CsCl Gradient Centrifugation Research Articles

A simplified CsCL protocol for lambda DNA purification: no enzymatic treatment/one phenol extraction

A modification of the CsCl gradient centrifugation method for DNA phage purification is presented. It avoids the enzymatic steps as well the need for a preliminary phage titration, a tedious process proposed in the majority of the protocols in use. The quality of the DNA obtained makes it amenable for additional manipulations like digestions, ligations, labelling, subcloning, etc.

Detection of Infectious Adeno-Associated Virus Particles in Human Cervical Biopsies

Recently we reported that DNA of the human oncogenic papillomaviruses (HPV) and the tumor suppressive human helper virus-dependent parvoviruses, adeno-associated viruses type 2 (AAV-2), colocalize in cervical epithelium. To analyze whether infectious AAV particles are present in cervical tissue, we examined cervical biopsies from 36 patients with HPV-related lesions (squamous intraepithelial lesions) for the presence of AAV DNA and of infectious AAV. From each patient specimens from the lesion and from adjacent normal epithelium were analyzed. After PCR analysis AAV DNA-containing samples were purified by CsCl gradient centrifugation. The presence of AAV virions in CsCl gradients was analyzed and infectivity of AAV was determined. In addition, the biopsies were tested for the presence of HPV DNA. AAV DNA could be detected in biopsies from 23 of 36 patients. AAV particles were found in 11 AAV DNA-positive biopsies from 7 patients (lesions and/or normal tissue, respectively). AAV particles were found to be infectious virions in 10 of the 11 cases. These results demonstrate for the first time that infectious AAV can be isolated from human cervical biopsies, indicating a possible sexual transmission of AAV.

Hepatitis C virus structural proteins assemble into viruslike particles in insect cells.

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of HCV has been hampered by the low level of viral particles in infected individuals, the inability to propagate efficiently the virus in cultured cells, and the lack of a convenient animal model. Due to these obstacles, neither the structure of the virus nor the prerequisites for its assembly have been clearly defined. In this report, we describe a model for the production and purification of HCV-like particles in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural proteins. In insect cells, expressed HCV structural proteins assembled into enveloped viruslike particles (40 to 60 nm in diameter) in large cytoplasmic cisternae, presumably derived from the endoplasmic reticulum. Biophysical characterization of viruslike particles by CsCl and sucrose gradient centrifugation revealed biophysical properties similar to those of putative virions isolated from infected humans. The results suggested that HCV core and envelope proteins without p7 were sufficient for viral particle formation. Analysis of particle-associated nucleic acids demonstrated that HCV RNAs were selectively incorporated into the particles over non-HCV transcripts. The synthesis of HCV-like particles in insect cells may provide an important tool to determine the structural requirements for HCV particle assembly as well as to study viral genome encapsidation and virus-host interactions. The described system may also represent a potential approach toward vaccine development.

Purification of DNA by Anion‐Exchange Chromatography

Column chromatography has evolved to provide a rapid and effective alternative to more laborious methods for preparing high-quality DNA, such as CsCl-gradient centrifugation. This unit describes the use of a column made of a unique anion-exchange resin that selectively binds nucleic acids, allowing rapid separation of DNA from contaminating RNA, proteins, carbohydrates, and metabolites. The procedure employs columns supplied by QIAGEN; other preparation methods are available from other suppliers. A crude nucleic acid sample (usually a cleared cell lysate) is applied to the QIAGEN tip under conditions that favor binding. Contaminants in the sample are washed from the column with a moderate-salt buffer, and DNA is eluted using a high-salt buffer.

Quantitative polymerase chain reaction assay for DNA repair within defined genomic regions

We have developed a quantitative assay to determine repair of structurally different DNA lesions at defined genomic sites. This assay depends on the fact that many different types of damage are repaired by the same nucleotide excision repair (NER) pathway which includes synthesis of short DNA fragments at the sites of damage. After exposure to damaging agents, cells are treated with 5-bromodeoxyuridine (BrdUrd) to label the regions undergoing repair with the presumption that regions that have been more efficiently repaired would incorporate more BrdUrd than regions that were less effectively repaired. Thus, the abundance of the different sequences in the BrdUrd-containing DNA would be a direct and quantitative measure for the repair rates of the corresponding regions. The BrdUrd-containing, repaired DNA was isolated by CsCl gradient centrifugation and immunoprecipitation with anti-BrdUrd antibody and was used as template in quantitative PCR in which the amount of the product was directly proportional to the amount of template. This approach was used to address the question whether DNA repair after UV-irradiation occurs in an uniform, random manner or with preferences for certain regions. We found out that there was a higher repair efficiency at the 5′-end of the mouse β-globin domain in Ehrlich ascites tumor cells.

