Methylated Albumin Column Research Articles

The proliferation of plasma cells from mouse bone marrow in vitro: II. Stimulation of IgG-producing cells by an RNase-sensitive thymocyte homogenate

A thymic humoral factor, previously demonstrated to promote the in vitro proliferation and differentiation of mouse bone marrow cells into IgG-forming plasma cells in the presence of antigen was shown to be RNase-sensitive but resistant to DNase and pronase. The plasma cell-stimulating activity could not be pelleted by 105,000 g ultracentrifugation, and it had a sedimentation value of 4–6 S by means of sucrose density gradient centrifugation. This activity was contained in an RNA fraction prepared by phenol extraction. The thymic activity of this preparation was eluted from a methylated albumin column at low ionic strength, and it coincided with the upper band by sucrose density gradient centrifugation. The extent of plasma cell proliferation was proportional to the concentration of the factor in the cultures, although the highest concentration of RNA tested was associated with a decrease in plasma cell proliferation. Xenogeneic rat and rabbit thymic RNA were more effective in stimulating the generation of plasma cells than syngeneic RNA. The administration of thymic RNA and protein antigen separately also induced proliferation, but to a lower extent than when they were administered together.

RNA in the degenerating silk gland of Bombyx mori

Nucleic acids in the degenerating posterior silk gland of Bombyx mori were analysed during the period from larval maturation to early pupal stage, by methylated albumin column chromatography and sucrose density gradient centrifugation. During the first half of the period, the amount of RNA decreased rapidly, but no accumulation of degradation products was detected. The ratio 26 S 17 S rRNA decreased slightly. A decrease of sRNA-like polynucleotide (∼4S RNA) was faster than that of rRNA. During the latter half of the period, rRNA continued to decrease, while ∼4S RNA increased in content. This probably resulted from the degradation of rRNA. There was a significant fall in the ratio of 26 S 17 S , suggesting that rRNA, at least in part, was degraded by the scheme of 26 S→17 S→∼ 4S. The possibility that a part of rRNA may be released outside the tissue without complete decomposition is discussed.

Charakterisierung von RNA aus synchron wachsender Bäcker-Hefe ( Saccharomyces cerevisiae) bei Trennung mit der Methylalbumin-Kieselgur(MAK)-Säule / Characterisation of RNA from Synchronously Growing Yeast Cells (Saccharomyces cerevisiae) as Separated by Columns of Methylated Albumin on Kieselgur (MAK)

Total RNA, extracted from synchronously growing yeast cells by the method of Georgiev-Mantieva, was separated by columns of methylated albumin on Kieselgur (MAK). By comparison with definited RNA's in discelectrophorese and after pulse labelling with [3H]methionine the following fractions are identified: t-RNA, 5.8S RNA, m-RNA, 18S and 25S R-RNA and their precursors 20S, 27S and 35S. High molecular fractions of RNA between 40S and 70S were also found.

The methylation of transfer RNA in Escherichia coli

Analysis of both RNA pulse-labeled in vivo with L-[Me- 3H]methionine and pools of methyl-accepting RNA from normally growing Escherichia coli indicates that methylation, by and large, takes place on molecules of the same size as mature tRNA rather than on longer precursor molecules. Two forms of such methyl-accepting tRNAs have been detected. One form, co-fractionating on methylated albumin—kieselguhr columns with mature tRNA, is apparently similar in structure to mature tRNA. The other form, fractionating similarly on methylated albumin columns to transfer RNA molecules lacking the modified nucleoside pseudouridine (prepared by substituting 5-fluorouracil for uracil in tRNA), apparently lacks this modified nucleoside. Using model substrates similar to the cellular substrates described above, kinetic studies were performed both in vitro and in vivo to determine whether there is a random or an ordered formation of the individual methylated bases in E. coli tRNA. Results indicate that, with the possible exception of 2-methyladenine formation, E. coli tRNA methylating enzymes act in random sequence in catalyzing the methylation of tRNA.

