Salt Wash Research Articles

Heterogeneity in the ?-subunit of translational initiation factor eIF-2 during brain development

We have detected by immunoblotting analysis of crude fractions from suckling and adult rat brain, resolved by two-dimensional isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis, the presence of two different forms of the beta subunit of polypeptide initiation factor 2 (eIF-2). These two forms differ in their apparent molecular weights and also in their isoelectric point values. Quantitation of both forms in the crude fractions shows that, the most basic form beta 1 (pI: 6.1, 52 kDa), is present in higher levels of the salt wash ribosomal fractions obtained from both, suckling and adult animals, than in the postmicrosomal fraction corresponding to the same animals. The most acidic form, beta 2 (pI: 5.9, 50 kDa), is present in the highest level in the postmicrosomal supernatant from adult animals. A close parallelism is found between beta 1 levels and eIF-2 activity.

Poliovirus translation initiation: Differential effects of directed and selected mutations in the 5′ noncoding region of viral RNAs

We have analyzed the translational defects of a number of mutations in the 5′ noncoding region of poliovirus type 1 RNA. These mutations fall into three categories: (1) two mutations which resulted in temperature sensitive (ts) viruses, (2) the second-site mutations responsible for the reversion of the two is viruses, and (3) mutations which were lethal to virus production. RNAs containing either of the is mutations translated in vitro at levels significantly lower than wild-type levels. RNAs containing the respective second-site reversions had corrected these translational defects to levels corresponding to their viral growth potentials. Unlike in vitro translation of wild-type poliovirus RNA, translation of the RNAs which gave rise to is mutant viruses was not stimulated by the addition of an S10 fraction from an uninfected HeLa cell extract to a rabbit reticulocyte lysate (RRL). In vitro translation of the mutant RNAs (corresponding to the is viruses) in a RRL was stimulated by factors present in a ribosomal salt wash (RSW) from a HeLa extract, although the levels of stimulation were only half those seen for wild-type. These results suggest that the stimulatory factors present in the RSW have a decreased affinity for the mutant RNA templates but can, to some extent interact, with such RNAs if provided in high enough concentration. The in vitro translation of RNAs containing either of the lethal mutations was not stimulated by factors present in the S10 or the RSW. Taken together, our data suggest a correlation between the ability of a genetically altered RNA to respond to translation stimulatory factors in vitro and the ability of that mutation to be recovered in infectious virus. In addition, we have identified the in vivo-selected reversion of translational defects for two different is viruses.

Bovine mitochondrial ribosomes possess a high affinity binding site for guanine nucleotides

Mammalian mitochondrial ribosomes possess a binding site for guanine nucleotides. GTP binds in unit stoichiometry and with high affinity (Kd = 15.3 +/- 2.8 nM) to the small subunit of bovine mitochondrial ribosomes. This binding activity survives high salt washes, indicating that the nucleotide binds to an integral site within this subunit. GDP also binds to the small subunit with high affinity (Kd = 17 +/- 5.8 nm) and in unit stoichiometry. The GTP binding activity can be competed with GDP but not appreciably by other nucleotides, indicating that both GTP and GDP bind specifically and to the same site. The non-hydrolyzable analogs of GTP, guanylyl-5'-imidophosphate, and guanylyl-(beta,gamma-methylene)- diphosphonate also bind to the small subunit, but with reduced affinity. These results indicate that mammalian mitochondrial ribosomes, unlike other ribosomes, are able to interact directly with guanosine triphosphate, suggesting that the bound GTP may be involved in a novel regulatory mechanism in mitochondrial protein synthesis.

