Evidence for en bloc incorporation of exogenous oligonucleotides into HeLa cell RNA.

3H uridine and 3H guanosine labeled oligonucleotides were separately produced by degradation of high molecular weight RNA by endogenous intracellular ribonucleases of HeLa cells. After incubation of this low molecular weight RNA with HeLa cells under conditions limiting labeled mononucleotide incorporation, significant label was found in high molecular weight 18s RNA, but not in 28s RNA. The label in the 18s RNA was alkali labile and remained with the 18s RNA peak under denaturation conditions. Actinomycin in the incubation mixture prevented the appearance of label in the 18s RNA. These results raise the possibility that exogenous oligonucleotide segments can be incorporated en bloc into RNA.     

Cucumber mosaic virus-associated RNA 5. VI. Characterization and denaturation-renaturation behavior of the double-stranded form.

The double-stranded form of cucumber mosaic virus-associated RNA 5 has been purified and further characterized. Its molecular weight determined by sedimentation equilibrium is 2.15 . 10(5). The buoyant density calculated from its symmetrical distribution in Cs2SO4, following isopycnic ultracentrifugation, is 1.615 g/cm3. The sedimentation rate of double-stranded cucumber mosaic virus-associated RNA 5 is slightly greater than that of cucumber mosaic virus-associated RNA 5; its electrophoretic mobility in polyacrylamide gel (2.4%) is less than that of cucumber mosaic virus-associated RNA 5. By the above standards the double-stranded cucumber mosaic virus-associated RNA 5 preparations used were found to be nomogeneous in size as well as density. Thermal denaturation monitored by means of ultraviolet light absorption produced multitransitional denaturation profiles. The average melting temperature (Tm) was 88 degrees C in 0.1 x SSC. Monotransitional denaturation profiles and slightly higher Tm values were obtained when resistance against ribonuclease digestion was measured. These denaturation experiments and other propertied led to the conclusion that double-stranded cucumber mosaic virus-associated RNA 5 and the double-stranded form of peanut stunt virus-associated RNA 5 are small double-stranded nucleic acids with several homostable base-pair regions, characterized by distinct G + C contents and Tm values.     

Characterization of RNA from equine infectious anemia virus.

The genome of equine infectious anemia virus, a nononcogenic retrovirus, has been characterized by velocity sedimentation, electrophoresis in polyacrylamide gels, buoyant density in CS2SO4, and susceptibility to nuclease digestion. The nucleic acid of purified virus was resolved by sedimentation analysis into a fast-sedimenting genome component, which comprises about two-thirds of the virion RNA, and a slow-sedimenting RNA, which is probably comprised of host-derived tRNA and a trace amount of 5S RNA. The fast-sedimenting RNA had a sedimentation coefficient of 62S and a molecular weight of 5.4 X 10(6) to 5.6 X 10(6), as determined by sedimentation velocity and electrophoretic mobility. Upon heat denaturation, [3H]uridine-labeled 62S RNA dissociated into material comprised of 90 to 95% single-stranded species, sedimenting predominantly at 34S, with a molecular weight of 2.7 X 10(6) to 2.9 X 10(6) and 5 to 10% 4S RNA. The 62S RNA was predominantly single-stranded but contained double-stranded regions, as indicated by partial resistance to RNase IA and SI nuclease and by a lower buoyant density in CS2SO4 than that of the single-stranded 34S RNA derived by heat denaturation. These data indicated that the viral genome consisted of two 34S subunits of single-stranded RNA held in a high-molecular-weight complex with 4S RNA by a mechanism involving a small degree of base pairing. Thus, the structure of equine infectious anemia virus RNA is similar to that of other retroviruses.     

Removal of RNase activity from DNase by affinity chromatography on agarose coupled aminophenylphosphoryl-uridine-2'' (3'')-phosphate.

