIF-4A, the prototype of the DEAD box protein family—the ATPase and the helicase activities of DbpA are not coupled. Moreover,
the RNA unwinding activity of DbpA is not specific for 23S rRNA, since DbpA is also able to unwind 16S rRNA hybrids. Furthermore,
we determined that the ATPase activity of DbpA is triggered to a significant extent not only by the 93 bases of the 23S rRNA
previously reported but also by other regions of the 23S rRNA molecule. Since all these regions of 23S rRNA are either part
of the ‘functional core’ of the 50S ribosomal subunit or involved in the 50S assembly, DbpA may play an important role in
the ribosomal assembly process.
... Data were normalized to the highest expression value of lscB, which was set to 100%. as compared to LscB under denaturing conditions could potentially be attributed to the apparent mass shift for two proteins with nearly identical molecular masses as described earlier [26]. Interestingly, the migration of LscB UpN A was significantly slower than that of LscB under native conditions. ...
... Debido a la importancia de esta bacteria en las infecciones nosocomiales, se ha desarrollado un conjunto de métodos que, además de la biotipificación, permiten identificar características fenotípicas y estructurales de la bacteria. Cabe destacar las pruebas de susceptibilidad antimicrobiana y el análisis del ADN plasrnídico y cromosomal (2,3,9,13,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Probablemente los métodos más efectivos para estudiar el S. epidermidis han sido el análisis de plásmidos y la electroforesis de campo pulsado (32). ...
... Serum samples were used to determine malondialdahyde (Albero et al., 1986)and the nitrite level in the collected serum samples was calculated according to the method described by Green et al. (1982). Total serum protein was measured according to the method described by Richardson (1977) and Polyacrylamide gel electrophoresis was made according to Hamesh and Rickwood(1990). ...
In this study, samples from cultured Common carp (Cyprinus carpio), Nile tilapia (Oreochromis niloticus) and African catfish(Clarias gariepinus) fishes were collected from Kafr el-Sheikh, Menofya, Behira and Sharkia Governorates in Egypt for detection of Pseudomonas aeruginosa infection. Isolation and identification of Pseudomonas aeruginosa was done by traditional methods then confirmed using regular PCR technique. Pseudomonas aeruginosa gave 956 bp product size specific for 16S rDNA. The experimental inoculation of Clarias gariepinus with Pseudomonas aeruginosa was fully demonstrated. The most common clinical signs were external haemorrhage and ulcer with mortality rate 40%. Histopathological changes revealed degeneration and necrosis in all internal organs associated with hyperplesia in the wall of the blood vessels. Chronic inflammatory cell infiltration and melanomacrophage cells were detected in all fish tissues. The effect on some oxidative stress and immunological parameters of experimentally inoculated Clarias gariepinus with Pseudomonas aeruginosa were studied. Results revealed that there were significant increase in lipid peroxidation product (malondialdehyde) , hypoprotineamia, hypoalbuniaemia and hypoglobulinaemia. In-vitro sensitivity test of isolated Pseudomonas aeruginosa iitalosi to different chemotherapeutic agents was conducted. In conclusion, this study showed that P.aeruginosa infection is common in some common cultured freshwater fishes in Egypt. It can be diagnosed easily using PCR technique.The experimental infection of Clarias gariepinus with P.aeruginosa was associated with mortality rate 40 % , severe histopathological changes in all organs of infected fishes, immunosuppression and oxidative damage of tissues which reflect the significant economic importance of Pseudomonas aeruginosa infection in cultured fishes. Colistin sulphate, Danofloxacin, Nalidixic acid, Oxolonic acid and Oxytetracyclinen are recommended for control of Pseudomonas aeruginosa infection in cultured fishes.
... Genotype score of individual was inferred by their parents' at the same polyacrylamide gels to avoid redundant (to expel from disturbing of nonspecific band by referring to the parental genotypes), the different microsatellite DNA fragment at the same loci on different polyacrylamide gels was distinguished by standard DNA marker (pBR322/Msp I). Allele size of microsatellite PCR amplification production was determined by method of Rickwood and Hames (1982) and referred to the standard DNA marker, so there was some error, but it was consistent and identical in all population, and the results was credible in this study. ...