Phosphorylation of DHBV Pre-S: Identification of the Major Site of Phosphorylation and Effects of Mutations on the Virus Life Cycle

Four potential serine/threonine phosphorylation sites [(S/T)-P motif], designated P1–P4, on the pre-S protein of duck hepatitis B virus (DHBV) have been mutated. Mutants include single (P2, P3, P4) and double amino acid substitutions (P1+P2, P3+P4) and one with all four sites mutated (4P). Serine at position 118 (P3) was identified as the major site of phosphorylation by Western blotting and radioimmunoprecipitation afterin vitrocell labeling with [35S]methionine or [33P]orthophosphate. Mutant virions generated by transfection of LMH cells were infectious bothin vitroin duck hepatocyte primary cultures andin vivoin Pekin ducks. Intracellular relaxed circular (RC) and covalently closed circular (ccc) DNA syntheses were not affected by the P3 mutation or even the quadruple mutant. Extracellular virus production was slightly increased when the P3 site was mutated. CsCl gradient centrifugation showed no clear difference between mutant and wild-type virus with respect to the ratios of enveloped virus and nucleocapsid particles in hepatocyte culture supernatants. Trypsin or V8 protease digestion with or without NP-40 indicated that phosphorylation of the pre-S domain is not involved in determining the transmembrane topology of DHBV large protein. This phenotypic analysis indicates that DHBV pre-S phosphorylation has no apparent effect on DHBV replication and formation of mature viral particles in duck hepatocyte primary culture and does not affect infectivity in ducklings.

Ribonucleoprotein particles of quiescent maize embryonic axes.

Certain RNA molecules are known to be sequestered and stored as ribonucleoprotein particles (RNPs) in many different tissues, particularly at some stages of metabolic quiescence. In this research RNPs from embryonic axes of mature maize seeds were isolated by sucrose and CsCl gradient centrifugation and characterized based on their RNA and protein contents. Two types of RNP particles of non-ribosomal nature were identified by northern blot analysis with specific probes: the 7S RNP and the signal recognition particle (SRP) particles which contain 5S rRNA and 7S RNA respectively. The proteins associated to these RNA molecules were the transcription factor TFIIIA-homologous protein associated to 7S RNP, and the p72, p68 and p54-GTPase proteins associated to SRP.

Downregulation of gastric mucin gene expression and its biosynthesis by dexamethasone in the human.

The effect of corticosteroids on the release and biosynthesis of gastric mucin remains unclear. We studied the effects of dexamethasone on biosynthesis of mucin and MUC1 gene expression in the human stomach in vitro. Gastric mucosal specimens, obtained from six subjects at gastrectomy, were cultured with various concentrations of dexamethasone. Biosynthesis of mucin was studied by labeling gastric mucosa for 2 h with [3H]-glucosamine. After purification of mucin by CsCl gradient centrifugation, radioactivity of intra- and extracellular samples was counted. MUC1 gene expression was studied by Northern and dot-blot analysis using a cDNA encoding human gastric mucin gene MUC1. The dexamethasone treatment decreased mucin secretion from the isolated mucosa in a time- and concentration-dependent manner, with maximal inhibition of secretion (32+/-6%) observed after 8 h and 10(-5) M. Dexamethasone treatment also decreased MUC1 levels (28+/-9%). The inhibitory effect was also observed in carbachol-evoked secretion. These findings suggest that a decrease in mucin biosynthesis by corticosteroids may be involved in steroid-induced gastric mucosal damage.