Severe degradation of high molecular weight RNA was shown to occur during incubation with commercially purified DNase. Most of the RNase activity could be removed by passage of the DNase through a column of agarose-coupled amino phenylphosphoryl-uridine-2' (3')-phosphate. Incubation with the treated DNase caused only minimal alteration of the sedimentation pattern of high molecular weight nuclear RNA, determined under partially denturing conditions. No impairment of DNase activity was detected.     

Novel form of non-muscle tropomyosin in human fibroblasts

The cytoskeletal extracts of cultured human fibroblasts were found to contain at least four distinct polypeptides, each of which demonstrated the resistance to denaturation and the acidic isoelectric point characteristic of tropomyosin. One of these, hscp 36 (heat-stable cytoskeletal protein having an apparent molecular weight of 36,000), cross-reacted efficiently with an antiserum to chicken skeletal muscle tropomyosin. Furthermore, the messenger RNA coding for hscp 36 was selected by a chicken complementary DNA clone containing a tropomyosin sequence. The abundance of mRNA coding for hscp 36 was found to be similar in both normal and simian virus 40 (SV40) transformed human fibroblasts. The apparent molecular weight of hscp 36 is different from non-muscle tropomyosins previously isolated from human sources, which show the apparent molecular weight of 30,000 normally associated with non-muscle tropomyosin. This, together with the complexity of the heat-stable cytoskeletal proteins present in human fibroblasts, suggests the existence of multiple genes coding for human non-muscle tropomyosins.     

Characterization of Bluetongue Virus Ribonucleic Acid

An improved purification procedure yielded bluetongue virus free from any single-stranded ribonucleic acid (RNA) component. Double-stranded RNA obtained from purified virus or isolated from infected cells was fractionated into 5 components by means of sucrose gradient sedimentation analysis, and into 10 components by electrophoresis on polyacrylamide gels. The size of these components vary from 0.5 x 10(6) to 2.8 x 10(6) daltons, with a total molecular weight estimate of about 1.5 x 10(7) for the viral nucleic acid. The denaturation of the genome and separation of the resulting fragments are also discussed.     

Structural studies of apolipoprotein B: physical properties of the protein in guanidine hydrochloride

Apolipoprotein B was isolated from human plasma low-density-lipoprotein without precipitation by diethyl ether/ethanol extraction of the protein in 6 M guanidine hydrochloride. The physical properties of this protein, which contained a residuum of approximately 7% phospholipid, were examined in 6 M guanidine solution under reducing conditions. The circular dichroism spectrum was indistinguishable from that of a random coil protein. Sedimentation equilibrium analyses of apolipoprotein B by the meniscus depletion method of Yphantis (1984, Biochemistry 3, 297-317) were complicated by heterogeneity and nonideality despite the low concentrations employed. 63 analyses of the weight average (Mw) and z average (Mz) molecular weight were made on the apolipoprotein B from 12 subjects. The Mw observed was a function of initial concentration, rotor speed, and a heterogeneity index (Mz/Mw). Multiple linear regression of apolipoprotein B molecular mass against these parameters suggested that an Mw of 540,000 +/- 110,000 would be observed under apparently ideal and homogeneous conditions. The sedimentation coefficient and intrinsic viscosity of the reduced protein at 25 degrees C in 6 M guanidine were 2.13 S and 116 ml/g, respectively; these values predict molecular weights of 640,000 and 250,000, respectively, if apolipoprotein B was fully denatured into a random coil. Lack of agreement between these estimates and with the sedimentation equilibrium analysis can best be explained by compactness of structure and incomplete denaturation to a random coil state. Furthermore, an irreversible temperature dependence of apolipoprotein B reduced viscosity indicated that residual structure remained in solutions of 6 M guanidine hydrochloride/20 mM dithiothreitol. Taken together, the physical data demonstrate that apolipoprotein is a single polypeptide of approximately 540 kDa, whose structure resists denaturation under conditions where most proteins exist as random coils.     

Three DNA polymerases of rat ascites hepatoma cells: properties of the enzymes and effect of RNA synthesis on the reactions.

