Cs2SO4 Density Gradients Research Articles

Characterization and localization of cryptic satellite DNAs in barley (Hordeum vulgare)

Three satellites on the heavy side of the main band and two satellites on the light side were isolated in a pure from by preparative ultracentrifugation of H. vulgare DNA in Ag(+)/Cs2SO4 density gradients. The satellites were characterised in terms of their buoyant densities in CsCl and their thermal dissocation temperature in both native and reassociated forms to Cot 4. In CsCl gradients, heavy satellites formed a single peak whereas light satellites resolved into more than one component. Thermal transitions of some satellites indicated the presence of more than one molecular species. The multicomponent nature of thermal denaturation profiles was evident on differential analysis. Radioactive RNAs complementary to the three heavy satellites of H. vulgare were localised by in situ hybridization onto its nuclei and chromosomes. One heavy satellite (H3) was found to be distributed on all chromosomes, although one pair showed less hybridization compared to the others. The other satellite (H1) appeared to be present in a much lower amount on the chromosomes.

Gene distribution and nucleotide sequence organization in the human genome.

Human DNA was fractionated by centrifugation in Cs2SO4 density gradients containing 3,6-bis(acetatomercurimethyl)dioxane (BAMD). Fractions were investigated in their analytical CsCl profiles and a number of specific sequences were localized in them. The results so obtained led to an improved understanding of the organization of nucleotide sequences in the human genome, as well as to the discovery that a class of DNA having a very high G + C content and not represented in the mouse genome, is particularly rich in genes and interspersed repetitive sequences.

RNA—PROTEIN CROSSLINK AND RNA CHAIN BREAK FORMATION ON UV‐IRRADIATION OF TOBACCO MOSAIC VIRUS

Abstract— The ability of UV‐irradiation (254 nm) to induce formation of RNA‐protein crosslinks in tobacco mosaic virus (TMV) particles have been studied by Cs2SO4 density gradient centrifugation, analytical centrifugation, nitrocellulose filter binding and two‐dimensional peptide mapping. RNA‐protein crosslinks were found to be formed on UV‐irradiation of TMV, but the parallel process of UV‐induced RNA chain breakage complicated their quantitation. Using speciall devised equations, the quantum yield of RNA‐protein crosslink formation was found to be 0.65 × 10−5 and that of RNA chain break formation 0.95 × 10−5.

Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells.

Heterogeneous nuclear RNA is normally complexed with a specific set of proteins, forming ribonucleoprotein particles termed hnRNP. These particles are likely to be involved in mRNA processing. We have found that the structure of hnRNP is profoundly altered during the heat shock response in Drosophila cultured cells. Although hnRNA continues to be synthesized at a near-normal rate during heat shock, its assembly into hnRNP is incomplete, as evidenced by a greatly decreased protein content of the particles in Cs2SO4 density gradients. RNA-protein cross-linking conducted in vivo (Mayrand and Pederson, Proc. Natl. Acad. Sci. U.S.A. 78:2208-2212, 1981) also reveals that hnRNA made during heat shock is complexed with greatly reduced amounts of protein. The block of hnRNP assembly occurs immediately upon heat shock, even before the onset of heat shock protein synthesis. Additional experiments reveal that hnRNP assembled normally at 25 degrees C subsequently disassembles during heat shock. The capacity for normal hnRNP assembly is gradually restored after heat-shocked cells are returned to 25 degrees C. Heat-shocked mammalian cells also show a similar block in hnRNP assembly. We suggest that incomplete assembly of hnRNP during heat shock leads to abortive processing of most mRNA precursors and favors the processing or export (or both) of others whose pathway of nuclear maturation is less dependent on, or even independent of, normal hnRNP particle structure. This hypothesis is compatible with a large number of previous observations.

Variation in DNA complexity inNicotiana glauca tissue cultures. I. pith tissue dedifferentiation ?in vitro?

Amplification of G + C-rich and A + T-rich fractions occurs in DNA fromNicotiana glauca pith explants in the first hours of theirin vitro culture. This amplification has been evidenced by derivative melting profiles and by analysis of fractions from Ag+-Cs2SO4 density gradients.

Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells.