Extrinsic polypeptides of spinach photosystem I

By combining Triton X-114 partitioning with alkaline-salt and chaotropic washings of thylakoid membrane vesicles and photosystem I particles, we have studied the protein subunit composition and organization of spinach photosystem I. Upon fractionation of photosystem I particles with Triton X-114, 6 polypeptides of 5.0, 8.2 (psaE), 10.5, 16.6 (psaG), 19.3 and 22.1 kDa (psaD) were considered to be extrinsic membrane proteins. By combining this partitioning with salt washes of thylakoid membranes, the polypeptides of 8.2, 11.6 (psaH), 19.3 and 22.1 kDa were directly shown to be stromally oriented and extrinsic while no extrinsic subunits were identified at the inner thylakoid surface. The 5.0, 8.2, 10.5, 17.2, 19.3 and 22.1 kDa polypeptides appear to have regulatory rather than catalytic functions as their release from photosystem I particles upon high salt-alkali treatment does not affect photosystem I-mediated electron transport.

Purification of E. coli 30S ribosomal proteins by high-performance liquid chromatography under non-denaturing conditions

High-performance ion-exchange chromatography was applied to the separation of proteins from the 30S ribosomal subunit under non-denaturing conditions. It was shown that a single chromatographic step only allows the purification of nine proteins. To increase the number of separated proteins, a prefractionation step was added that depends on the physical characteristics of the proteins to be purified. Sixteen out of 21 proteins could be purified by using prefractionation (gel permeation and lithium chloride salt washing). This method is well suited to preparing fresh samples on demand for optical studies owing to the simplicity of the buffers used and the amounts of proteins recovered in the eluted peaks (0.05–0.1 mg/ml).

Polyadenylated RNA sequences from vaccinia virus-infected cells selectively inhibit translation in a cell-free system: Structural properties and mechanism of inhibition

The mechanism of vaccinia virus-induced selective inhibition of host cell protein synthesis was studied in a nonpermissive (Chinese hamster ovary, CHO) and in a permissive mouse cell line (L cells). Small polyadenylated RNAs obtained from uninfected and infected cells were fractionated into six size classes by polyacrylamide gel electrophoresis. The RNAs from the first two largest fractions (>500 nucleotides, nt) were translated into some low-molecular-weight polypeptides, whereas, the RNAs from the remaining fractions (400–500, 300–400, 200–300, and 100–200 nt) had no translational activity in reticulocyte lysates. When these nontranslating polyadenylated short sequences (POLADS) were added to the cell-free system together with HeLa cell mRNAs, translation was inhibited from 70%, by the 400- to 500-nt fraction, to about 20%, by the 100- to 200-nt fraction. The degree of inhibition of protein synthesis was clearly dependent on the size of POLADS. The translation of vaccinia virus mRNAs in the cell-free system was inhibited by about 25% with the 400- to 500-nt fraction, by 5% with the 300- to 400-nt fraction, while the smaller size POLADS had no inhibitory effect. The inhibition of HeLa cell and vaccinia virus mRNA translation by POLADS was reversed by the simultaneous addition of oligo(dT) to the cell-free system. POLADS were also obtained from uninfected cells, but they inhibited the translation of HeLa cell and vaccinia virus mRNAs to a much lesser extent. The removal of the poly(A) moiety from POLADS by treatment with ribonuclease H and oligo(dT) abolished their inhibitory effect on HeLa cell mRNA translation. The average length of the poly(A) tails of POLADS obtained from infected cells was longer than that of POLADS from normal cells. Inhibition of HeLa cell mRNA translation mediated by POLADS in the cell-free system was reversed (∼70%) by addition of crude initiation factors (ribosomal salt wash, RSW). Significantly, inhibition of translation by POLADS was reversed (>90%) by addition of purified poly(A) binding protein (PAB). Purified initiation factor 4A (elF-4A) also reversed this inhibition, but to a lesser extent than RSW and PAB. Our results show that the translation of vaccinia virus mRNAs is resistant to POLADS, suggesting that POLADS, by virtue of their long poly(A) tails, may sequester PAB and thus, play a role in selective inhibition.

Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity.