Effect of DNA preparation from laver (Porphyra yezoensis) thalli on reproducibility of RAPD (random amplified polymorphic DNA) patterns

Laver (Porphyra yezoensis) DNAs were extracted from thalli with five different procedures and used for RAPD (random amplified polymorphic DNA) analysis as templates. Restriction enzyme-digestive DNAs were obtained with all procedures examined. However, RAPD patterns generated with these DNAs appeared highly irreproducible and were considerably different from each other. When DNAs purified with CsCl gradient centrifugation were used for RAPD analysis as templates, highly reproducible RAPD patterns were obtained, suggesting that unpurified DNAs extracted from thalli with all five extraction procedures contained an excess of RNA, polysaccharides and/or other materials which affected the RAPD reproducibility. Thus, results indicated that purification of DNA is essential to produce reproducible RAPD patterns of Porphyra DNA.

Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells.

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP.

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996). Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs. Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.

Rapid determination of plasmid copy number

Plasmid copy number, the number of expression vectors per host cell, is a key variable in recombinant microbial cultivation. Therefore, it would be very helpful, if the plasmid copy number could be determined during the operating process period. A rapid quantification of this important process variable would even open the possibility of its use in process control. However, current assays like gel electrophoresis, CsCl-gradient centrifugation, HPLC and other methods are time consuming and difficult to quantify. Indirect methods, like the correlation of copy number with e.g. the activity of an enzyme, coded on the plasmid, are prone to errors due to the production kinetics, turnover rate and protein denaturation. Here, a method is presented, which enables the plasmid copy number to be determined in less than 30 min. This novel procedure based on plasmid isolation by means of a commercial DNA-isolation kit and quantification by capillary electrophoresis, should allow the copy number to be used in process control.

The Development and Use of a DNA Polymerase Arrest Assay for the Evaluation of Parameters Affecting Intrastrand Tetraplex Formation

We show here that a K+-dependent block to DNA synthesis is a sensitive and specific indicator of intrastrand tetraplex formation that can be used, both to identify sequences with tetraplex-forming potential and to examine parameters that affect tetraplex formation. We show that tetraplex formation is determined by a complex combination of factors including the size and base composition of its constituent loops and stems. In the process of carrying out this study we have found that the number of sequences with the ability to form tetraplexes is larger than previously thought, and that such sequences are ubiquitous in eukaryote genomes.

Separation and Characterization of Two Related Giardiaviruses in the Parasitic ProtozoanGiardia lamblia

Giardiavirus encapsidates a 6.2-kb double-stranded (ds) RNA within a capsid that consists of a major 100-kDa capsid protein (p100) and a minor 190-kDa protein (p190). In this study, two nonhomologous 6.2-kb ds RNAs cohabiting inGiardia lambliatrophozoites were found to be separately encapsidated into two distinct virions, one (designated GLV[p100]) whose capsid consists of p100 and p190, and the other (designated GLV[p95]) whose capsid consists of a 95-kDa protein (p95) and a minor p190-equivalent protein. Both types of virions were enriched in the membranous fraction of a lysate from virus-infectedG. lambliacells. Separation of these virions was achieved by CsCl gradient centrifugation following osmotic rupture of the viral particles. By these treatments, the 6.2-kb ds RNA was removed from GLV[p100] whereas that in GLV[p95] remained unchanged, and the two 6.2-kb ds RNAs that had been purified by this protocol displayed differential hybridization properties to viral cDNA probes. Western blotting and peptide mapping experiments show that p100 and p95 were closely related proteins, but each had distinct amino acid sequences. Virus purification and pulse–chase experiments show that GLV[p100] was selectively secreted into the medium whereas GLV[p95] remained within the trophozoites ofG. lambliatoward the late phase of cell growth. Secretion of GLV[p100] was not inhibited by Brefeldin A. These findings demonstrate the cohabitation of multiple Giardiavirus species inG. lamblia.

Hyperoxia alone causes changes in lung proteoglycans and hyaluronan in neonatal rat pups.