Three different DNA polymerases have been isolated from rat ascites hepatoma cells [1--3]. The molecular weight of a DNA polymerase (polymerase C) purified from the soluble fraction of the cells was estimated to be 142 000 by sedimentation on a sucrose gradient, while the molecular weights of two DNA polymerases (polymerase P-1 and P-2) purified from nuclear membrane-chromatin fraction were estimated to be 117 000 and 44 000, respectively, by the same method. Under certain conditions, the poly (dT) strand of poly[(dA)-(dT)] was copied well by the polymerases, especially by the nuclear polymerases. Poly (dC) was a good template for the high molecular weight DNA polymerases C and P-1, but poly(dT) and poly(dA) were not effective templates. By addition of complementary oligoribonucleotides, the single-stranded deoxypolymers were copied by the high molecular weight polymerases C and P-1. When single-stranded fd phage DNA was used as template, the polymerization reactions by the high molecular weight polymerases were stimulated by the concomitant synthesis of RNA. This indicates that the oligoribonucleotide acts as a primer in these reactions.     

Physical and Biological Properties of Phage φ29 Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) molecules having a mean length of 5.8 mum were released from purified Bacillus subtilis bacteriophage phi29 with 2 m sodium perchlorate. Small 0.1 to 0.2-mum molecules were also detected in these DNA preparations. Since intact single chains annealed to form linear duplex molecules, phage phi29 DNA was found to be nonpermuted. The molecular weights of single chains of phi29 DNA were approximately half that of native DNA, as determined by analytical band sedimentation in CsCl, indicating that phi29 DNA is composed of two continuous polynucleotide chains. The molecular weight values of native and annealed phi29 DNA from sedimentation agreed with the molecular weight values obtained from electron microscopy. The infectivity of phi29 DNA was reduced to a low level by alkaline denaturation and was partially restored by annealing.     

Uncertainty in the determination of the molecular weight of poly(A)-containing RNA.

Estimates of the weight average molecular weight of mouse myeloma cell cytoplasmic poly(A)(+)RNA obtained by polyacrylamide-gel electrophoresis and by sucrose gradient sedimentation differ by a factor of two. This difference is not due to degradation of the RNA molecules during sedimentation nor to acid poly(A) double helix formation during electrophoresis. The difference is probably due, at least in part, to the presence of poly(A) sequences in the RNA molecules.     

Thermodynamic characterization of the human acidic fibroblast growth factor: evidence for cold denaturation.

The thermodynamic parameters characterizing the conformational stability of the human acidic fibroblast growth factor (hFGF-1) have been determined by isothermal urea denaturation and thermal denaturation at fixed concentrations of urea using fluorescence and far-UV CD circular dichroism (CD) spectroscopy. The equilibrium unfolding transitions at pH 7.0 are adequately described by a two-state (native <--> unfolded state) mechanism. The stability of the protein is pH-dependent, and the protein unfolds completely below pH 3.0 (at 25 degrees C). hFGF-1 is shown to undergo a two-state transition only in a narrow pH range (pH 7.0-8.0). Under acidic (pH <6.0) and basic (pH >8.0) conditions, hFGF-1 is found to unfold noncooperatively, involving the accumulation of intermediates. The average temperature of maximum stability is determined to be 295.2 K. The heat capacity change (DeltaC(p)()) for the unfolding of hFGF-1 is estimated to be 2.1 +/- 0.5 kcal.mol(-1).K(-1). Temperature denaturation experiments in the absence and presence of urea show that hFGF-1 has a tendency to undergo cold denaturation. Two-dimensional (1)H-(15)N HSQC spectra of hFGF-1 acquired at subzero temperatures clearly show that hFGF-1 unfolds under low-temperature conditions. The significance of the noncooperative unfolding under acidic conditions and the cold denaturation process observed in hFGF-1 are discussed in detail.     

