Cesium Chloride Gradient Research Articles

B-B′ proteins from small nuclear ribonucleoproteins have an endoribonuclease catalytic domain inactive in native particles

Native small nuclear ribonucleoproteins (snRNPs) purified by several conventional procedures or reconstituted in vitro have no ribonuclease activity. However, when these same snRNPs are centrifuged in cesium chloride gradients at low [Mg 2+] and in the presence of sarkosyl, an endoribonuclease is unmasked at the density of core particles (i.e. containing only the set of low molecular weight proteins common to all snRNPs), while an inhibitory component is released in soluble form. The nature of this inhibitor was not further investigated and the molecular events underlying this inhibition/activation process remained only a matter of speculation. On the other hand, evidence was obtained that the nuclease activity is carried by B-B′ on the basis of its comigration with B-B′ as well as with two of their cleavage products after SDS/polyacrylamide gel electrophoresis of snRNP proteins. One was identified by a B-B′-specific monoclonal antibody. Another one, especially prominent and migrating between D and E core proteins, was identified as the N-terminal half of B-B′ by microsequence analysis. Although tightly associated with core snRNPs, the activity is not dependent upon the presence of an snRNA. For the time being, the functional significance of this nuclease remains entirely elusive.

Transmissible mupirocin resistance inStaphylococcus aureus

The spread of two strains of Staphylococcus aureus with high level resistance to mupirocin is described. The resistance proved to be easily transferred to other S. aureus strains by filter mating experiments and on the skin of mice. No plasmid band corresponding to the resistance could be demonstrated by agarose gel electrophoresis or by caesium chloride gradient centrifugation but cleavage of 'chromosomal' DNA from resistant recipients showed bright bands of DNA absent from sensitive controls.

Stable expression of hepatitis B virus genome in a primate kidney cell.

Transfection of Vero cells with cloned hepatitis B virus (HBV) DNA resulted in the secretion of hepatitis B surface protein (HBsAg) and core proteins (HBc/eAg). Syntheses of both viral antigens in the transformed Vero cells continued for at least 50 days after cultivation. HBsAg particles were composed of many spherical and some filamentous particles containing both pre-S1 and pre-S2 domains. Restriction analysis of the transfected cellular DNA showed that one copy of HBV DNA was integrated in the cells. Some species of HBV-specific RNA were detected by Northern blot, and among these a major transcript was found to migrate at 2.1 kb by S1-mapping analysis. Viral core proteins present in the culture medium were fractionated and characterized by cesium chloride gradient and found to form particles.

DNA repair replication by soluble extracts from human lymphoid cell lines.

A system is described in which extracts from human cells can perform repair replication on DNA damaged by ultraviolet light or chemical carcinogens. Whole cell extracts from lymphoid cell lines are incubated with damaged plasmid DNA circles at 30 degrees C in the presence of ATP and the four deoxynucleoside triphosphates. Repair synthesis is monitored by the incorporation of alpha-32P-dATP into closed circular plasmid molecules. Analysis of the time course of the reaction suggests that the slowest step in repair is incision, rather than polymerization or ligation. The size of repair patches inserted into ultraviolet-irradiated DNA during a reaction was estimated by substitution of thymidine triphosphate with 5-bromodeoxyuridine triphosphate and sedimentation in alkaline cesium chloride gradients. Patches with heterogeneous sizes of less than 120 bases were observed.

Purification of DNA from the intracellular symbiotes of Sitophilus oryzae and Sitophilus zeamais (Coleoptera: Curculionidae)

DNAs from the endosymbiotes of two species of grain weevils, Sitophilus oryzae and S. zeamais, were separated from host-weevil DNA and purified by cesium chloride gradient centrifugation. Endosymbiote DNA was identified by hybridization with a probe of the gene encoding the 16S rRNA of Escherichia coli. Estimations of the CsCl buoyant densities of symbiote DNAs of S. oryzae and S. zeamais were similar ( ϱ = 1.712 and 1.714, respectively) corresponding to a mole per cent guanine + cytosine (GC%) of ≈52–55%. The GC%s of host-weevil DNAs were ≈33%.