Correlation of microsatellite heterozygosity with performance or heterosis was reported in wild animal populations and domestic animal populations, but the correlation with heterosis in a crossbreeding F pig population remained uncertain. To explore this, we had random selected and mated YorkshireMeishan (F, n = 82) and their reciprocal (G, n = 47) to F, and used the two straightbreds as control groups (Yorkshire = 34, Meishan = 55), and observed the heterosis of birth weight (BWT), average daily gain (ADG) and feed and meat ratio (FMR). Two Kinds of measurement-individual heterozygosity (IH) and individual mean d (lg value, ID) were used as index of heterozygosity and variance from 39 microsatellite marker loci to perform univariate regression analysis against heterosis. We detected significant correlation of IH with BWT in all of F (F+G) and in F. We observed significant correlation of ID with ADG in all of F (F+G), and with FMR in all of F (F+G) and in F. There was significant maternal effect on heterosis, which was indicated by significant difference of means and distribution of heterosis between F and G. This difference was consistent with distributions of IH and ID, and with difference of means in F and G. From this study, it would be suggested that the two kinds of genetic index could be used to explore the genetic basis of heterosis in crossbreeding populations but could not determine which is better.
... Subsequently, the amplification product was analyzed by GelRed-stained agarose gel electrophoresis with a 1X TBE buffer solution (Rickwood and Hames, 1990). Lastly, the DNA bands were observed and photographed under UV light in a gel documentation system (Kodak Gel Logic 200 ). ...
The majority of heifer calves born as hetero–sexual twin births are sterile freemartins. It is important that this condition be diagnosed at an early age because freemartin heifers can't be used as replacement stock. Different methods are available for the diagnosis of freemartinism, however molecular methods are preferred due to their accuracy and shorter duration of process. In the present study, freemartinism status of 40 female calves of heterosexual multiple births was investigated by multiplex polymerase chain reaction (multiplex PCR). The reaction was carried out using two primer sets for the Sry and K–casein genes of domestic cattle. The PCR product was analyzed by agarose gel (3%) electrophoresis, which allowed to identify the genotypes of the animals studied: normal males (453 bp and 163 bp), normal females (453 bp), and 39 (97.50%) study cases that showed chromosome chimerism (freemartin, 453 bp and 163 bp). Compared to the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique, this method (i.e., multiplex PCR) proved a far cheaper and quicker (approximately 27 minutes) way to diagnose freemartinism. All copyrights reserved to Nexus® academic publishers
... Data were normalized to the highest expression value of lscB, which was set to 100%. as compared to LscB under denaturing conditions could potentially be attributed to the apparent mass shift for two proteins with nearly identical molecular masses as described earlier [26]. Interestingly, the migration of LscB UpN A was significantly slower than that of LscB under native conditions. ...
Pseudomonas syringae pv. glycinea PG4180 is an opportunistic plant pathogen which causes bacterial blight of soybean plants. It produces the exopolysaccharide levan by the enzyme levansucrase. Levansucrase has three gene copies in PG4180, two of which, lscB and lscC, are expressed while the third, lscA, is cryptic. Previously, nucleotide sequence alignments of lscB/C variants in various P. syringae showed that a ~450-bp phage-associated promoter element (PAPE) including the first 48 nucleotides of the ORF is absent in lscA.
Herein, we tested whether this upstream region is responsible for the expression of lscB/C and lscA. Initially, the transcriptional start site for lscB/C was determined. A fusion of the PAPE with the ORF of lscA (lscBUpNA) was generated and introduced to a levan-negative mutant of PG4180. Additionally, fusions comprising of the non-coding part of the upstream region of lscB with lscA (lscBUpA) or the upstream region of lscA with lscB (lscAUpB) were generated. Transformants harboring the lscBUpNA or the lscBUpA fusion, respectively, showed levan formation while the transformant carrying lscAUpB did not. qRT-PCR and Western blot analyses showed that lscBUpNA had an expression similar to lscB while lscBUpA had a lower expression. Accuracy of protein fusions was confirmed by MALDI-TOF peptide fingerprinting.
Our data suggested that the upstream sequence of lscB is essential for expression of levansucrase while the N-terminus of LscB mediates an enhanced expression. In contrast, the upstream region of lscA does not lead to expression of lscB. We propose that lscA might be an ancestral levansucrase variant upstream of which the PAPE got inserted by potentially phage-mediated transposition events leading to expression of levansucrase in P. syringae.
