Kieselguhr Column Research Articles

Base composition and column chromatography studies of ribonucleic acid differentially extracted from pea roots with sodium lauryl sulfate or p-amino salicylate

Three-day-old pea roots, aseptically grown, were incubated for 2 hr in 32P phosphate and extracted by the phenol method in the absence of sodium lauryl sulfate or p-amino salicylate. One or the other of these compounds was then added and the residue was re-extracted. RNA obtained by the re-extraction with either compound was of higher specific activity and contained a higher mole percent AMP than the RNA from the first extraction. A small part of the AMP-rich RNA eluted with DNA during chromatography on methylated albumin kieselguhr columns, but most of it adhered to the columns. Presence of unlabelled carrier nucleic acids had no measurable effect on the elution pattern.

Abscission: The Role of RNA Synthesis

Ethylene stimulated the incorporation of (32)P into RNA in the abscission zone of bean explants (Phaseolus vulgaris L. var. Red Kidney). The enhancement was observed in all fractions separated by methylated albumin kieselguhr column chromatography, although the magnitude of the increase was not the same for each fraction. Differential extraction of the nucleic acids indicated that the ethylene stimulation was confined to the fraction extracted with sodium lauryl sulfate, with the increase mainly in Fraction III (Ribosomal RNA) and Fraction IV (Messenger RNA). Actinomycin D, which blocks ethylene-stimulated abscission, inhibited (32)P incorporation into all column fractions. 5-Fluorouracil, which blocked 50% of the ethylene-enhanced (32)P incorporation, did not inhibit ethylene-enhanced abscission. The results indicate that ethylene may regulate abscission through control of specific RNA's.

Correlation between replication and degradation of cellular DNA in polyoma virus-infected cells

Although approximately only 20% of the cells in polyoma virus-infected mouse embryo cultures synthesize viral antigen, approximately 93% of the cells synthesize DNA at some time during the infective process. The synthesis of DNA is not synchronized and in some of the cells in the cultures there is a delay in the onset of DNA synthesis. This delay is dependent upon the stage of the growth cycle of the cell at the time of infection. In all the cells in the infected cultures, the DNA is degraded during the later stages of the infective process and elutes from methylated albumin kieselguhr columns in a position characteristic of DNA with a molecular weight approximately that of viral DNA; this degradation occurs only after the DNA has replicated in the infected cells.

Dormin (Abscisin II), inhibitor of plant DNA synthesis?

Dormin (abscisin II), inhibits growth of Lemna minor cultures. At 1 part per million (3.8x10(-6)M), the culture appears nearly completely dormant but can be revived readily by transferring it to fresh medium free of dormin. The cytokinin benzyladenine, but not auxin or gibberellin, will counteract the dormin effect. Quantitative restoration of normal growth by cytokinin, however, can be achieved only if the dormin concentration does not exceed a critical level. Separation, after phenol-detergent extraction, of nucleic acids on methylated albumin kieselguhr columns showed suppression of nucleic acid synthesis by dormin in all fractions. Inhibition of the synthesis of the DNA fraction seems to precede that of RNA. Cytokinins reverse the process. They promote synthesis of all nucleic acid fractions, but again DNA seems to lead. Further work on the interaction of dormin with growth-promoting hormones might be facilitated by adopting the Monod model of allosteric transition, with, for example, DNA polymerase as the protein, dormin as the inhibitor, and cytokinin or other growth promoters as activators.

Effects of Phosfon-S on Nucleic Acid Metabolism in Pisum sativum Alaska

Phosfon-S, a substance which inhibits stem elongation, alters nucleic acid metabolism in Pisum sativum Alaska. Methylated albumin kieselguhr (MAK) columns were used to fractionate (32)P-labeled nucleic acids. Phosfon-S treatment of the plants resulted in a decrease in soluble RNA and an increase in ribosomal RNA. Specific activities of the various nucleic acid fractions were lower as a result of treatment. The nucleic acids from treated tissues were more resistant to RNase degradation, and endogenous RNase activity was lower in treated tissues. When RNase treated nucleic acids were fractionated on MAK columns, the DNA-RNA fractions from treated plants had a higher specific activity than that of the control, which was not true before nuclease treatment. Spectrophotometric examination of this fraction revealed a difference in absorption spectra, possibly indicating a Phosfon-S nucleic acid complex. It is suggested that these alterations in nucleic acid metabolism could in turn alter a wide variety of metabolic processes, resulting in retarded growth.