Heterogeneous nuclear RNA is normally complexed with a specific set of proteins, forming ribonucleoprotein particles termed hnRNP. These particles are likely to be involved in mRNA processing. We have found that the structure of hnRNP is profoundly altered during the heat shock response in Drosophila cultured cells. Although hnRNA continues to be synthesized at a near-normal rate during heat shock, its assembly into hnRNP is incomplete, as evidenced by a greatly decreased protein content of the particles in Cs2SO4 density gradients. RNA-protein cross-linking conducted in vivo (Mayrand and Pederson, Proc. Natl. Acad. Sci. U.S.A. 78:2208-2212, 1981) also reveals that hnRNA made during heat shock is complexed with greatly reduced amounts of protein. The block of hnRNP assembly occurs immediately upon heat shock, even before the onset of heat shock protein synthesis. Additional experiments reveal that hnRNP assembled normally at 25 degrees C subsequently disassembles during heat shock. The capacity for normal hnRNP assembly is gradually restored after heat-shocked cells are returned to 25 degrees C. Heat-shocked mammalian cells also show a similar block in hnRNP assembly. We suggest that incomplete assembly of hnRNP during heat shock leads to abortive processing of most mRNA precursors and favors the processing or export (or both) of others whose pathway of nuclear maturation is less dependent on, or even independent of, normal hnRNP particle structure. This hypothesis is compatible with a large number of previous observations.

Ultraviolet light-induced crosslinking of HnRNA to informofer proteins in vitro

RNA-protein interaction in the 30S subunits of rat liver hnRNP has been studied by crosslinking of informofer proteins to hnRNA induced by UV irradiation. Irradiation of 30S particles with 254 nm UV light in doses of 1 1 x 10(5) erg/mm2 leads to the extensive crosslinking hnRNA to informofer proteins. The crosslinked material was analyzed either by resedimentation in a 15-30% sucrose gradient in the presence of 3 M guanidine-HCl and 1 M NaCl or by centrifugation in a Cs2SO4 density gradient containing guanidine-HCl and sarkosyl. The crosslinked complexes sedimented at about 25S in the sucrose gradient and proved to be heterogeneous in isopycnic centrifugation experiments. The proteins of the crosslinked complexes were analyzed by polyacrylamide gel electrophoresis. Proteins with Mr values of 70 000, 58 000, 43 000 and 40 000 appeared to be crosslinked with hnRNAs of the 30S particles. In the unirradiated 30S particles after centrifugation in the CS2SO4 density gradient containing guanidine-HCl and sarkosyl two minor proteins were observed with Mr values of 70 000 and 58 000, banded in density zones characteristic for free RNA.

A novel species of double stranded RNA in mitochondria of Saccharomyces cerevisiae.

A double stranded RNA species has been detected in guanidine hydrochloride extracts of mitochondria from respiratory competent cells of Saccharomyces cerevisiae. This novel mitochondrial RNA, termed mtdsRNA, has been purified in a Cs2SO4 density gradient where it bands at a density of 1.58 g/ml. The mtdsRNA runs as a single slow moving band on agarose gels. Its double stranded RNA character was evidenced by its sensitivity to digestion by RNase III, but not by RNase H, or DNase I. Moreover the mtdsRNA hybridized to each separated strand of a petite mtDNA. It is concluded that mtdsRNA contains long transcripts derived from most regions of yeast mtDNA, because 1) its weight-average length as determined by electron microscopy was 4.5 micrometer (about 14 kb, or 20% of the wild type mtDNA genome), and 2) it hybridized to each of a series of eight petite mtDNA probes carrying sequences derived from widely different segments of mtDNA. It is proposed that prolonged transcription of both strands of yeast mtDNA can occur and that mtdsRNA arises from hybridization of these long complementary transcripts.

The polyribosomal poly(A)-binding protein is highly conserved in vertebrate species: Comparison in duck, mouse and rabbit

In eukaryotic cells mRNA is present in the form of messenger ribonucleoprotein complexes (mRNP particles). When polyribosomes, from a variety of cell types and tissues, are dissociated with EDTA or puromycin, the released mRNA is found tightly associated with 3 major proteins o fM r ~ 43 000-52 000 and 72 000-78 000 (review [1]). The larger of these proteins was shown to interact with the 3'-polyadenylate sequence of mRNA [2,3] since it may be recovered with poly(A) following digestion of polyribosomes or purified mRNP with ribonucleases A and T1. From density measurements of the poly(A) RNP particle it may be estimated that ~4 5 copies of this protein are associated with the average-sized poly(A) fragment [4]. The affinity of the poly(A)-binding protein for poly(A) is very high since it resists not only 0.5 M KC1 [1] but also centrifugation through Cs2SO4 density gradients [5,6] and affords protection ofpoly(A) against ribonucleases and nucleases [7 10]. Furthermore, this protein binds not only poly(A) but also regions of poly(A) adjacent and non-adjacent to poly(A) [81. A protein of the same M r as the polyribosomal poly(A)-binding protein has been observed to be associated with the 3'-poly(A)region of nuclear pre-

Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells.

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA-protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell.

Nuclear ribonucleoprotein particles probed in living cells.