Characterization of the DNA in Drosophila melanogaster.

DNA has been quantitatively extracted from Drosophila melanogaster at various stages of embryonic development and analyzed by isopycnic centrifugation in CsCl and by fractionation on methylated albumin columns. The DNA is composed of three main classes of DNA, as defined by their buoyant density, rho, in CsCl: a bulk DNA, rho = 1.699 g cm(-3), and two satellite DNAs, rho = 1.685 g cm(-3) and rho = 1.669 g cm(-3). These three types of DNA persist throughout the development of the insect. In the unfertilized egg, 80% of the total DNA consists of the satellite DNAs; this amount decreases to 18% during the first three hours after fertilization and then remains constant through embryogenesis. There is a concomitant increase of the satellite DNA's with the bulk DNA after blastoderm formation.

Comparison of mammalian mitochondrial ribosomal ribonucleic acid from different species.

Mitochondrial ribosomal RNA species from mouse L cells, rat liver, rat hepatoma, hamster BHK-21 cells and human KB cells were examined by electrophoresis on polyacrylamide-agarose gels and sedimentation in sucrose density gradients. The S(E) (electrophoretic mobility) and S values of mitochondrial rRNA of all species were highly dependent on temperature and ionic strength of the medium; the S(E) values increased and the S values decreased with an increase in temperature at a low ionic strength. At an ionic strength of 0.3 at 23-25 degrees C or an ionic strength of 0.01 at 3-4 degrees C the S and S(E) values were almost the same being about 16.2-18.0 and 12.3-13.6 for human and mouse mitochondrial rRNA. The molecular weights under these conditions were calculated to be 3.8x10(5)-4.3x10(5) and 5.9x10(5)-6.8x10(5), depending on the technique used. At 25 degrees C in buffers of low ionic strength mouse mitochondrial rRNA species had a lower electrophoretic mobility than those of human and hamster. Under these conditions the smaller mitochondrial rRNA species of hamster had a lower electrophoretic mobility than that of human but the larger component had an identical mobility. Mouse and rat mitochondrial rRNA species had identical electrophoretic mobilities. Complex differences between human and mouse mitochondrial rRNA species were observed on sedimentation in sucrose density gradients under various conditions of temperature and ionic strength. Mouse L-cell mitochondrial rRNA was eluted after cytoplasmic rRNA on a column of methylated albumin-kieselguhr.

Highly polymerous nuclear DNA from calf thymus isolated by gel filtration in 4 M guanidine hydrochloride

It has previously been shown that concentrated solutions of guanidine hydrochloride (GHCI) in an SSC medium (0.15 M NaCl; 0.015 M sodium citrate) at room temperature do not cause irreversible denaturation [l] to DNA but severely denature proteins [2] Denatured protein components of cell nuclei, viruses, and bacterial particles are soluble in this medium. By separating DNA and protein components by gel filtration [3] , followed by an additional purification (if necessary) on a methylated albumin column (MAC) [4], highly polymerous, pure DNA can be obtained. In this paper, certain physico-chemical characteristics of nuclear thymus DNA isolated by gel filtration in 4 M GHCl are compared with the properties of DNA prepared according to Kay [5].

A low molecular weight ribonucleic acid synthesized by Escherichia coli in the presence of chloramphenicol: Characterization and relation to normally synthesized 5 s ribonucleic acid

