Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Use of aqueous isopropanol to remove detergent from gels

Blank, A.; Sugiyama, R.H.; Dekker, Charles A.

doi:10.1016/0003-2697(82)90347-5

Cited by 215 publications

(104 citation statements)

References 17 publications

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“…Although numerous attempts at protein renaturation (3,4,13) were made following size-fractionation by SDS-PAGE, we failed to renature the nuclease fraction of the gp120 preparation. However, including DNaseI as a control nuclease that migrated to the correct position (33 kDa) and demonstrated activity after renaturation confirmed that the system was working.…”

Section: Attempted Detection Of Nuclease Activity Of Gp120 Preparatiomentioning

confidence: 99%

“…Nuclease detection in the gp120-BSA preparation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to a well-described method (15) with some minor modifications (3,4), and using DNase I (Boehringer Mannheim) with molecular weight of 34 000 Da as a positive control. In addition, because nuclease renaturation of the gp120-BSA preparation (but not the DNase I) proved impossible after SDS-PAGE despite the use of numerous renaturation techniques (3,4), we attempted to detect the nuclease in the gp120 preparation in non-denaturing gels (16)(17)(18).…”

Section: Detection Of Nuclease Activity Of Recombinant Gp120mentioning

confidence: 99%

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Identification of Endonuclease Activity in HIV-1 gp120 Preparations Produced Using Baculovirus Expression Systems

Davidoff

Mendelow

1997

BioTechniques

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Experiments undertaken with commercially available recombinantly produced human immunodeficiency virus Type 1 (HIV-1) gp120 demonstrated that the resuspended lyophilized protein, a product of the baculovirus expression system, had intrinsic nuclease activity. This nuclease activity was distinguishable from the moleculargrade bovine serum albumin that it was constituted in. The activity was thermolabile in that if the preparation was heated to 100°C for 10 min, the activity was abolished, although this did not happen when it was stored at -20°C. The nuclease activity was also INTRODUCTIONThe successful use of the insect cell/baculovirus expression strategy in the large-scale production of recombinant proteins of mammalian, insect, plant and bacterial origin has revolutionized biology, biotechnology and chemistry. This highly efficient expression system has been well characterized (6,9,11), and its ubiquitous utilization and exploitation is testament to the many factors intrinsic to the system that contribute to its popularity. These include high protein expression levels, ease and speed of genetic engineering, accommodation of large DNA inserts, post-translational protein processing (e.g., glycosylation) similar to mammalian cells and ease of insect cell growth in suspension (11).Despite years of intensive investigation and speculation concerning the specific virological and immunological mechanisms of cell death involved in human immunodeficiency virus Type 1 (HIV-1) infection (13), certain of these still remain elusive. Nevertheless, there is mounting and conclusive evidence that the process of apoptosis is at least contributory to T-cell depletion (1,10,19,20).Since the gp120 protein has been incontrovertibly linked to apoptotic cell death mechanisms in HIV-1 disease pathogenesis, and because there are dangers inherent in the purification of gp120 from cultures infected with the virus and from blood derived from HIV + donors, gp120 is often produced in the insect/baculovirus system for subsequent use in research and commercial enterprises.In this study, which was aimed at the evaluation of putative apoptosis-blocking strategies in cultures of peripheral blood mononuclear cells (PBMC) exposed to cross-linked gp120/anti-gp120 complexes, we noted contaminating endonuclease activity in culture supernatants containing recombinant gp120 protein that had been produced in the baculovirus expression system. While not disputing the role of apoptosis in Tcell depletion in AIDS, this report is aimed at drawing attention to the baculovirus-associated endonuclease activity, which may confound the interpretation of studies addressing this phenomenon in vitro. MATERIALS AND METHODS Use of Special ReagentsBaculovirus-produced recombinant gp120 IIIB and IgGpurified rabbit anti-gp120 antibodies were obtained from Intracel (London, England, UK or Cambridge, MA, USA) and were reconstituted in sterile double-distilled water. Molecular biology-grade bovine serum albumin (BSA), which contained no deoxyribonucleases, ribonuc...

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Section: Attempted Detection Of Nuclease Activity Of Gp120 Preparatiomentioning

confidence: 99%

Section: Detection Of Nuclease Activity Of Recombinant Gp120mentioning

confidence: 99%

Identification of Endonuclease Activity in HIV-1 gp120 Preparations Produced Using Baculovirus Expression Systems

Davidoff

Mendelow

1997

BioTechniques

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“…Subsequent to washing, these gels were incubated in sodium acetate buffer above for 2 h at 37°C with gentle agitation in a rocking-hybridization oven (Shake 'NЈ Bake, Boekel Scientific, Feasterville, PA). Following incubation, gels were fixed in 7.5% (v/v) aqueous acetic acid, stained with toluidine blue O (0.2% (w/v) in 10 mM HEPES, pH 8.5), and de-stained using multiple changes of deionized water (10). Nuclease activity in these gels was readily apparent as distinct, clear/colorless bands of substrate hydrolysis in an otherwise uniformly stained, dark blue background of unhydrolyzed polynucleotide substrate.…”