Virus-ribosome complexes from cell-free translation systems supplemented with cowpea chlorotic mottle virus particles

When particles of cowpea chlorotic mottle virus (CCMV) were added to cell-free extracts from wheat germ, the encapsidated viral genome was translated into polypeptides similar to the translation products specified by unencapsidated viral RNA (as shown before by M. J. Brisco, R. Hull, and T. M. A. Wilson, 1986, Virology 148, 210–217). The rate of protein synthesis observed upon addition of virus particles was much slower than that of extracted RNA and the quantity of protein formed was only 10% of that of extracted RNA. Using sucrose and cesium-chloride gradient analysis, virus-ribosome complexes, containing up to four ribosomes per virus particle, were isolated from translation mixtures supplemented with CCMV particles. These complexes, with densities intermediate of those of virus (1.36 g cm −3) and ribosomes (1.58 g cm −3), were analyzed and quantified in the electron microscope. Less than 5% of the particles was found in association with ribosomes. To verify whether these complexes were involved in the process of cotranslational disassembly, tobacco mosaic virus was analyzed with the same techniques and methods. The results found for TMV were similar to those found for CCMV except that virus-ribosome complexes with up to 20 ribosomes per virus particle were observed. The implications of the process of virion-directed translation for the structure of the particle as well as the role of this process in vivo are discussed.

Synthesis and secretion of rat pancreatic proteins by Xenopus laevis oocytes.

An in vivo translation system, the Xenopus laevis oocyte, was employed to study the synthesis and secretion of pancreatic proteins. RNA was purified from normal and diabetic rat pancreas and normal rat liver by use of guanidine isothiocyanate lysis and cesium chloride gradient centrifugation. The presence of functional mRNA was documented by translation in a reticulocyte lysate that yielded precursors of all major secretory proteins, i.e., slightly higher Mr than proteins synthesized in situ by pancreatic acini. Mature X. laevis oocytes were then microinjected with either total RNA or purified mRNA. When oocytes were subsequently incubated with 35S-methionine, pancreatic secretory proteins or hepatic albumin could be immunoprecipitated from oocyte lysate with specific polyclonal antibodies against amylase, trypsin, ribonuclease, and albumin. Amylase was shown to be enzymatically active. Moreover, oocytes released pancreatic secretory proteins into the medium when injected with pancreatic RNA in a time-dependent manner. Only the mature form of amylase was secreted and secretion was not regulated by secretagogues. When a comparison was made after injection of RNA from diabetic pancreas known to contain altered amounts of individual mRNAs, there was a decrease in amylase and an increase in trypsinogen synthesis in oocytes that was comparable to the results of cell free translation. The oocyte expression system, therefore, should be useful not only for studies of protein synthesis but also for processing and secretion.

Association of mitochondria DNA with viral DNA in purified preparations of poxviruses

The presence of cellular material in purified preparations of Shope fibroma virus (SFV) and two orthopoxviruses (vaccinia and Indiana), was investigated. Mitochondria were observed in purified preparations of SFV by electron microscopy and mitochondrial (mt) DNA was identified in restricted viral DNA by Southern blot hybridization with cloned mouse mt DNA. Mitochondrial DNA was also detected in vaccinia and SFV DNAs extracted from purified virions treated with DNase I followed by core isolation. The viral and mt DNAs could be separated on the basis of their size by agarose gel electrophoresis, but not by their buoyant density by centrifugation in cesium chloride gradients. These findings led us to re-examine previously reported results showing some homology between SFV, a leporipoxvirus and Indiana, an Orthopoxvirus (Berkowitz and Pogo, Virology 142, 437–440, 1985) using cloned fragments of SFV DNA instead of the entire viral DNA. The results indicated that cross-hybridization between SFV and Indiana DNAs was due in part to mt DNA but they also revealed an unrecognized region of homology between the two poxvirus genera.

A rapid procedure for isolating mitochondrial DNA

A technique for the rapid isolation of mitochondrial DNA (mtDNA) from animal tissues is described that eliminates the time-consuming separation of nuclear and mtDNAs using cesium chloride gradient ultracentrifugation. The procedure utilizes digestion of the nuclear DNA with DNase, after which lysis of mitochondria and subsequent extraction of proteins results in relatively pure mtDNA. Up to 5 μg of mtDNA per gram of liver tissue resulted, a suitable yield for five digests with restriction enzymes and staining with ethidium bromide.