Isolation and partial characterization of a 700 kilodalton human colon carcinoma associated antigen

An antigen of high molecular weight (CA-3) was isolated from the cytosol fraction of GW-39 human colon tumor cells by antibody affinity chromatography. CA-3 was characterized by an acidic pI value of 4.5-4.9, a molecular weight of 700 kilodaltons and a sedimentation coefficient of 13S. It contained all of the commonly occurring amino acids and had an acidic to basic amino acid ratio of 1.4. CA-3 was resistant to dissociation by reducing agents as well as by sodium dodecylsulfate. Quantitation of CA-3 by a radioimmunoassay employing rabbit anti-CA-3 antiserum revealed a marked elevation of CA-3 in the cytosol extracts of human primary colon carcinoma in comparison to normal colon. The molecular properties of CA-3 are compared to those of carcinoembryonic antigen, high molecular weight colon specific antigen CSAp and two other high molecular weight proteins, fibronectin and conglutinin. Colon antigen CA-3 appears to be different from these other molecules in terms of its molecular weight, sedimentation value, isoelectric point and amino acid composition.     

Physical properties of single- and double-stranded coliphage ribonucleic acid

1. The physical characteristics of single- and double-stranded coliphage RNA with regard to their sedimentation behaviour in gradients of sucrose in high or low ionic conditions were examined. The effect of heat on their sedimentation characteristics was also determined. 2. Single-stranded coliphage RNA was found to exist in three different forms having sedimentation coefficients 28s, 20s and 12s. The latter two were interchangeable, depending on ionic strength. All three were almost equally infectious to spheroplasts. 3. Double-stranded coliphage RNA was found to be non-infectious to spheroplasts and had sedimentation coefficients 15s and 12s. Thermal denaturation gave rise to infectious single-stranded 12s RNA. 4. Four possible hypotheses on the mechanism of replication of coliphage RNA are discussed.     

The measurement of plant polyadenylic acid by hybridisation with radioactive polyuridylic acid

Summary Saturation hybridisation of polyadenylic acid with [³H]polyuridylic acid is described. Under conditions of [³H]poly(U) excess, poly(A) is detected in the RNA of a number of higher plants. The ribonuclease resistant hybrids melt sharply when subjected to thermal denaturation. Plant RNA which contains poly(A) sequences detected by [³H]poly(U) hybridisation is polydisperse in molecular weight. Data presented shows that the amount of poly(A) in plant RNA is variable. This technique is useful for the qualitative and quantitative detection of poly(A) sequences in higher plant RNA.Abbreviations A.R. Analar Reagent - Poly(A) Polyadenylic acid - Poly(U) Polyuridylic acid - Oligo(dT)-cellulose oligo(deoxythymidylate)-cellulose - Tm melting temperature - SSC standard saline citrate     

Sunflower seed protein: size and charge heterogeneity in subunits of the globulin fraction

The subunit heterogeneity of the globulin fraction of sunflower seeds was investigated by two dimensional electrophoresis, using isoelectric focusing in the first dimension and sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension. Under non reducing conditions, intermediary subunits B, C and D (molecular weight 54 000, 48 000 and 40 000, respectively) were focused within a pI range 5.4-6.0 but intermediary subunits A (molecular weight 60 000) focused within a pI range 6.3-6.8. Under reducing conditions the electrophoretic patterns show that intermediary subunits consist in large "acidic" and small "basic" subunits linked by disulphide bonds. The large subunits of B species are more acidic and less heterogeneous than the corresponding subunits of the A species. These results confirm that helianthinin had a "legumin-type" structure.     

The origin of the polydispersity in sedimentation patterns of rapidly labelled nuclear ribonucleic acid