... Slightly sigmoid calibration curves, relating the migrated distances of electrophoretic bands to hyperbolic transformations of the fragment sizes, were deduced from a fine set of molecular weight markers ("Raoul I", Appligene) electrophoresed alongside the samples. The applied hyperbolic functions of the type L = a : (M+ b) + c (where L is the length of a fragment and M its migrated distance), proposed by Rickwood and Hames (1982), were defined by three selected bands of the marker, two of them enclosing just the range of the bands in the pattern to be analyzed and the third one close to the middle of the pattern. ...
Using a novel nonaqueous procedure, chloroplast DNA was isolated from 318 individual adult rice plants, representing 247 accessions and the breadth of the diversity in section Oryza of genus Oryza. Among them, 32 different cpDNA restriction patterns were distinguished using the restriction endonucleases EcoRI and AvaI, and they were further characterized by restriction with BamHI, HindIII, SmaI, PstI, and BstEII enzymes. The differences in the electrophoretic band patterns were parsimoniously interpreted as being the result of 110 mutations, including 47 restriction site mutations. The relationships between band patterns were studied by a cladistic analysis based on shared mutations and by the computation of genetic distances based on shared bands. The deduced relationships were compared with earlier taxonomical studies. The maternal parents for BC genome allotetraploids were deduced. Within species, cpDNA diversity was found larger in those species with an evolutionary history of recent introgression and/or allotetraploidization. Occasional paternal inheritance and recombination of cpDNA in rice was suggested.
... A comprehensive overview of all recent developments in technological, theoretical, and practical aspects has been given in many reviews and books. In particular, many unique nanostructures, such as nanopillars Yasui et al. 2007), nanowalls (Yasui et al. 2011b), nanofilter arrays (Han and Craighead 2000;Fu et al. 2006;Fu et al. 2007;Mao and Han 2009), nanochannels (Li et al. 2003;Cross et al. 2007;Pennathur et al. 2007), nanoparticles (Doyle et al. 2002;Tabuchi et al. 2004;Zeng and Harrison 2007;Zeng et al. 2008;Nazemifard et al. 2010), and nanofence array (Park et al. 2012) have been developed for the separation of biomolecules, and these nanostructure-based analytical tools have a potential to play an important role toward switching from conventional randomly ordered polymeric matrices, which involve time-and labor-intensive and manual operations (Rickwood and Hames 1990;Viovy 2000;Watson et al. 2004;Landers 2008), to highly ordered sieving structures. ...
Recent developments of nanofabrication techniques have created a trend switching from randomly ordered polymeric matrices, such as gel, to highly ordered sieving nanostructures in the separation of biomolecules. These nanostructures have enormous potential for fast separation of biomolecules, while nanostructure-based separation techniques suffer from critical scaling problems; they are efficient in handling less than nanoliter amounts of sample fluids, but most biomolecule samples are available in a liquid volume that is over several microliter, leading to a reduction in sensitivity and resolution. In this study, we developed a nanopillar array chip integrated with an easy and rapid on-line stacking method and achieved fast DNA separation with high sensitivity and high resolution. The developed on-line stacking method is based on the balance of two forces driven by electric fields: electroosmotic flow (EOF) and electrophoresis. The EOF mobility from the microchannel to the nanopillar-channel is drastically decreased, while, on the other hand, electrophoresis has constant mobilities in the whole length of the channels. The on-line stacking was realized at the well-balanced position of the two forces, and the on-line stacking using the nanopillar array chip can also be achieved within 10 s by just applying electric voltages without any other special reagents and materials. After applying on-line stacking using the nanopillar array chip, the relative fluorescence intensity increased 1,000-fold, and the resolution was twice as good as that without on-line stacking.
This chapter presents a procedure described earlier by Jinny Paul—a simple, practical, and cheap technique for long-term nonphotographic storage of Coomassie blue-stained protein separation pattern found in an electrophoresis gel.
Tool and techniques of chromatography is written according to the curriculum of various
universities for both bachelor’s and master’s courses in pharmacy and allied science conducted
globally. It contains detailed principle, methods and application on Indian system of medicinal,
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patent attorneys, government scientists and regulatory personnel, and other seeking information
concerning the design, manufacture, and control of pharmaceutical dosage forms and new drug
discovery. It can form the basis for the modern revision of syllabi of courses of B. Pharm, M.