Characterization of DNA from the Dinoflagellate Woloszynskia bostoniensis1

ABSTRACTSome physical and chemical properties of DNA isolated from the dinoflagellate Woloszynskia bostoniensis were determined. Analytical cesium chloride gradient centrifugation gave a major component and a minor component banding at 1.719 and 1.693 g/cm, respectively. Thermal denaturation in 0.1 SSC showed a broad transition with a Tm of 70.5° C. Derivation of this curve indicated that two components were present having Tm values of 66° C and 70° C. Base composition analysis showed a GC content of 48.1% and a high degree of thymine replacement by 5‐hydroxymethyluracil. Two minor bases, identified as 5‐methylcytosine and N6‐methyladenine, were also detected. Reassociation kinetics showed a typical eukaryotic reassociation pattern with 45% repetitive and 55% single copy sequences.

Small-scale purification of bacteriophage λ DNA by an airfuge centrifugation step in cesium chloride gradients

A rapid and efficient procedure for purifying bacteriophage λ DNA is described. This small-scale purification involves isolation of bacteriophage particles on cesium chloride gradients. Using an Airfuge ultracentrifuge, the centrifugation step can be readily achieved in 90 minutes. The method allows a 1-day purification of up to 12 independent λ DNA (20–40 μg each). The recovered DNA, essentially devoid of RNA and DNA contaminants, is efficiently cut by restriction endonucleases and can serve as starting material for the ligation of DNA fragments in other cloning vehicles.

Purification and characterization of bovine herpesvirus-1 isolates and virus DNA utilizing bovine embryonic lung cells

Techniques are described for the culture of bovine embryonic lung (BEL) cells by the primary explant technique and mild enzymatic disaggregation of lung tissue. Preparations of bovine herpesvirus-1 (BHV-1) isolates with titers of 109 plaque-forming units/ml are purified by sucrose and cesium chloride gradient centrifugation after replication in primary cultures of BEL cells. BHV-1 isolated by velocity sedimentation bands in 35% sucrose and sediments at a density of 1.23 g/cc in cesium chloride density gradients. The DNAs of all virus isolates have a velocity sedimentation coefficient of approximately 54S. Virus DNAs from all isolates sediment to equilibrium in cesium chloride at 1.730 g/cc. Differences between virus isolates can be demonstrated by digestion of virus DNA with the restriction endonucleaseKpnI and by polyacrylamide gel electrophoresis of virion polypeptides. These techniques are useful for production of purified high titer virus free of host cell contaminants.

Interaction of Hoechst 33258 with Repeating Synthetic DNA Polymers and Natural DNA

Fluorescence, circular dichroism and sedimentation through cesium chloride gradient techniques were performed to study the physical properties of the binding of the bisbenzimidazole dye Hoechst 33258 (H33258) to natural DNAs and synthetic polynucleotides of defined repeating units. These studies show that Hoechst 33258 exhibits at least two modes of interaction with duplex DNA: (1) a strong base pair specific mode which requires at least 4 consecutive AT base pairs and (2) a weaker mode of binding which is significantly reduced in the presence of high salt (0.4 M NaCl) and exhibits no apparent base specificity. The H33258 binding was found to be sensitive to the substitutions in the minor groove elements of a series of synthetic polynucleotides supporting the model of H33258 binding in the minor groove of the DNA with AT rich sequences. Similar mode of binding was predicted in natural DNAs by methylation of dye-DNA complexes. Footprint analysis of the complex of dye to a pBR322 fragment also supports that a minimum of 4 consecutive AT base pairs are required for H33258 binding to DNA.

Characterization of the effect of aluminum metal on poliovirus

Metallic aluminum has been studied for possible use in conjunction with or as an alternative to conventional soluble disinfectants as applied to drinking and wastewater. Acid-washed aluminum was incubated with [35S]methionine-labeled poliovirus type 1 (LSc) and the counts per min (cpm)/plaque-forming units (pfu) ratio was determined. After 2 h, only 0.013% of the cpm remained in solution, indicating viral adsorption onto the surface of the aluminum. After 76 h, 93% of the cpm returned to solution, while infectivity dropped from 2.2×107 pfu/ml to undetectable levels. This suggests that infectious viruses were adsorbed onto the aluminum surface and released from the surface of the aluminum as non-infectious particles. Analysis by electron microscopy, cesium chloride gradient and polyacrylamide gel electrophoresis indicates that either dissociation or destruction of the viral capsid proteins occurs during incubation with aluminum, which results in viral inactivation.