Coated pits will assemble onto purified plasma membranes that are attached to a poly-L-lysine coated substratum (Moore, M. S., Mahaffey, D. T., Brodsky, F. M., and Anderson, R. G. W. (1987) Science 236, 558-563; Mahaffey, D. T., Moore, M. S., Brodsky, F. M., and Anderson, R. G. W. (1989) J. Cell Biol. 108, 1615-1624). To better understand the assembly reaction, we have purified both clathrin triskelion and AP-2 subunits from bovine brain and assayed for their ability to bind to the cytoplasmic surface of attached membranes. Two types of membranes were analyzed: those washed with a high pH buffer that selectively removes triskelions and those washed with a high salt buffer that removes both the AP-2 and the triskelion subunits. We found that purified AP-2 subunits bind with high affinity (Kd approximately 3 x 10(-8) M) to salt stripped membranes. Binding is saturable and abolished by treating membranes with less than 20 micrograms/ml of elastase. When membranes were treated with elastase before the salt wash and then salt washed and assayed for AP-2 binding, normal binding was seen, which indicates that the presence of clathrin-coated pits protects the binding site from the protease. Membranes that had rebound AP-2 did not bind purified triskelions, even though high pH buffer-washed membranes that bear endogenous AP-2 bound triskelions with high affinity (Kd approximately 3 x 10(-9) M) and supported lattice assembly. We conclude that coated pit assembly is initiated by the binding of AP-2 to an integral membrane protein but that the AP-2 complex must be activated by an unknown process before the coated pit lattice will assemble.

Inhibition of the initiation of hepatic protein synthesis during ethionine mediated ATP depletion in vivo: Modification to ribosomal subunits, evidence of impaired ternary complex formation and a subcellular redistribution of eIF-2 alpha

Acute ethionine intoxication is known to induce a reversible hepatic injury in female rats by reducing the level of hepatic ATP. The injury indirectly impairs the initiation of hepatic protein synthesis, with resultant polysome disaggregation. Administration of adenine rapidly restores the ATP levels and protein synthesis. Analysis of liver polysome and ribosomal subunits reveals that polysome disaggregation occurs following 3 h of the intoxication, and reaggregation occurs following the administration of adenine. Inactive hepatic ribosomes accumulate as monomers and disomes when analysed by sucrose gradient sedimentation in low-salt buffers. High-salt buffers dissociate the inactive ribosomes into the component 40 S and 60 S subunits. The level of higher density, 1.48 g/cc, 40 S subunit increases during the inhibition of protein synthesis, while the lower density, 1.41 g/cc, 40 S subunit species does not change significantly. Hepatic micosomal and cytosolic extracts examined for their ability to support the formation of the ternary complex of eIF-2-GTP and [ 35S]Met-tRNA i demonstrate that during acute ethionine intoxication, ternary complex formation in the two extracts decrease 65% and 85%, respectively. These changes are coincident with polysome disaggregation. Administration of adenine to reverse the intoxication restores the ternary complex forming ability of the cytosolic extract, but does not affect the activity of the microsomal salt wash extracts. Mixing experiments indicate the accumulation of an inhibitor of ternary complex formation in the microsomal salt wash fraction. The application of quantitative western blotting demonstrates that the level of antigenic eIF-2 alpha in the microsomal salt wash extract increases 31% during the inhibition. These observations are consistent with the idea that the inhibition of the initiation of hepatic protein synthesis induced by ethionine is mediated by eIF-2 alpha phosphorylation. The latter results in an inhibition of ternary complex formation, redistribution of eIF-2 to the microsome fraction, polysomal disaggregation, and accumulation of inactive ribosomal subunits.

A fractionated reticulocyte lysate retains high efficiency for protein synthesis

The rabbit reticulocyte lysate is a highly efficient system for protein synthesis in vitro, and therefore has been used frequently in studies of translational control. However, when the lysate is fractionated into ribosomes and soluble proteins by centrifugation, there is a severe loss of activity, thereby making the system less suitable for identifying components involved in translational control. We have devised a new method of cell fractionation which employs rapid (20-min) centrifugation to pellet ribosomes. The post-ribosomal supernatant (S-100), high salt-washed ribosomes, and the high salt wash are all required in the reconstituted protein synthesis assay, and greater than 70% of the activity of the unfractionated lysate is attained. Proteins in the high salt wash have been further fractionated by ammonium sulfate precipitation, and the assay system has been used to measure the activities of eIF-4B and eIF-4F. This highly active fractionated lysate system should be useful in measuring the specific activities of translational components and may be appropriate for detecting changes due to covalent modifications such as phosphorylation.