Pharm, B. Sc. and M. Sc. with specialization in chemistry and pharmaceutics.
Dieses Kapitel beschreibt die Grundlagen der elektrophoretischen Trenntechniken, und die verschiedenen Methoden der Zonenelektrophorese in freier Lösung und in stabilisierenden Medien für die Trennung von Proteinen und Nukleinsäuren. Bei den Techniken in freier Lösung wird die trägerfreie Elektrophorese sowohl für präparative als auch analytische Trennungen verwendet, die Kapillarelektrophorese und Mikrochip-Elektrophorese nur für analytische Zwecke. Bei der Elektrophorese in nicht restriktiven Gelen wird der Reibungswiderstand der Gelmatrix vernachlässigbar gering gehalten, sodass die elektrophoretische Mobilität nur von den Nettoladungen der Probenmoleküle abhängig ist. Obwohl bei der Elektrophorese im restriktiven Gel unter nativen Bedingungen die elektrophoretischen Mobilitäten sowohl von der Anzahl der Nettoladungen als auch vom Molekularradius abhängen, kann die Methode auch zur physikochemischen Charakterisierung von Proteinen benutzt werden. Mit der Diskelektrophorese erzielt man einen schonenden Probeneintritt in die Trennmatrix und sehr hohes Auflösungsvermögen. Zur weiteren Verbesserung der Trennleistung kann man Gele mit linearen oder exponentiellen Porengradienten herstellen. Durch Zugabe von ionischen Detergenzien zum Trenn- und Laufpuffer erzeugt man denaturierende Bedingungen, um Auftrennungen nur nach Molekülgrößen zu erhalten. Mit Hilfe von zahlreichen methodischen Modifikationen können die Anwendungsbereiche der Zonenelektrophorese erheblich erweitert werden.
Lariat ethers and hydraphiles have been studied by the use of gel electrophoresis to evaluate the complexation of a 10 kilobase plasmid DNA. Both receptor systems show behaviour that reflects differences in side or spacer chain lengths. In addition, the formation of complexes or aggregates (identified by transmission electron microscopy, TEM) is pH dependent. Some detergents and polyamines can affect the formation and/or deaggregation of the complexes which TEM shows form particles of a relatively uniform size (~100–150 nm) and density for a specific receptor.
Als analytisches Verfahren in der Biochemie ist die Polyacrylamid-Gelelektrophorese (PAGE) bezüglich ihrer universellen Einsetzbarkeit bis heute unübertroffen.
Electrophoresis is one of the most powerful and fundamental techniques for separation and analysis of proteins and nucleic acids which has been widely accepted for both preparative and analytical purposes (Andrews 1981; Hames and Rickwood 1981; Rickwood and Hames 1982; Maugh II 1983), especially in the fields of recombinant DNA and nucleic acid sequencing. There are only a few investigations that do not utilize electrophoretic techniques (Rickwood and Hames 1982; Weissbach and Weissbach 1986).
The analysis of a protein is usually an attempt to characterize its structure and/or determine its degree of purity. Proteins of unknown structure are purified to apparent homogeneity and then analyzed to determine structure. The success of the structural analysis is, of course, critically dependent on the level of purification achieved.
If the structure of the protein is already known, then the analysis is performed to determine the level of purity, and it is, therefore, simply a critique of the method(s) used for the purification. To give an example of this situation, when a company wishes to test an improved method it has developed for the production of a currently manufactured protein whose structure is well known, the purity of the protein is analyzed simply to evaluate the new purification process. The improved purification process could lead to increased profits either due to increased product purity or to improved process throughput.
The various methods employed for the analysis of proteins will be discussed in the background section along with an assessment of the relative importance of the various techniques. Overlap of some material previously discussed is unavoidable because methods used for preparative purification of proteins (e.g., chromatography) can also be used to analyze the protein for impurities.