Specific changes in composition and content of lung extracellular matrix (ECM) proteoglycans (PGs) and hyaluronan (HA) have been observed during the acute response to damage in several forms of injury including infant respiratory distress syndrome (IRDS). These ECM components are thought to modulate the healing response. Hyperoxia, a contributing factor to IRDS, is known to damage both adult and developing lung, however, the extent and pattern of impairment depends on lung maturity. We hypothesized that exposing neonatal rats to hyperoxia alone might result in changes in lung HA, as well as in age-specific changes in lung PGs, similar to those shown to occur in IRDS. In control rats, lung HA decreased over the first 10 days of life, whereas rats exposed to hyperoxia exhibited a time-dependent, time-limited increase in both lung HA and lung wet weight. Histochemistry showed the HA in hyperoxia-exposed lungs to be accumulated in perivascular cuffs of medium sized arteries, and in the alveolar walls. Rats were then exposed to normoxia or hyperoxia for 7 days beginning at either 3 days of life (neonatal) or 21 days (adolescent), and lung tissue was cultured in the presence of [35S]-sulfate to label newly synthesized PGs. Proteoglycans were extracted, and analyzed by isopycnic CsCl gradient centrifugation, sequential enzymatic deglycosylation, size chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). When controlled for total protein extracted, 63% more label was incorporated into large molecular weight material in the tissue exposed to hyperoxia, with a 95% increase in incorporation in the most dense fraction, D1. [35S]-Sulfate incorporation into chondroitin and dermatan sulfate in hyperoxic tissue specifically increased 116% (242% in the D1 fraction), while incorporation into heparan sulfate remained essentially unchanged. There was a nearly fivefold increase in [35S]-sulfate incorporation into chondroitin sulfate chains in the D1 fraction. When the D1 fractions of extracts of treated and control rat lungs were compared on SDS-PAGE, a large chondroitin sulfate proteoglycan (CSPG; core protein of 195 kDa) was upregulated in the D1 fraction from hyperoxic tissue of neonatal rats, but was not detected in the lungs of adolescent animals exposed to hyperoxia. This CSPG and four additional large CSPGs were noted to be upregulated on western blotting by a polyclonal antibody directed against the G1 domain of the aggrecan protein core. We conclude that hyperoxia alone causes an increase in lung HA and lung water, and speculate that this contributes significantly to the clinical syndrome of IRDS. In addition, several large CSPGs are upregulated by hyperoxic exposure in a developmentally specific manner. We speculate that this increase in CSPGs may interfere with the normal developmental sequence of events, contributing to hypoalveolarization.

Evidence for the Covalent Binding of SHAP, Heavy Chains of Inter-α-Trypsin Inhibitor, to Hyaluronan

We previously showed that serum-derived 85-kDa proteins (SHAPs, serum-derived hyaluronan associated proteins) are firmly bound to hyaluronan (HA) synthesized by cultured fibroblasts. SHAPs were then identified to be the heavy chains of inter-alpha-trypsin inhibitor (ITI) (Huang, L., Yoneda, M., and Kimata, K. (1993) J. Biol. Chem. 268, 26725-26730). In this study, the SHAP.HA complex was isolated from pathological synovial fluid from human arthritis patients. The SHAP.HA complex was digested with thermolysin, followed by CsCl gradient centrifugation. The HA-containing fragments thus obtained were further digested with chondroitinase AC II and subjected to TSK gel high performance liquid chromatography (HPLC). Peptide-HA disaccharide-containing fractions (the SHAP.HA binding regions) were further purified by reverse phase HPLC. Major peaks were analyzed by protein sequencing and mass spectrometry (electrospray ionization mass spectrometry and collision induced dissociation-MS/MS). By comparison with the reported C-terminal sequences of the human ITI family, the peptides were found to correspond to tetrapeptides derived from the C termini of heavy chains 1 of and 2 of inter-alpha-trypsin inhibitor (HC1 and HC2), and heavy chain 3 of pre-alpha-trypsin inhibitor (HC3), respectively, and a heptapeptide from HC1. Mass spectrometric analyses suggested that the C-terminal Asp of each heavy chain was esterified to the C6-hydroxyl group of an internal N-acetylglucosamine of HA chain. This report is the first demonstration to give evidence for the covalent binding of proteins to HA.