Contacts between heterogeneous nuclear RNA (hnRNA) and protein in nuclear ribonucleoprotein particles have been photochemically crosslinked in intact HeLa or Friend erythroleukemia cell by irradiation with 254-nm light at doses of 10(1) to 10(5) ergs/mm2 (1 to 10(4) microJ/mm2). The resulting crosslinked particles were isolated and compared with conventional hnRNA . protein (hnRNP) preparations. By the criteria of nuclear fractionation behavior, sedimentation coefficients, nuclease digestion profiles, and RNA-to-protein ratio measured by banding in Cs2SO4 density gradients, the hnRNP particles crosslinked in vivo are identical to nonirradiated particles. Gel blot hybridization of RNA from Friend cell hnRNP crosslinked in vivo reveals that beta-globin RNA sequences remain both intact and hybridizable after the irradiation procedure. The crosslinked hnRNA--protein bonds are stable in 8 M urea/0.5% sodium dodecyl sulfate and withstand centrifugation in Cs2SO4 gradients of initial density 1.50 g/cm3. These results establish that hnRNA is tightly complexed with nuclear proteins in vivo and that hnRNP particles isolated by nuclear fractionation represent native structures.

Biophysical properties of virions of human adenovirus of the type 6 and its DNA.

A study has been made of human adenovirus type 6 (Ad 6) which belongs to the C subgroup of the nononcogenic adenoviruses. The buoyant density of the Ad 6 virions in CsCl gradient was 1.3545 +/- 0.0010 g/cm3. The DNA content determined from the buoyant density values and direct phosphorus analysis was 11.2 +/- 0.5 and 11.5 +/- 0.5 per cent, respectively. The S20,w0 and D20,w0 values for the Ad 6 virions were 795 +/- 18S and (3.255 +/- 0.035) X 10-8 cm2/s. The hydrodynamic parameters of the viral particle and its volume as well as the specific partial volume, the content and the molecular weight of the DNA have yielded a molecular weight of the virions of (203.6 +/- 10.0) X 10(6). The sedimentation constant and the intrinsic viscosity of the DNA have been found to be 32.7 +/- 0.7S and 93.5 +/- 5.0 dl/g, respectively. The molecular weight of the DNA found from these results is (23.4 +/- 0.8) X 10(6). The buoyant density of the Ad 6 DNA measured in CsCl and Cs2SO4 density gradients was 1.7128 +/- 0.0010 and 1.427 +/- 0.001 g/cm3, respectively. The GC content found from the buoyant density and the melting temperature (90.5 +/- 0.1 degrees C in 1 SSC and 75.0 +/- 0.1 degrees C in 0.1 SSC) was 52.6 +/- 1.0 per cent, that is, it is considerably lower than the GC content of 56-60 per cent which, according to the "Green's rule", should be typical of the DNAs of all the human adenoviruses of the subgroup C (30).

Influence of A-T content on the fractionation of DNA restriction fragments by RPC-5 column chromatography.

The properties of the fractionated Hae III fragments of pRZ2 DNA (Patient, R.K., Hardies, S.C., and Wells, R.D. (1979) J. Biol. Chem. 254, 5542-5547) were studied in an effort to determine why several of the fragments bind more tightly to RPC-5 than expected on the basis of their length. The purified fragments were analyzed for their nucleotide composition by direct determination of their constituent mononucleotides and by analytical CsCl and Cs2SO4 density gradient analyses. A-T-rich fragments elute at higher salt concentrations than fragments of equivalent size which are not A-T-rich. In addition, denaturation mapping studies by electron microscopy indicate that an A-T-rich run within an otherwise G-C-rich fragment can give rise to delayed elution. At least one other factor influences the separation of DNA restriction fragments by RPC-5 chromatography. Some of the fragments in this digest which elute later than predicted from their size either contain known genetic regulatory sites or bind regulatory proteins.

Notizen: Methylmercury-Induced Sedimentation Heterogeneity of T7 Bacteriophage DNA

Single-stranded and methylmercurated T7 DNA is composed of two species as evidenced from the sedimentation pattern displayed during band-sedimentation in self-generating density gradients as well as during equilibrium-sedimentation in CS2SO4 density gradients. Under the given experimental conditions, viz., at pH 9.18 and in presence of 0.1 mM CH3HgOH, the two species band in CS2SO4 with a density difference of 0.008 g/ml. The ratio of the sedimentation coefficients of the two species is sw, 20 (fast)/sw, 20 (slow) = 1.63 at pH 9.18 and in presence of 1.6 mM CH3HgOH. Both native and denatured T7 DNA behave as monodisperse systems in the absence of CH3HgOH.

Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag+/Cs2SO4 density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag+/Cs2SO4 density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm3 and a heavy shoulder at density 1,703 g/cm3 are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag+ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag+/Cs2SO4 gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag+/Cs2SO4 satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag+/Cs2SO4 gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag+/Cs2SO4 gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.