The purification scheme for α-galactosidase has progressed through several steps, including its induced synthesis, batch capture, and column purification. This isolation and purification of α-galactosidase has been an attempt to simulate an actual purification because we have not assumed any prior knowledge of the enzyme structure. The exercises described in this chapter represent the last step in our purification process. The IEX purified α-galactosidase will be analyzed by native polyacrylamide gel electrophoresis. The purpose of the analysis is to evaluate the purity of the enzyme and also to be the final purification by extracting the enzyme from the gel (if the extraction step is determined to be necessary).
Die Wanderung geladener Teilchen im elektrischen Feld wird zur Trennung komplexer Gemische von Biomolekülen ausgenutzt. Die Trennmethoden werden unter dem Begriff Elektrophorese zusammengefasst. Da die meisten Biomoleküle Ladungen tragen, gehören Elektrophoresen zu den wichtigsten analytischen Methoden. Für die Untersuchung und Charakterisierung komplexer Mischungen sowie zur Überprüfung ihrer Einheitlichkeit sind sie unentbehrlich. Die wesentlichen Unterschiede in den Trennungsmethoden ergeben sich aus dem für jedes Biomolekül charakteristischem Verhältnis von Ladung zu Masse bzw. aus ihrer Molekülgröße und -form.
The basic technology required for analyzing PCR products is electrophoresis (Sambrook et al. 1989; Rickwood and Hames 1990). Electrophoresis, which separates DNA fragments according to their size, may either be carried out on slab gels or through capillaries. If slab gels are used, two different carrier substances are available, agarose or polyacrylamide. The method of electrophoresis that is chosen depends on the type of a nalysis and resolution with which the scientific question is being asked or on the type of answer that is expected (Fig. 5.1).
This article describes the structural and functional properties of polynucleotides, beginning with a description of the hierarchical levels of structure exhibited by the natural polynucleotides, DNA and RNA. This is followed by a description of modern methods for characterizing polynucleotides and their complexes, including electrophoresis, calorimetry, and spectroscopy. Enzymatic and chemical methods for synthesizing oligo- and polynucleotides are then reviewed. This leads to a description of synthetic polynucleotide analogues, which find widespread use in biotechnology and clinical diagnostics. Molecular mechanisms for sequence-specific recognition of DNA and RNA are described, followed by a survey of methods for preparation and properties of functional polynucleotides, broadly defined as those sequences which fold into a well-defined three-dimensional structure that exhibits a function, such as selective binding to a ligand or catalysis of a chemical reaction. The article concludes with a summary of recent work in which polynucleotides are used as construction materials for the assembly of nanomaterials.
******This paper has been withdrawn by the corresponding author on August 09, 2015******
Abstract: Drought stress was generated by PEG 6000. Water potentials were: zero as the control, and –0.15, –0.49, –1.03, and –1.76 MPa as treatments. After 24-h treatment the total soluble protein content of 2 maize (Zea mays L.) cultivars (704 and 301) was determined and SDS-PAGE gel electrophoresis in the first dimension was performed. By decreasing water potentials, total soluble protein content first increased, and then decreased in the roots and leaves of both varieties. The decrease in total soluble protein content in the roots of both varieties was equal, but in the leaves of cv. 301 it was greater than in cv. 704. In drought
conditions the decrease in root and shoot fresh weight in cv. 704 was greater than in cv. 301. With water potential –1.76 MPa, the accumulation of dehydrin-like 38, 50, 57, and 65 KDa M.W. root proteins and 15, 17, 20, 27, 30, 37, 54, and 59 KDa M.W. leaf proteins increased. However, the expression of 15, 19, and 27 KDa M.W. root proteins, and 22 KDa M.W. leaf protein was induced in both varieties. The accumulation of dehydrin-like proteins in the roots and leaves of cv. 704 was higher than in cv. 301. There was no relationship between protein changes and drought tolerance.
Key Words: Dehydrin-like proteins, Drought stress, Maize, Polyethylene glycol 6000, Protein content, SDS-PAGE
A simple, convenient, and inexpensive method for long-term non-photographic storage of information present in electrophoresis gel, based on protein blotting patterns, is presented here.