Lethal Mutations in a Yeast U6 RNA Gene B Block Promoter Element Identify Essential Contacts with Transcription Factor-IIIC

The B block promoter element is the primary binding site for the RNA polymerase III transcription initiation factor TFIIIC. It is always located within the transcript coding region, except in the Saccharomyces cerevisiae U6 RNA gene (SNR6), in which the B block lies 120 base pairs downstream of the terminator. We have exploited the unique location of the SNR6 B block to examine the sequence specificity of its interaction with TFIIIC. The in vitro and in vivo effects of all possible single base pair substitutions in the 9-base pair core of the B block were determined. Five mutant alleles are recessive lethal when present at a low copy number; these alleles identify crucial contacts between TFIIIC and the B block promoter element. Transcript analysis reveals that lethal B block substitutions reduce U6 RNA synthesis at least 10-fold in vivo and 20-fold in vitro. One viable B block mutant strain has one-third the wild type amount of U6 RNA and exhibits reduced levels of the U4-U6 RNA complex required for spliceosome assembly. The locations of lethal single and double point mutations leads us to propose that two domains of TFIIIC contact overlapping sites on the B block element.

Identification of a Satellite Double-Stranded RNA in the Parasitic Protozoan Trichomonas vaginalis Infected with T. vaginalis Virus T1

Co-infection by a 0.5-kb small double-stranded (ds) RNA together with Trichomonas vaginalis virus (TVV) genomic 4.6-kb dsRNA is commonly observed in a number of T. vaginalis isolates. By molecular cloning and primer extension experiments, the 497-bp cDNA sequence of a 0.5-kb dsRNA co-infecting with TVV-T1 in T. vaginalis T1 isolate was elucidated. Consistent with the replication cycle of a typical dsRNA virus, a plus-strand viral RNA beginning at +1 of the 0.5-kb dsRNA was identified in infected T. vaginalis T1 cells by primer extension and Northern hybridization studies. The 0.5-kb dsRNA was separately encased in TVV capsits from the viral genomic dsRNA, as shown by protein analysis and electron microscopic examination of viral particles purified by multiple rounds of CsCl gradient centrifugation. The riboprobes transcribed from a cloned cDNA of the 0.5-kb dsRNA exhibited strong hybridization to a small dsRNA in a T. vaginalis T9 isolate, which harbors a TVV-T9 distantly related to TVV-T1, but the same probes showed very little hybridization to the viral genomic dsRNA of both TVV-T1 and TVV-T9. Very little sequence homology between the 0.5-kb dsRNA and the 4.6-kb dsRNA in TVV-T1 was found by computer-assisted analysis, suggesting that the small dsRNA in T. vaginalis T1 is not derived from the genome of TVV-T1 or other distantly related T. vaginalis viruses. These results suggest that the small dsRNAs in T. vaginalis are satellite RNAs of T. vaginalis virus.

Purification of infectious pancreatic necrosis virus by anion exchange chromatography increases the specific infectivity

An improved method for the isolation and purification of infectious pancreatic necrosis virus (IPNV) is described. Virions released into the clarified growth medium are adsorbed to an anion exchange resin of diethylaminoethyl cellulose at pH 8.1. IPNV together with the likewise released and accumulated excess pool of the precursor to the major capsid protein, ICP62, are eluted at a salt concentration between 100 and 125 mM NaCl. The bovine serum albumin content of the growth medium supplement also elutes close to this position. Upon one step of combined sucrose- and CsCl-gradient centrifugation the recovered viruses display lower levels of aggregation, higher specific nucleic acid contents and an approximately 350% higher specific infectivity as compared with pools of viruses processed in parallel and isolated according to the established method relying on precipitation with poly(ethylene glycol).

Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell.

Hepatitis B virus (HBV) nucleoprotein complexes were isolated from nuclei of the human hepatoblastoma cell line HepG2.2.15. Under conditions of physiological ionic strength, the complexes sedimented at a rate corresponding to about 82 S. They contained viral DNA, histone, and nonhistone proteins. For DNA a circular, covalently closed structure was shown both by CsCl gradient centrifugation and electron microscopy. Spread preparations revealed the typical "beads-on-a-string" appearance of nucleosomally organized DNA. The average number of nucleosomes was 18, resulting in a biochemical repeat unit of HBV chromatin of approximately 180 base pairs of DNA. This value was confirmed by experiments analyzing the structure of the HBV chromatin by the use of micrococcal nuclease. Electron microscopy demonstrated that exposure to high ionic strength conditions resulted in removal of nucleosomes from the complexes, but also revealed proteinaceous structures remaining bound to viral DNA molecules. The nature of these residual proteins is discussed. Since native nucleoprotein complexes could be precipitated with HBV-core antibodies, core protein appeared to be one of the nonhistone proteins.