A low molecular weight RNA synthesized by Escherichia coli cells during exposure to chloramphenicol (100 μg/ml.) has properties which are similar, though not identical, to those of normally synthesized ribosomal 5 s RNA. We call it Cam-5 s RNA. Radioactive Cam-5 s RNA is annealed to E. coli DNA in a manner similar to that of normal 5 s RNA, and this hybridization can be inhibited in a competitive fashion by normal non-radioactive 5 s RNA. Cam-5 s RNA can replace normal 5 s RNA, when larger ribosomal subunits are reconstituted from lithium chloridetreated ribosomal cores and lithium chloride-split proteins. By thermal denaturation, the secondary structure of Cam-5 s RNA can be reversibly changed in a fashion similar to that of normal 5 s RNA, but none of the forms of Cam-5 s RNA coincide with the corresponding forms of normal 5 s RNA when they are co-chromatographed on columns of methylated albumin—kieselguhr. When 5 s RNA and Cam-5 s RNA are subjected to limited degradation by ribonuclease T 1 and fractionated by acrylamide gel electrophoresis, similar large partial digestion fragments are obtained from both RNA's. The over-all base compositions of Cam-5 s RNA and normal 5 s RNA are quite similar, and no methylated or other modified nucleotides could be detected in alkaline hydrolysates of Cam-5 s RNA which were fractionated in two dimensions by electrophoresis and descending chromatography. Two-dimensional fractionations of Cam-5 s RNA completely digested by pancreatic or T 1 ribonuclease demonstrate that the primary structure of Cam-5 s RNA is essentially identical to that of normal 5 s RNA except for the presence of different 5′-terminal nucleotide sequences: in Cam-5 s RNA, the normal 5′-terminal sequence pUpG- is absent and the following sequences are found, in a combined yield of one mole: pApUpUpUpG-, pUpUpUpG- and pUpUpG-. The RNA chains (Cam-5 s RNA) which have these 5′-terminal sequences are slightly longer than the 5′-terminus of normal 5 s RNA and probably represent precursor forms of mature 5 s RNA.

Aspects of nucleic acid metabolism during development of the brine shrimp Artemia salina

Encysted embryos of the brine shrimp, Artemia salina, contain at least five species of RNA which are resolvable by chromatography on columns of methylated albumin of Kieselguhr (MAK). During development the proportion of each species within the embryo remains relatively constant. Quantitative extraction data along with 32P i and adenosine- 3H incorporation studies indicate that RNA synthesis during development of Artemia is most intense immediately after hatching, whereas very little RNA synthesis, if any, occurs beyond 36 hours development at 30°C. DNA synthesis begins shortly after hatching and it complete by 60 hours. During the period of intense RNA synthesis (20–36 hours), certain tRNA species and heavy RNA (hRNA) appear to be the most metabolically active RNA fractions. The exact function of hRNA during development of Artemia salina remains to be elucidated.

Contribution a l'etude d'acides ribonucleiques a marquage rapide de la racine de lentille: Isolement et caracterisation, action d'une hormone sur leur synthese et sur celle de peroxydases

Chromatography on methylated albumin columns of RNA's from Lentil roots, shows the presence of rapidly labelled RNA's tenaciously bound onto the column and which cannot be eluted by high NaCl molarities. Elution at 35°, with sodium dodecyl sulfate, gives two peaks (α and β). At 70°, a third elution peak (γ) is obtained. For short labelling periods, RNA's of the α peak are uridine rich (UMP/AMP approx. 1.2), heterodisperse and may have an important percentage of high-molecular-weight species. RNA's from the β peak are adenine rich (UMP/AMP approx. 0.4). Their modal sedimentation constant is about 12 S. The mean life of the uridine-rich RNA's of the α peak is lower than 1 h; that of the RNA's from the β peak is higher than 2 h. RNA's from α peak are essentially, or exclusively, of nuclear origin. On the other hand, polysomic messenger RNA's are present in the β peak. A close similitude exists between uridine-rich RNA's described in animal cells and the nuclear rapidly labelled RNA's of Lentil roots contained in the α peak. Short treatment (45 min) of Lentil roots with indoleacetic acid increases the amount of nuclear uridine-rich RNA's. A prolonged treatment (5 h) causes an increase in various rapidly labelled RNA's, probably polysomic messenger RNA's. Treatment of excised roots with indoleacetic acid leads, after a lag period of about 5 h, to the synthesis of two peroxydases associated with the ribosomes. The mean life of their messenger RNA's is about 2.5 h.