Distribution in cesium chloride gradients of proteoglycans of chick embryo brain and characterization of a large aggregating proteoglycan

Proteoglycans were extracted from 14-day chick embryo brains, which had been labelled in vitro with [ 35S]sulfate or 3H-labelled amino acids. 4.0 M guanidinium chloride (containing proteinase inhibitors) extracted 94% of the 35S-labelled glycoconjugates. Following cesium chloride equilibrium centrifugation, the proteoglycans in each fraction were characterized by chromatography on Sepharose CL-2B. The most dense fraction (D1), which contained no detectable non-proteoglycan proteins, contained a large, aggregating chondroitin sulfate proteoglycan in addition to small chondroitin sulfate and heparan sulfate proteoglycans. The less dense fractions (D2–D6) contained both small chondroitin sulfate and heparan sulfate proteoglycans. Removal of hyaluronate from the D1 sample by digestion with Streptomyces hyaluronidase in the presence of proteinase inhibitors showed that aggregation of the large chondroitin sulfate proteoglycan is hyaluronate-dependent. Aggregation was restored by re-addition of hyaluronate. Reduction and alkylation, which blocked aggregation of a cartilage A1 proteoglycan, did not interfere with aggregation of the large brain proteoglycan.

An electron microscopical investigation of faecal small round viruses.

A retrospective study of small round featureless viruses (SRVs) initially identified by negative-staining electron microscopy of stool samples was performed. A variety of technique, including immunoelectron microscopy and caesium chloride gradient centrifugation, was applied in an attempt to classify further these viruses. Over a four-year period, 64 SRV-positive samples were reported (1.8% of the stool samples sent for electron microscopy and 6.2% of the total number of positive samples), of which 53 were available for further study. A significant degree of misclassification was found. Viruses previously identified as SRVs were shown to be astrovirus (n = 14), calicivirus (n = 2), and "Norwalk-like" virus (n = 1). The majority of the 36 remaining samples were identified as parvovirus-like (n = 27) (75%), 14 of which were associated with the presence of adenovirus particles. Enteroviruses (n = 3) and hepatitis A virus (n = 1) were infrequently detected. The remaining viruses (n = 5) could not be adequately classified. Parvovirus may be the predominant SRV associated with acute diarrhoeal disease in childhood.

Timing of proto-oncogene replication: a possible determinant of early S phase sensitivity of C3H 10T1/2 cells to transformation by chemical carcinogens.

The temporal order of replication of several genes was studied in 10T1/2 cells synchronized by release from confluence-induced arrest of proliferation followed by treatment with 2 micrograms/mL aphidicolin for 24 h. DNA subjected to bromodeoxyuridine substitution for 1- or 2-h intervals spanning the S phase was separated from the remaining DNA in cesium chloride gradients, filtered onto nitrocellulose in a slot-blot apparatus, and hybridized with various 32P-labeled probes. Ha-ras was among the first genes replicated at the onset of the S phase. The myc proto-oncogene replicated later but within the first hour of the S phase. The replication of Ki-ras, raf, and mos was detected between hour 1 and 2 of the S phase. The dihydrofolate reductase gene replicated early (0-2 h) and the myb proto-oncogene replicated in mid-S phase (2-4 h). An immunoglobulin VH sequence and the beta-globin gene replicated late in 10T1/2 cells, 4-6 h after removal of aphidicolin. Replicating DNA is preferentially adducted by chemical carcinogens, and replication of damaged proto-oncogenes before they are repaired may activate their transforming potential. Therefore, the observed replication of proto-oncogenes during the early S phase may underlie the enhanced sensitivity of 10T1/2 cells to chemically induced transformation at this point in the cell cycle.

Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica.