Allomorphic Forms of Bacteriophage φX-174 Replicative DNA

The bacteriophage øX-174 replicative form of DNA exists in two configurations, as judged by electron microscopy and sucrose density gradient sedimentation, and on methylated albumin-coated kieselguhr column chromatography. The predominant 21S form of the DNA is a tightly twisted circle. Treatment with deoxyribonuclease causes a single scission in one of the two strands, resulting in an open circle which sediments at 16S.

Synthesis of ribosomal RNA during growth and division in lilium

The synthesis of RNA has been examined in the microspore of lily buds during division and later development into mature pollen grains. RNA synthesis is most active just before division (G2) and after the microspore nuclear division. Column chromatography using a methylated-albumin Kieselguhr column has shown that ribosomal RNA makes up the major portion of RNA being synthesized during this period, when nucleoli are large and active. All of the conventional cytological criteria have failed to demonstrate a nucleolus in growing pollen tubes. RNA is synthesized in pollen tubes but the abrupt drop in specific activity at the ribosomal peaks and the base ratios of this RNA do not correspond to ribosomal RNA. The conclusion seems apparent that little, if any, ribosomal RNA is being synthesized in the pollen tube nuclei in the absence of nucleolar activity.

Modification of leucyl-sRNA after bacteriophage infection

Aminoacyl-sRNA's of Escherichia coli B with and without bacteriophage T2 infection have been examined using methylated albumin kieselguhr column chromatography. Out of 17 aminoacyl-sRNA's examined, only one, leucyl-sRNA, was found to be clearly different before and after the infection. This alteration lies in sRNA and not in leueyl-sRNA synthetase, and is one of the earliest events after the infection. Phage T4 and T6 infections show the same modification of leueyl-sRNA, whereas infection with T1, T3, T5 and T7 and λ induction do not induce the modification. Leucyl-sRNA was examined in Shigella dysenteriae 60 and in various strains of E. coli after T2 infection, and in permissive and nonpermissive hosts after infection with various early amber mutants of T4. In all cases, the same modification of the leueyl-sRNA was observed. It is not excluded that the leucyl-sRNA change may take some role in the arrest of host-protein synthesis

Identification of the pigment in amniotic fluid of erythroblastosis as bilirubin

The yellow pigment found in amniotic fluid obtained from pregnancies with fetusesaffected with erythroblastosis behaves in exactly the same manner as does bilirubin on spectrophotometric scanning and diazotization after chloroform extraction and when developed by reverse phase kieselguhr column chromatography.

Species specificity of tyrosine transfer RNA's from Escherichia coli and yeast

Transfer RNA's from Escherichia coli and yeast were fractionated on a protamine-coated kieselguhr column. The elution patterns from the column were similar to that from methylated albumin columns. Using these fractionated tRNA's, it was shown that yeast tyrosine RNA oxidized by periodate inhibited competitively the formation of yeast tyrosyl RNA by purified yeast tyrosine enzyme. The K m value for tyrosine RNA was 3·5×10 −7 M and the K 1 value for oxidized yeast tyrosine RNA was 5·1×10 −7 M . On the other hand, yeast tyrosine enzyme could not synthesize E. coli tyrosyl RNA from E. coli tyrosine RNA. However, native tyrosine RNA of E. coli did not show an inhibitory effect for yeast tyrosyl RNA synthetase. This suggests that E. coli tyrosine tRNA is unable to interact with yeast tyrosine enzyme. Oxidized E. coli tRNA behaves in a similar manner. It was also found that yeast tyrosine RNA did not interact with E. coli tyrosyl RNA synthetase. Based on these results, two possible mechanisms of species specificity of tRNA's are suggested.

Differences in lysine-sRNA from spore and vegetative cells of Bacillus subtillis.