RAPD-PCR method and isozyme analysis were used to obtain information of genetic relationship among cucumber varieties. Such information is urgently utilized to support plant breeding program of cucumber. Research was done at the Biotechnology Laboratory of Plant Pest and Diseases, Faculty of Agriculture of Brawijaya University, Malang and Molecular Biology Laboratory, Faculty of Matemathic and Natural Sciences of Brawijaya University, Malang. DNA isolation was done using CTAB method by additional NaCl modification. Sixteen primers from operon were employed to amplify DNA genome by RAPD-PCR. Two enzymes, Esterase and AAT were chosen for isozyme analysis. Clad 97 Program was used for analyzing the data and results in data grouping based on proximity value. Cluster analysis based on isozyme data indicated that there was an adequate lower genetic variation in cucumber, where seven of nine tested varieties showed proximity value of 1.00. Eleven of sixteen primers in RAPD-PCR analysis produced DNA bands. Relativity analysis by using RAPD-PCR method showed high enough of genetic variation. Relativity analysis by using both methods showed that variety 07 was the furthermost. The proximity value between varieties 01 and 02 was 0.916667, these varieties have the higest proximity value among allvarieties.© 2008 Jurusan Biologi FMIPA UNS SurakartaKey words: Cucumis sativus L., RAPD-PCR, isozyme.
The polymorphism of six microsatellites was investigated in four indigenous pig breeds (Erhualian, Tongcheng, Qingping and Wannanhua) and three introduced breeds (Large White, Landrace and Duroc) in China. The genetic variations within and among populations were analyzed. The results showed that genetic diversity of Chinese indigenous pig breeds is higher than that of the introduced pig breeds. The clustering of seven breeds is approximately consistent with their geographical distribution. Estimated time of breed divergence ranged from 653 to 1856 years.
Nanostructures based on nanotechnologies have opened up a novel research field for the fast analysis of biomolecules with ultrahigh resolution, including the analysis of single biomolecules. Nanostructures for electrophoretic separation, especially, are an exciting topic among researchers in many areas, and their designs are widely expected to contribute to the goal of developing a single separation tool for a wide range of biomolecules. In this review, nanopillar, nanowall, and nanowire devices are introduced for fast separation of DNA molecules and protein samples, and the numerous advantages of these devices are described. This review also outlines the fabrication processes for nanostructures, including “top-down” and “bottom-up” nanofabrication approaches. Besides describing the fast separation of biomolecules, the electroosmotic flow (EOF) suppression effect, and its related online concentration technique in nanopillar devices, is reviewed. The nanowall devices have the unique feature that longer DNA molecules migrate faster than shorter ones, and that is completely different from the separation behavior of DNA molecules based on nanopillar devices. The feasibility is shown for self-assembly of the nanowire structure embedded in a microchannel on a fused silica substrate, as a means to separate DNA molecules. Applications of a newly-fabricated 3D network structure with spatial density control for the fast separation of a wide range of DNA molecules are also given.
Maryati KT, Sugiyarto. 2009. Characterization of white grub (Melolonthidae; Coleoptera) in salak plantation based on morphology and protein banding pattern. Bioteknologi 6: 80-87. This research aims to find out the white grub (Melolonthidae; Coleoptera) variability based on the morphological characteristic and protein banding pattern found in "salak pondoh" farm in Regencies of Sleman, Yogyakarta and Magelang, Central Java. Each area has five sampling points. Morphological analysis on white grub was conducted using descriptive method and analysis on protein banding pattern was conducted using qualitative analysis based on the presence or absent of band pattern on the gel, and qualitatively based on the relative mobility value (Rf) of protein. The result indicated that the white grub in Sleman and Magelang, based on morphology characteristic is only one species, namely Holothricia sp. Based on the protein banding pattern, the white grub sample have differences of protein band number and protein molecular weight. Abstrak. Maryati KT, Sugiyarto. 2009. Karakterisasi lundi putih (Melolonthidae: Coleoptera) pada pertanaman salak berdasarkan ciri morfologi dan pola pita protein. Bioteknologi 6: 80-87. Penelitian ini bertujuan untuk mengetahui keanekaragaman lundi putih (Melolonthidae; Coleoptera) berdasarkan ciri morfologi dan pola pita protein yang ditemukan di lahan pertanaman salak pondoh di Kabupaten Sleman, Yogyakarta dan Kabupaten Magelang, Jawa Tengah. Pada masing-masing wilayah diambil lima titik sampling. Analisis morfologi lundi putih digunakan metode deskriptif, dan analisis pola pita protein digunakan analisis kualitatif berdasarkan muncul tidaknya pola pita pada gel, dan secara kuantitatif berdasarkan nilai mobilitas relatif protein (RF). Hasil penelitian menunjukkan bahwa sampel lundi putih di Kabupaten Sleman dan Magelang, berdasar karakter morfologi hanya satu spesies yaitu Holotrichia sp. Karakter pola pita protein sampel lundi putih dari Sleman dan Magelang mempunyai perbedaan jumlah pita protein dan berat molekulnya. Kata kunci: salak, lundi putih, morfologi, pola pita protein.