Effects of salt wash on the structure of the prolamellar body membrane and the membrane binding of NADPH-protochlorophyllide oxidoreductase

Effects of salt wash on the structure of the prolamellar body membrane and the membrane binding of NADPH-protochlorophyllide oxidoreductase

Radiation-Damage Rims in Quartz from Uranium-Bearing Sandstones

ABSTRACT Using cathodoluminescence (CL) mode of a scanning electron microscope (SEM), radiation damage in quartz is made visible in uranium-vanadium-bearing sandstones located in the Salt Wash Member of the Morrison Formation (Jurassic) from the Colorado Plateau (U.S.A.). Detrital quartz grains surrounded by uranium minerals display radiation-damage rims. Three concentric rims with thicknesses of 37 µm, 24 µm and 16 µm have been observed. The rims are similar to halos surrounding uranium- and thorium-rich accessory minerals observed by Owen (1988). They are due to alpha particles, which create damage when penetrating a medium. The width of the rims agrees well with the Bragg-Kleeman rule and corresponds to the most energetic alpha particles in the 238U decay series Sandstones entirely cemented by vanadium clays also exhibit radioactive rims, which demonstrates that uranium has been removed during diagenesis. These results have important implications for the uranium ore genesis in the Salt Wash sandstones, because they imply an ancient episode of uranium leaching prior to recent weathering. Therefore, cathodoluminescence in sandstones may be useful in recognizing ancient zones or episodes of accumulation and leaching of alpha-emitting nuclides from the uranium series.

4847199 Agglutination immunoassay and kit for determination of a multivalent immune species using a buffered salt wash solution: Brian Snyder, Robert Belly assigned to Eastman Kodak Company

4847199 Agglutination immunoassay and kit for determination of a multivalent immune species using a buffered salt wash solution: Brian Snyder, Robert Belly assigned to Eastman Kodak Company

Authigenic Barite as an Indicator of Fluid Movement through Sandstones Within the Colorado Plateau

ABSTRACT Sandstones of the Salt Wash Member, Late Jurassic Morrison Formation, contain abundant authigenic barite on the east side of the northern Colorado Plateau (mean = 0.5 g barite/kg rock) and systematically less barite (mean = 0.02 g barite/kg rock) in the western part. The area containing abundant barite coincides approximately with the extent of bedded evaporites in the underlying Pennsylvanian Hermosa Formation. A genetic link between the barite and solutes from the Hermosa Formation is supported by sulfur isotope data. Most barites from the abundant barite zone have 34S values that range from +8 to +14 per mil, which is similar to the range of bedded gypsum and anhydrite in the Hermosa Formation. Sulfate derived from t e evaporites probably entered the Morrison Formation in solutions that moved along faults. Measured 87Sr/86Sr ratios of barite in the abundant barite zone are indicative of fault-controlled solution flow. Strontium contents and 87Sr/86Sr ratios (0.7103 to 0.7084) in barite decrease with increasing distance from faults. These trends reflect mixing of radiogenic strontium in the saline waters that ascended faults (87Sr/86Sr = 0.7100), with strontium originally contained in early diagenetic carbonate cements within the sandstones (87Sr/86Sr = 0.7080). Because measured 87Sr/86Sr values exceed the ratios in Hermosa anhydrite, it is concluded that radiogenic strontium was added to the rising brines by reaction between the evaporite-derived saline solutions and arkosic san stones along the flow path. Morrison sandstones within the abundant barite zone were altered by saline waters that added sulfate and strontium. Barite formation in this zone was limited by the availability of dispersed barium in the host rock. The zone of sparse barite on the western side of the Colorado Plateau was not affected by the same warm saline waters. Instead, intraformational redistribution of small amounts of barium, strontium, and sulfate formed the relatively sparse barite in this area.