Ribonucleic acids and ribonucleoproteins from small oöcytes of Xenopus laevis

1. 1. More than 80 % of the RNA of small oöcytes of Xenopus laevis is 4-S RNA. 2. 2. Several constituents distinguishable by means of methylated-albumin column chromatography are found in this RNA and are present in two different ribonucleoproteic fractions, the sedimentation constants of which are, respectively, 5–6 S and about 20 S. 3. 3. The ribonucleoproteic fractions are also heterogeneous, as shown by CsCl gradient analyses and [ 3H]uridine incorporation; they appear from electron microscopic observations to be composed of many fibrils about 100 Å long, like those one can observe “in situ” in sections of small oöcytes. 4. 4. At this stage, oöcytes contain very few ribosomes and ribosomal RNA. 5. 5. The possible nature and function of this low molecular weight RNA are discussed.

Comparison of Lysyl-Transfer Ribonucleic Acid Species from Vegetative Cells and Spores of Bacillus subtilis by Methylated Albumin-Kieselguhr and Reversed-Phase Chromatography

Lysyl-transfer ribonucleic acid (tRNA) species from a spore-forming strain of Bacillus subtilis (168 trp2(-)) and an early blocked asporogenous mutant (spoA 12) were compared on reversed-phase and methylated albumin-kieselguhr columns. Lysyl-tRNA species from spores and the asporogenous mutant in stationary phase both exhibited altered chromatographic profiles compared to that of log-phase cells. The major peak in spore lysyl-tRNA species eluted later than that characteristic of vegetative cells, whereas the major peak of the lysyl-tRNA species from the asporogenous mutant in stationary phase eluted earlier. Although the early eluting lysyl-tRNA species was observable on methylated albumin columns, the late eluting peak was not detectable by that column technique. By using a shallower gradient on an RPC-2 column, the resolution of all lysyl-tRNA species increased. Several subspecies were revealed. The chromatographic comparisons clearly show that both the spore-forming strain and the asporogenous mutant undergo relative increases in different lysyl-tRNA species when grown to late stationary phase. No new species seem to be involved but rather altered amounts of minor species existing in log-phase cells. The experiments also demonstrate the usefulness of reversed-phase columns for such comparisons.

Synthesis of stable RNA by a stringent Escherichia coli strain during specific amino acid deprivation

By definition amino‐acid starvation of an RCstrEscherichia coli strain causes simultaneous arrest of RNA and protein synthesis. However, it has recently been demonstrated that under such conditions mRNA synthesis does not stop and this result showed that RNA synthesis is not a coordinated process. The present study, which was carried out to determine the effects of amino‐acid starvation on net RNA synthesis in an RCstrE. coli strain, has led to the following results:a) during amino‐acid starvation net RNA synthesis is reduced to about 5% of the normal level;b) ribosomal RNA appears in 50 S and 30 S ribosomes and in 38 S and 21 S particles which seem to be precursors of 50 S and 30 S ribosomes, respectively;c) analysis by chromatography on methylated albumin column and by electrophoresis on polyacrylamide gels shows that the 38 S particle is devoid of 5 S RNA;d) a 5 S RNA appears in the supernatant fraction of ribosome preparations. This RNA, which is slightly different from the true 5 S RNA, is called 5 S stringent RNA. With respect to its chromatographic behaviour on G‐100 Sephadex and methylated albumin column, its mobility on disc electrophoresis, it is identical to 5 S relax RNA and 5 S chloromycetin RNA which are synthesized when protein synthesis is blocked either by amino‐acid starvation of a RCrelE. coli strain or by addition of chloromycetin;e) from the relative quantity of the different classes of RNA it is concluded that RNA synthesis is not coordinated and from the appearance of rRNA in ribosomal particles, a mechanism concerning the regulation of RNA synthesis is proposed.

Characterization of rapidly labelled rat liver ribonucleic acid showing high affinity for columns of methylated albumin on kieselguhr.