1. A study was made of the sedimentation properties of purified preparations of the rapidly labelled RNA in the nucleus and the cytoplasm of the HeLa cell. The sedimentation of the rapidly labelled nuclear RNA was very sensitive to changes in ionic strength and bivalent cation concentration. Under the conditions usually used in sucrose-density-gradient centrifugation the rapidly labelled nuclear RNA showed extreme polydispersity, and much of it sedimented more rapidly than the 28s RNA. At low ionic strength and after removal of Mg(2+), however, the rapidly labelled nuclear RNA sedimented as a single peak at about 16s. The conversion of the polydisperse material into the 16s form did not involve degradation of the RNA, since the effect could be reversed by increasing the ionic strength of the solution. 2. The cytoplasm did not contain any RNA that showed polydisperse sedimentation under the usual conditions of sucrose-density-gradient centrifugation, or that had the same sensitivity as the rapidly labelled nuclear RNA to changes in ionic strength. All the radioactivity in the cytoplasmic RNA sedimented with the 28s, 16s and 4s components over a wide range of physical conditions, but these components did contain a labelled fraction with some of the features of the rapidly labelled nuclear RNA on columns of methylated albumin on kieselguhr. 3. In both nucleus and cytoplasm the RNA detected by ultraviolet absorption could also be converted into a 16s form by removal of bivalent cations at low ionic strength; this effect was again, within certain limits, reversible. The nuclear RNA as a whole was more susceptible to changes in ionic strength than the cytoplasmic RNA. 4. It thus appears that all the RNA in the cell, except the 4s RNA, can be prepared, without degradation, as a single peak sedimenting at about 16s. The relationship of these various 16s components to each other is discussed.     

Length distributions of single-stranded DNA in Chinese hamster ovary cells.

The distribution of lengths of single-strand DNA in Chinese hamster ovary cells in the G₁ phase of the cell cycle has been observed for various conditions of cell lysis and incubation of the lysates. The method of analysis was band sedimentation through a self-generating density gradient, a technique developed originally for the analytical ultracentrifuge, but modified here for the preparative ultracentrifuge so that measurements of sedimentation coefficients could be made under conditions that minimize shearing of the single-stranded DNA. The effect of rotor speed dependence of the sedimentation coefficient is considered in developing the relation between the sedimentation coefficient and molecular weight for this technique.Special precautions were taken to ensure that complete separation of long single strands took place upon alkaline denaturation, to preclude the possibility of anomalous sedimentation due to interstrand entanglement. Bromodeoxyuridine was incorporated into the DNA in the last round of replication. Advantage was taken of the increased sensitivity to ultraviolet irradiation for the production of single-strand breaks in DNA strands substituted with bromodeoxyuridine. After irradiation the bromodeoxyuridine-substituted strand could be completely separated from the complementary strand in alkaline sedimentation profiles without any apparent breakage in the unsubstituted strand.The conditions of lysis, chosen to minimize the degradation of DNA in the lysates, included lysis at pH 9.3 with Pronase and lysis at high pH (10.8 and 12.0). Sedimentation analysis was performed at various time intervals after incubation at 4 °C or 37 °C. Lysis and incubation at pH 12.0 produced a continuous single-strand breakdown of the DNA in the lysate. Analysis of the sedimentation profiles indicates that these alkaline-induced breaks are randomly distributed. However, lysis and incubation at pH 10.8 and at pH 9.3 with Pronase produced stable sedimentation profiles with number-average molecular weights of 1.7 × 10⁸ and 6.0 × 10⁷, respectively. Analysis of the single-strand DNA sedimentation profiles for these lysates indicates that the distribution of lengths of single-stranded DNA is non-random, i.e. that the distributions may represent regular subunits of chromosomal DNA structure. Suggestive evidence is presented that the approximately 60-μm units are structurally alternated in the two chains. The possible origin of the discontinuities between the subunits is also discussed.     

Molecular-weight determination of animal-cell RNA by electrophoresis in formamide under fully denaturing conditions on exponential polyacrylamide gels.

A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA - RNA hybrids and double-stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45 degrees and 55 degrees C, respectively. In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 10(4) - 10(7). Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights the size of animal cell was reexamined. Precursor tRNA from HeLa cells migrates according to a molecular weight of 4.1 x 10(6). Nascent precursor mRNA has molecular weights of up to 5 x 10(6) in the case of duck erythroblasts and of up to 10(7) in HeLa cells. This seems to represent the largest size of non-viral animal-cell RNA molecules.