Establishment of adherence by Entamoeba histolytica is mediated by a 170-kD Gal/GalNAc inhibitable lectin and is required for cytolysis and phagocytosis of mammalian target cells. We studied the biochemical mechanisms of the in vitro interaction between rat and human colonic mucins and axenic E. histolytica trophozoites. Crude mucus prevented amebic adherence to Chinese hamster ovary (CHO) cells by up to 70%. Purification of the colonic mucins by Sepharose 4B chromatography, nuclease digestion, and cesium chloride gradient centrifugation resulted in a 1,000-fold enrichment of the inhibitory mucins. Purified rat mucin inhibited amebic adherence to and cytolysis of homologous rat colonic epithelial cells. Oxidation and enzymatic cleavage of rat mucin Gal and GalNAc residues completely abrogated mucin inhibition of amebic adherence. The binding of rat 125I-mucin to amebae was galactose specific, saturable, reversible, and pH dependent. A monoclonal antibody specific for the 170-kD amebic Gal/GalNAc lectin completely inhibited the binding of rat 125I-mucin. Rat mucin bound to Affigel affinity purified the amebic lectin from conditioned medium. Colonic mucin glycoproteins act as an important host defense by binding to the parasite's adherence lectin, thus preventing amebic attachment to and cytolysis of host epithelial cells.

Heparan sulfate proteoglycan synthesis and metabolism by mouse uterine epithelial cells cultured in vitro.

Characterization and metabolism of heparan sulfate glycosaminoglycans and proteoglycans (HSPGs) synthesized by primary cultures of mouse uterine epithelial cells are reported. HSPGs were detected in both the medium and in the cell-associated fraction, whereas glycosaminoglycans containing little or no protein (free glycosaminoglycans) were found primarily in the cell-associated fraction. The cell-associated HSPGs were relatively large (Kav = 0.1 on Superose 12), had a buoyant density in cesium chloride gradients of 1.45-1.55 g/ml, and contained heparan sulfate chains that fell into two size classes, exhibiting Kav values on Superose 12 of 0.2-0.5 and 0.7-0.8, respectively. The free glycosaminoglycan chains displayed a Kav on Superose 12 of 0.6-0.7. The secreted HSPGs were smaller (median Kav on Superose 12 of 0.28) than the cell-associated HSPGs. More than 90% of the cell-associated HSPGs contained hydrophobic portions, as evidenced by their ability to bind to octyl-Sepharose. In contrast, only 10-15% of the secreted HSPGs bound to octyl-Sepharose. HSPGs were detected at both apical and basal cell surfaces/extracellular matrices by indirect immunofluorescence in vitro and in utero and by accessibility to external proteases in vitro. It was estimated that 60-70% of the total cell-associated HSPGs were exposed at the cell surface. The HSPGs released from the cell surface by proteases were slightly smaller than the intact HSPGs and lacked the hydrophobic properties of the latter. These observations suggested that the cell surface HSPGs contain a small, hydrophobic domain that functions in the attachment of HSPGs to cells. The free glycosaminoglycans appeared to be primarily intracellular and were not secreted. The cell-associated HSPGs turned over rapidly (t1/2 = 1.5 h) and appeared to be the precursors to the free glycosaminoglycans. Metabolic turnover of the free glycosaminoglycan pool was a relatively slow process (t1/2 = 10-12 h).(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid isolation of animal mitochondrial DNA in a small fixed-angle rotor at ultrahigh speed.

In recent years, mitochondrial DNA (mtDNA) sequence analysis has become a powerful tool for the investigation of the evolutionary and population biology of various animal species (Wilson et al., 1985). Most such studies have relied on physical purification of mtDNA, typically by ultracentrifugation in cesium chloride gradients. Thorough descriptions of such methods are given by Lansman et al. (1981) and Wright et al. (1983). Practical concerns over the expensive instrumentation and extensive time required by such methods have been expressed by several workers (e.g., Grill et al., 1983; Powell and Zufiiga, 1983). Several of our colleagues have expressed doubt that purified mtDNA samples can be generated in a reasonable time in numbers sufficient to perform large-scale population studies. We describe here an ultracentrifugal procedure that yields homogeneous mitochondrial DNA from as many as i0 samples in as little as 10 hr. The procedure uses a relatively inexpensive tabletop ultracentrifuge and relies on a self-forming cesium chloride gradient generated in a small fixed-angle rotor that operates at a speed of 100,000 rpm and a relative centrifugal force of 436,000g.