Most of the rapidly labelled RNA from rat liver submitted to column chromatography on methylated albumin on kieselguhr remains tightly bound to the column and can only be recovered by elution with m-ammonia. The tightly bound RNA is composed mainly of DNA-like RNA. The binding capacity is dependent not only on base composition but also on molecular size: the heavier RNA molecules show a greater affinity to the column than do the lower-molecular-weight components. Rapidly labelled mouse liver and Saccharomyces cerevisiae RNA show similar behaviour to rat liver RNA on columns of methylated albumin on kieselguhr.

Intranuclear accumulation of RNA resembling the smaller ribosomal RNA component.

ABSTRACT A study was made of the nuclear RNA in HeLa cells with particular reference to the rapidly labelled fractions. It was found that if cells were incubated at a high density, that is, under ‘step-down’ conditions, there was a rapid accumulation of RNA in the nucleus. The fraction of the nuclear RNA which includes rapidly labelled RNA and which binds tightly to columns of methylated albumin on kieselguhr increased in amount and reached levels which permitted enough of the material to be isolated for direct measurement of its base composition. This was found to be very similar to that of 16s ribosomal RNA. When cells growing logarithmically were treated with low concentrations of actinomycin D and then incubated in the presence of [“[3H]uridine it was found that an RNA fraction which bound tightly to methylated albumin on kieselguhr again accumulated in the nucleus. This fraction resembled that which accumulated under ‘step-down’ conditions. It contained over 85 % of the total radioactivity in the nuclear RNA and again had a base composition very similar to 16s ribosomal RNA. Since nucleolar RNA synthesis was inhibited by the concentrations of actinomycin D used, it appeared that an RNA closely resembling 16s ribosomal RNA was synthesized outside the nucleolus. Sedimentation patterns on sucrose density gradients and thermal denaturation profiles lent support to the view that the RNA which binds tightly to columns of methylated albumin on kieselguhr probably represents ‘nascent’ 16s ribosomal RNA.

A scintillation probe for efficient counting of low energy beta rays in the elution from liquid or gas chromatographs

A counting system is described for the continuous scanning of soft beta ray emitters in the eluate from liquid or gas chromatographic columns. A relatively small portion of the column stream is diverted through the counting system while the main portion is passed through a light absorbance measurement and recording device to a fraction collector. The stream to be counted is mixed with liquid scintillator by cavitation and then nebulized into the counting chamber using a suitable gas to give a counting efficiency equivalent to that of vial counting. Radioactive counts and absorbance measurements can be plotted simultaneously on a chart recorder. The convenience and efficiency of the system was demonstrated by the recording of the counting and absorbance data for an aqueous elution of s-RNA, DNA and DNA-like RNA from a methylated albumin column.

Synthesis of replicative form deoxyribonucleic acid and messenger ribonucleic acid by gene IV mutants of bacteriophage S13.

Gene IV mutants of bacteriophage S13 are known to be blocked in infectious replicative form (RF) DNA synthesis, producing only a small fraction of the RF formed by wild-type phage. This investigation shows that gene IV mutants form only parental RF and are blocked in the synthesis of any progeny RF, either infectious or noninfectious. This was determined by density labeling of RF in cells treated with mitomycin C to suppress host deoxyribonucleic acid (DNA) synthesis. RF synthesis was also studied in untreated cells, using methylated albumin columns to separate RF from host DNA. In this case it was also found that synthesis of progeny RF by gene IV mutants is negligible. It has been found by DNA-ribonucleic acid (RNA) hybridization experiments that gene IV mutants form at least as much or more messenger RNA than wild-type phage. Therefore, parental RF alone can form messenger RNA in appreciable amounts.

The in vitro product of poliovirus polymerase.

Two types of ribonucleic acids (RNA) were synthesized between 2 and 4 h after infection of HeLa cells with the Mahoney strain of type 1 poliovirus. One type of RNA eluted from methylated albumin (MAK) columns with 0.8 M NaCl and was partially resistant to ribonuclease (RNase). The second species of RNA eluted from MAK columns with 0.9 M NaCl and was sensitive to RNase. The in vitro product of poliovirus polymerase was a mixture of single- and double-stranded RNA.