To investigate the mutational origin and nature of genetic diversity in groundnut, mutants derived from Dharwad Early Runner and others along with natural types, and intra and interspecific derivatives belonging to all the four botanical types were assessed for their morphological, biochemical and molecular diversity. Similarity among the natural and mutant categories suggests the key role of mutations in creating enormous diversity in terms of different subspecies and botanical types of groundnut. Behaviour of the Dharwad Early Runner derived mutants and several unusual features are indicative of the non-Mendelian turnover mechanisms. Multiple gene differences between the mutants and their parents, paternal inheritance and tissue-specific expression of glutamate oxloacetate transaminase isozyme, response of mutants to 5-azacytidine (a demethylating agent) and limited molecular diversity compared to enormous morphological diversity suggests the possible involvement of epigenetic mechanisms in the differentiation of groundnut into different subspecies and botanical varieties.
The monoterpenes are the most important contribution to the olfactory profile of wine due to their low odour threshold. These and other aroma-active substances do not generally exist in a free form but are conjugated to mono- or disaccharides, thereby forming water-soluble and odourless complexes. Enzymes that cleave the sugar moieties from the precursors can, therefore, have a major impact on the sensory profile of wine, as they release the volatile aroma compounds. For this reason, we searched for wine yeasts producing glycosidases which are active under oenological conditions. A collection of 100 wine yeasts were screened for glycosidase activities in whole cells and in culture supernatants. Kinetic parameters were determined spectrophotometrically with synthetic model substrates, and hydrolysis of natural glycosides was detected by thin-layer chromatography. A yeast isolate, AS1, was identified as a new Wickerhamomyces anomalus strain which hydrolysed a number of synthetic and natural glycosides under oenological conditions. Citronellol- and nerol-glucosides, among the most frequently occurring aroma precursors in wine, were also cleaved. In contrast to a commercial β-glucosidase, whole cells of W. anomalus AS1 catalysed deglycosylation of arbutin and salicin directly in a white and a red wine. Besides the formation of intra- and extracellular glucoside hydrolases, strain AS1 exhibited arabinosidase and xylosidase activities which are also essential for the release of flavour compounds. Even with limited functionality at oenological conditions, the glycoside hydrolase activities of W. anomalus AS1 may improve aroma development, provided that the reaction occurs over a longer period, as it is the case during wine-making.
Four enzymes with phospholipase A1 (PLA1) activity were purified from the fruiting bodies of the basidiomycete Armillaria ostoyae. The enzymes (PLA1-1, -2, -3 and -4) showed similar isoelectric points (4.3, 3.9, 4.0 and 4.0) and apparent molecular masses in the range of 35–47 kDa. Mass spectrometric analyses of proteolytic fragments revealed sequences homologous to α/β-hydrolase fold enzymes. The enzymes share one conserved region with fungal phospholipases B and the active site sequence with bacterial esterases and PLA1s. PLA1-1 cleaves phospholipids and lysophospholipids with an optimum activity at pH 5.3. In contrast, PLA1-2, -3 and -4 are characterized by broad pH optima in the slightly acidic to neutral range and are additionally capable of hydrolyzing mono- and diglycerides as well as fatty acid methyl esters. All enzymes favor glycerol-based lipids with a single medium-sized fatty acid moiety in the sn-1 position but show reduced activity towards the corresponding 1,2-diacyl derivatives with bulky long-chain or inflexible saturated fatty acid moieties in the sn-2 position. The enzymes prefer zwitterionic phospholipid substrates and are unable to hydrolyze triglycerides. From the selectivity of these broad-spectrum α/β-hydrolase fold enzymes towards the different classes of their substrates a regiospecific steric hindrance and a head group recognition are concluded.