Satellite DNA in differentiating chick tissues.

Embryonic chick DNA from different tissues was examined for differences which might indicate specific DNA amplification in somatic cells. The problem was approached by determining the DNA compositional heterogeneity and searching for possible variation in different tissues of the 12-day chick. Neural retina, muscle, and whole decapitated (general) chick DNA were analyzed in CsCl and Cs2SO4 density gradients. While overloaded CsCl gradients showed a main band (rho = 1.701 g/cm3) and a heavy shoulder (rho = 1.716 g/cm3), overloaded Cs2SO4 gradients displayed a main band (rho = 1.426 g/cm3) and a discrete heavy satellite (rho = 1.447 g/cm3). This satellite, comprising approximately 1% of the whole cell DNA, appeared to be of nuclear origin and not related to mitochondrial DNA, which was found to have a density of 1.426 g/cm3 in Cs2SO4. No differences were found in the densities of the main band or the satellite DNA in the DNA samples isolated from the different tissues. However, the method of DNA isolation was found to be of crucial importance when comparing satellite DNA's among different tissues.

Interaction between DNA and Escherichia coli protein omega. Formation of a complex between single-stranded DNA and omega protein.

Escherichia coli omega protein is found to form a complex with single-stranded DNA. The complex is stable in buoyant CsCl or Cs2SO4 density gradients. Addition of Mg(II) to the concentrated salt solutions, however, leads to the dissociation of the complex, even in the presence of EDTA in molar excess over Mg(II). The dissociated omega retains its enzymatic activity; the DNA recovered from the dissociated complex is indistinguishable from the original DNA. Exposure of the complex to alkali results in the cleavage of the DNA. This cleavage generates a 3'-hydroxyl DNA terminus, and the omega protein is found linked to the 5'-terminus, presumably covalently. Pronase digestion of the complex results initially in the removal of approximately 30% of the protein. A significant fraction of the residual complex is still stable in concentrated salt solutions, and can be dissociated by Mg(II). Extensive digestion with pronase results in the removal of the protein and the cleavage of the DNA chain.

The organization of DNA sequences in the mouse genome.

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag+--Cs2SO4 density gradient centrifugation.--According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2X10(4) copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).--Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides.--Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides.--The organization of mouse genome analyzed by Ag+--Cs2SO4 density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.

Circular ribosomal DNA during ribosomal magnification in Drosophila melanogaster

DNA from excised testes of Drosophila melanogaster males undergoing ribosomal DNA magnification shows two peaks on an ethidium bromide/CsCl density gradient: a minor peak that bands to a density of 1·570 g/cm3 and a main peak corresponding to 1·540 g/cm3 density. The minor peak is absent when DNA is extracted from testes of wild-type males or of males where rDNA magnification has gone to completion. Electron micrographs show that DNA at the minor peak is circular in shape. Ribosomal RNA/DNA hybridization experiments on the gradient and preparation of pure rRNA/DNA at minor peak hybrids on a Cs2SO4 density gradient indicate that most of these circular molecules are DNA complementary to rRNA. These results support the model that requires the formation of rDNA extra-copies for rDNA magnification (Ritossa, 1972) .

Isolation of messenger ribonucleoproteins in cesium sulfate density gradients: Evidence that polyadenylated and non-polyadenylated messenger RNAs are associated with protein

Centrifugation of L-cell polyribosomes at 25°C in Cs2SO4 density gradients containing EDTA or low concentrations of Mg2+ fractionates them into particles containing mRNA and particles containing rRNA. Precipitation of the particles in the gradients can be avoided by the addition of 15% dimethyl-sulfoxide. Under these conditions mRNA was found over a density range of 1·3 to 1·5 g/cm3. This finding suggested that it was present in the form of ribonucleoprotein particles. rRNA, on the other hand, banded near the density of pure RNA (1·57 g/cm3), whereas the protein removed from the ribosomes banded near the top of the gradient at the density of pure protein (1·1 to 1·2 g/cm3). When the mRNA was recovered and analyzed by means of chromatography on oligo(dT)-cellulose and polyacrylamide gel electrophoresis, it was found that not only poly(A)-containing mRNA, but also histone mRNA and non-histone poly(A)-lacking mRNA were present. When the proteins recovered from this region of the gradient were analyzed by means of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, five major components were found having approximate molecular weights of 103,000, 87,000, 76,000, 36,000 and 30,000. Additional experiments showed that histone mRNA, although present in the messenger ribonucleoprotein-containing region, did not reach its equilibrium density under the conditions of centrifugation routinely used, and was apparently not associated with protein. It is concluded that poly(A)-containing mRNA and non-histone poly(A)-lacking mRNA exist in polyribosomes in the form of ribonucleoprotein particles.