Chromatographic studies on RNA synthesis in the midgut of the silkworm, Bombyx mori, infected with a cytoplasmic-polyhedrosis virus

RNA synthesis in the midgut of the silkworm, Bombyx mori, infected with a cytoplasmic-polyhedrosis virus was investigated by means of the incorporation of 32P, using methylated-albumin column chromatography. In an early stage of infection, 32P was incorporated into cellular RNA within 24 hr after the isotope injection, but the incorporation into viral RNA (v-RNA) could not be recognized. Twenty-four hr later, when the typical symptoms of the disease start to appear, the v-RNA fraction becomes detectable spectrophotometrically, which is in fact substantiated by 32P incorporation into v-RNA. At 72 hr after the virus inoculation per os, when the virus multiplication level is very high, only 4 hr after 32P injection the radioisotope was incorporated into all RNA's including v-RNA, and during the successive 12 hr marked increase of incorporation into v-RNA was observed. The specific radioactivity of v-RNA at 16 hr after 32P injection reached about threefold that of ribosomal RNA. On the basis of these results, v-RNA synthesis during multiplication of the virus was discussed in connection with the cellular nucleic acid metabolism.

A specific modification next to the anticodon of phenylalanine transfer ribonucleic acid.

By mild treatment of tRNAPheyeast with acid (pH 2.9, 37°, 3–4 hours) the base Y+, which according to RajBhandary, Khorana et al. is located right next to the anticodon, can be excised without breaking the chain. Y+ and the acid treated tRNAPhe (=tRNAPheHCl) were characterized and the conversion of tRNAPhe to tRNAPheHCl was investigated in detail. 1 Y+ is a rather hydrophobic compound of unknown structure. Y+ and ribose were found when the nucleoside Y was treated with acid. Some physical and chemical properties of Y+ are reported. Y+ can be isolated easily in pure form from oligonucleotides, tRNAPheYeast, or even unfractionated tRNAyeast (not tRNAcoli) by mild acid treatment and subsequent extraction into chloroform. 2 tRNAPheHCl and tRNAPhe have very similar molecular sizes and shapes since their Sephadex elution profiles were overlapping. The facile separation of tRNAPhe and tRNAPheHCl by counter current distribution, on columns of methylated albumin on Kieselgur or benzoylated DEAE-celluose is due, at least in part, to the removal of a hydrophobic residue from an exposed region of the tRNAPhe. 3 tRNAPheHCl can be charged with Phe to the same extent as tRNAPhe. Since also the Michaelis constant of phenylalanyl tRNA synthetase is the same for both tRNAs (1.5×10−7 M) and the maximal velocity of charging is decreased by only one third in tRNAPheHCl, it can be concluded, that Y and probably the whole anticodon loop are not essential parts of the synthetase recognition region of tRNAPhe in homologous charging reactions. 4 It was found that tRNAPheHCl and tRNAPhe from which the –CpCpA endgroups had been removed do not differ in their acceptor capacities for CTP and ATP. 5 In the ribosome systems, Phe-tRNAPheHCl, does not bind to poly (U) or other polynucleotides and does not transfer its amino acid onto growing peptide chains. The complete loss of codon recognition is attributed largely to a change in the conformation of the anticodon loop. The ultraviolet spectra of an Y-containing hexanucleotide and of its acid conversion products are in agreement with the notion of a strong interaction between Y+ and its neighbouring bases, as are the rate differences in the liberation of Y+ from Y [1.5 times faster] and the hexanucleotide, respectively. 6 The rate of tRNAPheHCl formation, as measured by methylated albumin on Kieselgur-column chromatography and binding assays, is the same as the rate of Y+-liberation from the hexanucleotide. The acid induced conversion of tRNAPhe to tRNAPheHCl is a completely selective reaction affecting Y as the only nucleoside of tRNAPhe.