Human Rhinovirus (HRV) infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups) that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C) able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis.
An activity that inhibited the accumulation of TMV antigen in tobacco leaf disks was observed in the growth medium of uninfected tobacco cell suspension cultures. Levels of inhibitory activity were highest in medium from actively growing cells. The activity was partially heat-labile and protease-sensitive, and appeared in the high molecular weight fractions following ultrafiltration and dialysis. Growth medium was analysed by SDS-PAGE and found to contain two main protein bands corresponding to 14 and 40 kDa. These proteins were partially purified by ammonium sulphate precipitation and column chromatography. Both purified protein fractions had inhibitory activity and contained carbohydrate. The proteins were associated with the protoplast and not with cell wall material. Both proteins effluxed from protoplasts into the surrounding medium in the absence of bursting, indicating that they may normally be secreted. Incorporation of radiolabels into both viral coat protein and RNA was inhibited by growth medium. A polysaccharide fraction from growth medium also had inhibitory activity.
Reaction of methyl 2-deoxy-2-C-(3-bromoacetoxypropyl)-α-D-arabinofuranosides, prepared from methyl 2,3-anhydro-α-D-ribofuranoside, with oligodeoxyribonucleotide (21mer) in acetonitrile-H2O (pH 7) and subsequent treatment with piperidine resulted in the cleavage of the nucleotide chain at the position G, A, and C.
Ribosomes in lysates prepared from the mycelia of Aspergillus giganteus MDH 18894, which are actively secreting α-sarcin, do not contain the α-sarcin lesion. However, the addition of exogenous α-sarcin to these same lysates results in cleavage of the 26 S rRNA of the 60 S ribosomal subunit, characteristic of the cytotoxic action of α-sarcin. We conclude that A. giganteus ribosomes are not inherently resistant to the action of α-sarcin but are protected in vivo by producing α-sarcin in an inactive form and/or by the efficient cotranslational secretion of the toxin.
Short model homo-oligomeric deoxynucleotides ranging in length from 12- to 24-mer were separated using a 20 mM Tris(hydroxymethyl)aminomethane-N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid buffer containing 4% hydroxyethy cellulose of low viscosity as the polymer additive pH 7.0). The separation was performed using a DB-17-coated capillary. The influence of instrumental parameters such as field strength and temperature was evaluated. It required a separation voltage of 12 kV at reversed polarity and a temperature of 25°C. Efficiencies of up to 2.5×106/m were obtained. The most important parameters influencing the separation were the concentration and the viscosity of the polymer used. Different viscosity grades of hydroxyethy cellulose were evaluated for their loading time on the capillary and their ability to separate p(dA)12–18 and p(dA)19–24. The use of low viscosity-grade hydroxyethy cellulose at a relatively high concentration (4%) made it possible to replace the buffer after every run and permitted the use of hydrodynamic injection of oligonucleotide samples. The entangled polymer solution system was found to be applicable on automatic capillary electrophoresis (CE) equipment. For quantitation, the use of an internal standard has been shown to improve both migration time and peak area repeatability. This method using low viscosity-grade hydroxyethy cellulose has been demonstrated to have the repeatability, linearity and selectivity required for stability studies of oligonucleotides.
We have purified RsrI endonuclease (R·RsrI), an isoschizomer of EcoRI, from Rhodobacter sphaeroides strain 630. The enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and size-exclusion high-performance
liquid chromatography. RsrI endonuclease is a dimer over the concentration range of 0.05 to 1.4 mg/ml. The reduced and denatured molecular weight of
the enzyme is 30,000 Da. R·RsrI, like R·EcoRI, catalyzes the cleavage of duplex DNA and oligodeoxyribonucleotides between the first two residues of the sequence GAATTC.
R·RsrI exhibits a KM of 14 nM and a kcat of 6.5 min.1 when reacting with pBR322 DNA at 25°C. R·RsrI differs from REcoRI in its N-terminal amino acid sequence, susceptibility to inhibition by antibodies, sensitivity to N-ethylmaleimide, isoelectric
point, state of aggregation at high concentrations, temperature lability, and conditions for optimal reaction. R·RsrI displays a reduction of specificity (“star activity”) under conditions that also relax the specificity of R·EcoRI.
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