The changes in the syntheses of macromolecules that aecompany sporulation of Bacillus subtilis have been the subject of several recent reports. In brief, the available data show that as a rapidly growing culture of B. sublilis departs from strict logarithmic growth, the net synthesis of RNA is abruptly arrested although the protein and DNA contenit approximately double after that time.' De novo RNA synthesis, however, measured by the incorporationi of labeled precursors, coiitinues throughout the stationary and sporulation phase but is just offset by a degradation of cellular RNA. The IRNA synthesized during this time appears not only in the messeniger fraction but also in soluble and ribosomal fractioiis.'-3 Despite this active turniover of soluble and ribosomal IRNA durilng sporulation, the RNA composition of B. subtilis spores appears to be only slightly different from that of vegetative cells. The ratio of soluble to ribosomal RNA increases during stationary phase, but the base compositions of these species are not signifieantly different from their counterparts in vegetative cells.4I However, Doi6 has observed an asymmetry between the elution profiles from methylated albumin kieselguhr columns of soluble RNA from vegetative cells and spores, indicating an alteration of this fraction during sporulation. In the present communication, the abilities of sRNA from spores and vegetative cells of B. subtilis to accept several amino acids are compared. Of the six amino acids tested, all but lysine were incorporated into spore sRNA to a lesser extent than into vegetative sRNA. The incorporation of lysine into sporal sRNA was more than twice that observed when vegetative sRNA was employed as an aeceptor. Comparison of the elution profiles of spore and vegetative lysyl-sRNA shows that spore sRNA contains a component not found in appreciable amounts in vegetative slRNA.

RNA of low molecular weight in KB cells infected with adenovirus type 2

Chromatographic analysis on methylated albumin—kieselguhr columns of newly formed, radioactive RNA from KB cells infected with adenovirus type 2 revealed an unusual radioactive component which was eluted following 4 s RNA. This RNA was present predominantly in the 100,000 g ribosomal supernatant cell fraction, and was distinguished from 4 s, 16 s and 28 s RNA's on the basis of its behavior on methylated albumin columns, base composition, sedimentation velocity and behavior on Sephadex G100 columns. This RNA component has certain characteristics in common with a “5 s” RNA associated with ribosomes from uninfected KB cells and a variety of other organisms. The effect of deoxy-fluorouridine and actinomycin D on the metabolism of this unusual RNA suggests that DNA synthesis and transcription of DNA into RNA are required for its formation.

Isolation and Characterization of Ribonucleic Acid from Cerebral Cortex of Rat

Abstract Ribonucleic acid isolated from cerebral cortex of mature male and female rats was fractionated by chromatography on methylated albumin Kieselguhr columns or by sucrose gradient centrifugation. Three principal fractions were obtained with analytical ultracentrifuge s20,w0 values of 4, 17, and 28. By comparison with ribonucleic acid prepared from purified rat brain ribosomes, the 17 S and 28 S fractions were shown to be predominantly ribosomal ribonucleic acid. Differences in base analyses between the three fractions were small but significant. Compositions of all fractions were markedly complementary with G ≈ C and A ≈ U.

Chromatographic studies of RNA synthesis in tobacco leaf tissues infected with tobacco mosaic virus

RNA synthesis in tobacco leaf tissues infected with tobacco mosaic virus (TMV) was investigated by chromatography with methylated albumin kieselguhr columns. When the relative rates of host- and TMV-RNA synthesis in the infected tissues were compared by exposing the tissues to P 32 for 30–60 minutes at different times after inoculation, there were observed distinctive changes in the pattern of RNA synthesis in the infected leaf tissues as infection time elapsed. Although, at earlier stages of infection, there was an increase of host-RNA synthesis which seemed to be induced by virus infection, it was less at later times. The relative rate of TMV-RNA synthesis was highest at 48 hours after inoculation. Both host- and TMV-RNA synthesis continued at 96 hours after inoculation, though lower. In the infected leaf tissues treated with actinomycin D and labeled with P 32 or both P 32 and tritiated guanosine, host-RNA synthesis was markedly suppressed whereas TMV-RNA synthesis was not. This result confirms previously reported suggestions that TMV-RNA synthesis is independent of host DNA.

Fractionation of deoxyribonucleic acid from phage-infected bacteria.