Association of CD2 and T200 (CD45) in mouse T lymphocytes

A monoclonal antibody (mAb 12-15) reactive with the mouse CD2 was found to co-precipitate a high-molecular-weight glycoprotein from mouse thymocyte, splenic lymphocyte, Con A blast, and T cell tumor detergent lysates which was identified as the leukocyte common T 200 glycoprotein (CD45). The reactivity was specific for CD2 since antibodies to CD3 did not co-precipitate the T200 glycoprotein. mAb 12-15 did not react with immunoaffinity-purified T200 glycoprotein, ruling out the possibility that the antibody detected a cross-reactive epitope. Biochemical data indicated that the association of CD2 with T200 was not generated during lysis of the cell and that the molecular complex was non-covalently linked since it could be destroyed by high salt washing or boiling in SDS. Distribution analysis in Triton X114-H2O revealed that, in contrast to free T200 molecules, the complexed T200 was enriched in the detergents phase. To investigate the CD2-T200 association in more detail at the cell surface, modulation of CD2 and T200 was studied. Modulation could be induced on Con A blasts by monoclonal antibodies followed by cross-linking with a FITC-conjugated second antibody. Within 24 h the expression of CD2 or T200 was reduced to approximately 10-20% of the initial value on the majority of cells. However, two-color fluorescence showed that modulation of CD2 did not lead to co-modulation of CD3 or T200. A possible physiological role of CD2-T200 complexes is discussed.

A nuclear cap binding protein from HeLa cells

We have identified a cap binding protein in a HeLa nuclear extract using a gel mobility shift assay probed with capped RNA. Subcellular fractionation of HeLa cells revealed that the majority (about 70%) of the cap binding activity is present in the nuclear extract, about 20% is in the cytoplasmic S100 fraction, and almost none in the ribosome-high salt wash fraction, indicating that the protein in active form localizes mainly in the nuclei. Competition experiments with various cap analogues showed that the G(5')ppp(5')N-blocking structure as well as the methyl residue at the N7 position of the blocking guanosine is important for the binding of this protein, and that the trimethylguanosine cap structure which exists at the 5' termini of many snRNAs is not recognized by this protein. Immunoprecipitation experiments using various anti-snRNP antibodies suggested that this protein is partially associated with U2 snRNP. We purified this protein to near homogeneity from a HeLa nuclear extract by several chromatographic procedures including capped RNA-Sepharose chromatography. The purified protein shows molecular weight of 80 kilodaltons, as judged by SDS gel electrophoresis, and binds specifically to the cap structure.

Identification of a 90-kDa polypeptide which associates with adenovirus VA RNAI and is phosphorylated by the double-stranded RNA-dependent protein kinase

Interferon treatment of mammalian cells induces a double-stranded (ds) RNA-dependent protein kinase known as DAI. When activated, DAI phosphorylates the alpha-subunit of eukaryotic initiation factor eIF-2, impairing its ability to be recycled and leading to the inhibition of protein synthesis. We have identified a novel DAI substrate in the ribosomal salt wash of rabbit reticulocyte lysates. This substrate is a 90-kDa polypeptide which has been purified to apparent homogeneity. It can be cross-linked by ultraviolet irradiation to adenovirus VA RNAI, a small RNA polymerase III transcript RNA which acts as an inhibitor of DAI. As assayed by a nitrocellulose filter binding assay, the 90-kDa polypeptide is also able to associate with authentic double-stranded RNA, but not single-stranded RNA, made in vitro. Thus, this newly identified substrate of DAI appears to have affinity for dsRNA structures and may be involved in dsRNA-regulated processes in the reticulocyte. Polyclonal and monoclonal antibodies directed against the 90-kDa polypeptide co-precipitate DAI, suggesting that these two proteins may exist as a complex.

Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: evidence for phosphorylation

Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol.

Sequences in the 5' Non-coding Region of Human Rhinovirus 14 RNA that Affect in vitro Translation

A subgenomic cDNA clone from human rhinovirus 14 (HRV-14), comprising the 5' non-coding region and the first 1182 nucleotides of the coding sequence, has been inserted into a vector under the control of the T7 promoter, and RNA was transcribed. Deletions in the 5' non-coding sequence modulated viral polyprotein synthesis significantly in a reticulocyte lysate system. Removal of the first 491 nucleotides had little effect, but deletion of a further 55 nucleotides (491 to 546) significantly increased the efficiency of the translation process. Further deletion to nucleotide 621 almost abolished translation, suggesting an essential role for the 546 to 621 nucleotide sequence. The efficiency of the translation process can also be influenced by the addition of ribosomal salt wash prepared from uninfected HeLa cells.

In vitro methylation of Escherichia coli 16S ribosomal RNA and 30S ribosomes.

Treatment of synthetic 30S particles lacking all of the normally methylated nucleotides with S-adenosyl-[3H]methionine and either an S100 or ribosomal high salt wash extract resulted in ribosome-dependent incorporation of [3H]methyl groups into trichloroacetic acid-insoluble material. No incorporation was observed when naturally methylated isolated 30S particles were used, showing that methylation at unnatural sites did not occur. Enzymatic hydrolysis of the labeled RNA to nucleosides followed by HPLC analysis identified the [3H]methylated residues. Activities for the formation of N6-methyladenosine, N6-dimethyladenosine, 5-methylcytidine (m5C), 3-methyluridine, and N2-methylguanosine were found. Fractionation by ammonium sulfate partially resolved the different activities. All of the fractions with m5C activity were 6-8 times more active on synthetic unmethylated 16S RNA than on synthetic 30S ribosomes, whereas the N2-methylguanosine activity preferred 30S ribosomes to 16S RNA by a factor of more than 10. The N6-methyladenosine and N6-dimethyladenosine activities were 30S ribosome-specific. The m5C activity present in the 55-85% ammonium sulfate fraction of the high salt wash yielded a maximum of 1.0 mol of m5C per mol of 16S RNA, although two m5C residues, positions 967 and 1407, are found in vivo. RNase protection by hybridization with the appropriate oligodeoxynucleotide identified the methylated residue as C-967. Methylation of m5C-967 did not require prior methylation of G-966, and methylation of A-1518 and A-1519 was not dependent on prior methylation of G-1516.

Formation of large, sedimentable transcription complexes with VARNA genes and other related genes

We have used an Eppendorf centrifuge for isolation of transcription complexes assembled on VARNA genes and other related genes with NTP-depleted cell-free extracts. Similar to the 5 S rRNA gene, sedimentable, stable transcription preinitiation complexes could be assembled from two VARNA genes, two EB virus-specific EBER genes, four human tRNA genes, and one human Alu-family RNA gene, suggesting that the 5 S rRNA-specific transcription factor, TFIIIA, was not required for formation of these sedimentable, stable preinitiation complexes. Parameters affecting assembly of these complexes were sequences in circular DNA templates, sizes and sequences of linear DNA templates, temperature and incubation time. These complexes were stable at from 4 to 37 degrees C, and somewhat stable to salt wash. From results of effects of various mutations on assembly of these sedimentable complexes, we concluded that they were transcription machineries. Addition of the supernatant and partially purified factors to salt-washed complexes stimulated their transcription, we concluded that these sedimentable complexes were minimal transcription machineries containing suboptimal quantities of loosely bound transcription factors, TFIIIB, and RNA polymerase III. DNase 1 footprints of these sedimentable preinitiation complexes showed that two regions were protected, from +34 to +80 including the B block promoter element, and from +98 to +105. Similar DNase 1 footprints were also obtained from salt-washed complexes and stable preinitiation complexes isolated by molecular sieve column chromatography.