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confidence: 99%

“…Rev 5Ј-GGATCCTTAAGCGTAATCTG-GAACATCGTATGGGTAATACAAACATCCACC-3Ј (containing a BamHI restriction site shown in boldface; stop codon in boldface italics; and the HA epitope sequence (underlined)). The conditions used for these PCR were as follows: a hot start at 94°C for 5 min, followed by 10 (18). Briefly, mid-log phase trophozoites (ϳ1.5 ϫ 10 5 cells ml Ϫ1 ) were detached by chilling on ice for 10 min, pelleted by centrifugation at 500 ϫ g at 4°C for 10 min, washed twice in ice-cold PBS and once in electroporation buffer (120 mM KCl, 0.15 mM CaCl 2 , 25 mM HEPES, 2 mM EGTA, and 5 mM MgCl 2 , 10 mM K 2 HPO 4 /KH 2 PO 4 , pH 7.6).…”

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confidence: 99%

Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

McGugan

Joshi

Dwyer

2007

Journal of Biological Chemistry

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The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.The ancient eukaryotic human parasite, Entamoeba histolytica, is the causative agent of invasive amebiasis in humans infecting 10% of the world population and resulting in over 100,000 deaths annually, chiefly from amebic liver abscess (1). Infection is acquired through the ingestion of the quadranucleate cyst form of the organism from fecally contaminated food or water supplies. Excystation occurs in the small intestine, releasing immature trophozoites, which move into and colonize the nutrient-poor large intestine.As ancient eukaryotes, Entamoeba parasites contain only rudimentary endoplasmic reticulum and Golgi apparatus (2, 3). Despite this, vesicle trafficking pathways appear to be essential to the pathogenesis of these parasites as (i) uptake and digestions of nutrients by mature trophozoites in the large intestine, (ii) invasion of the intestinal epithelium, (iii) delivery of cyst wall components during encystation, and (iv) dissemination and establishment of extra-intestinal infections all rely on endocytosis and secretion. In addition, these parasites lack many traditional eukaryotic biochemical pathways, including the de novo biosynthesis of purine and pyrimidine nucleobases. As a result, these parasites are completely dependent upon their host for the supply of these essential preformed nutrients. In that regard, it has been reported previously that Entamoeba are capable of salvaging both purines and pyrimidines from the extracellular milieu during in vit...

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“…Nuclease detection in renatured SDS-PAGE gels containing nucleic acids was performed essentially as described elsewhere (Rosenthal & Lacks, 1977;Blank et al, 1982). Ten micrograms of salmon testes DNA (Fluka) and 40 mg 16S and 23S rRNA from E. coli (Roche) per ml of the resolving gel were used.…”

Section: Methodsmentioning

confidence: 99%

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

Accetto

Avguštin

2007

Microbiology

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Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1 Available tools for genetic analysis in the anaerobic rumen bacterium Prevotella bryantii are limited to only two known systems for gene delivery, and no genes, with the exception of plasmid maintenance and selection genes, have been successfully expressed from plasmids in any species of the genus Prevotella until now. It is shown here that nucB, a newly cloned nuclease gene from P. bryantii, can be controllably expressed from shuttle vector pRH3 in P. bryantii strain TC1-1, depending on the tetracycline concentration in the growth medium. nucB expression is also growth-medium dependent and this regulation presumably takes place at the translational level. His-tagged NucB was purified from P. bryantii TC1-1 culture supernatant and was shown to degrade DNA as well as RNA; it is most likely a minor 36 kDa P. bryantii non-specific nuclease. INTRODUCTIONMembers of the bacterial genus Prevotella have been found in the rumen as well as in the oral cavity and large intestine of man and animals (Edwards et al., 2004;Paster et al., 2001;Duncan et al., 2003;Eckburg et al., 2005; Leser et al., 2002). In the rumen they are symbionts contributing to the degradation of plant cell-wall polysaccharides (Miyazaki et al., 2003) and protein metabolism (Walker et al., 2003), whereas oral prevotellas are implicated in dental plaque establishment (Kolenbrander et al., 2002), periodontitis (Paster et al., 2001), advanced caries (Martin et al., 2002), oro-pharyngeal abscesses (Brook, 2004) and noma, a grotesque childhood orofacial gangrene, now confined mostly to sub-Saharan Africa (Enwonwu et al., 2006). The genetic tools that would make possible more thorough studies of prevotellas are still undeveloped despite their ecological and medical importance. This appears to be primarily due to the large phylogenetic distance between prevotellas and other well-characterized micro-organisms, and as a consequence of the rather specific genetic elements, exemplified by promoters, containing distinct 27/233 consensus sequences that are recognized by unusual primary s factor (Vingadassalom et al., 2005). Nevertheless, in two studies (Shoemaker et al., 1991;Accetto et al., 2005) the plasmid replicons and tetQ selectable marker derived from Prevotella and related colonic Bacteroides strains were used successfully for shuttle-vector introduction into the xylan-degrading ruminal species Prevotella bryantii, which forms a distinct phylogenetic lineage apparently somewhat closer to oral prevotellas than the other ruminal Prevotella species (Avguštin et al., 2001). Based on the conjugal transfer protocol developed in the first study, a P. bryantii B 1 4 carboxymethylcellulase gene, fused to a cellulose-binding domain of Thermomonospora fusca cellulase, and driven by a Prevotella ruminicola 23 xylanase promoter, was introduced into P. bryantii B 1 4. The gene was expressed in Bacteroides uniformis 1108, but not in P. bryantii B 1 4, possibly ...

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Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Use of aqueous isopropanol to remove detergent from gels

Cited by 215 publications

References 17 publications

Identification of Endonuclease Activity in HIV-1 gp120 Preparations Produced Using Baculovirus Expression Systems

Identification of Endonuclease Activity in HIV-1 gp120 Preparations Produced Using Baculovirus Expression Systems

Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

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