1. DNA has been isolated in 90% yield from T5-infected cultures of Escherichia coli ;pulse'-labelled with [(3)H]thymidine. It had a buoyant density in caesium chloride solution identical with the DNA of mature T5 phage, and no components of unusual buoyant density were detected. 2. The DNA preparation was resolved into two major components of differing specific activity on a column of kieselguhr coated with methylated serum albumin. The DNA of high specific activity could be eluted from the column only with 2n-ammonia, and the firm binding did not appear to be due to an artifact of preparation. 3. A similar fractionation into two DNA components of differing specific activity was observed when the ;pulse'-labelled culture was lysed with sodium dodecyl sulphate and the lysate rocked with phenol. The DNA of high specific activity was found in the interface precipitate between the phenol and aqueous layers. 4. The amounts of DNA in the two fractions were measured at different times after infection and the radioactivity content of each was determined at various times after a short ;pulse' of [(3)H]thymidine. The interface fraction contained the replicating phage DNA, and the DNA from mature phage particles appeared in the aqueous fraction. 5. Analogous results were obtained with T2-infected E. coli. In the presence of chloramphenicol the DNA in the interface fraction was not converted into DNA extractable into the aqueous layer. Since chloramphenicol prevents the condensation of DNA into phage heads, it is suggested that any DNA in extended configuration is trapped inside the rigid-layer framework of the cell wall. 6. Treatment with lysozyme released much of the DNA from the interface precipitate. This DNA was firmly bound by the chromatographic column and had the same buoyant density in caesium chloride solution as normal T5-phage DNA. Sucrose-gradient sedimentation studies showed that it was heterogeneous and that as much as 60% sedimented faster than T5-phage DNA.

SEQUENTIAL SYNTHESES OF VARIOUS MOLECULAR SPECIES OF RIBONUCLEIC ACID IN THE EARLY PHASE OF CONIDIA GERMINATION IN ASPERGILLUS ORYZAE

Ribonucleic acid synthesis in early stages of germination of Aspergillus oryzae conidia was studied using orthophosphate 32P or 14CO2 as precursor. RNA was fractionated by the methylated albumin-coated kieselguhr column chromatography or by the sucrose density gradient centrifugation analysis.RNA synthesis occurred at any time of germination so far observed and rRNA or its precursor was synthesized at the initial step of germination. sRNA synthesis occurred a little latter than the onset of germination and mRNA synthesis became apparent after 60min of incubation. Even in non-germinating medium (phosphate buffer), conidia could synthesize rRNA. It was suggested that the synthesis of rRNA or its precursor may be one of the starting reactions being driven at the very initial step of germination.

Fate of transforming DNA in the Haemophilus influenzae transformation system

Highly transformable Haemophilus influenzae bacteria were exposed to transforming DNA during one or two minutes. Unabsorbed DNA was removed by centrifugation and washing of the recipient cells. These were thereafter re-incubated in various media for various periods and then processed. Extracts were analyzed by means of dialysis, methylated albumin kieselguhr column chromatography, CsCl gradient centrifugation, sucrose gradient centrifugation and counting of biological activity and radioactivity in order to obtain information about the fate of the absorbed transforming DNA. The DNA preparations studied were either unlabeled or labeled with bromouracil, 32 P or [ 3 H]thymidine. The results strongly suggest that transforming DNA penetrates into the bacteria without being degraded. Within 10 minutes, however, about 75% is depolymerized; of this, roughly two-thirds are excreted as dialyzable material. The surviving DNA has so far retained its double-stranded form and its initial molecular weight. Incorporation of this DNA into the chromosome proceeds from the 10th to between the 30th and 60th minute, depending upon the conditions employed. The fact that the label is also incorporated into the bacterial genome rules out the so-called copy-choice DNA recombination scheme. Single-stranded DNA has been looked for in the extracts but it was not found. It is tentatively concluded that DNA recombination in H. influenzae can be described by the breakage-rejoining hypothesis, in contradistinction to the single-stranded DNA incorporation thought to take place in the Diplococcus pneumoniae transformation system.

The isolation of methyl deficient phenylalanyl transfer RNA from [formula omitted][formula omitted

The study of the role of methylated bases in transfer RNA (t-RNA) has been made possible by the discovery by Mandel and Borek (1961) that methyl deficient s-RNA is synthesized by a “relaxed” methionine requiring mutant of E.coli during methionine starvation. Preparations of s-RNA from methionine starved cells and from normal cells have been compared but no significant differences in the ability of these RNA's to accept (Starr, 1963; Littauer et al., 1963; Peterkofsky et al. 1964) and to transfer amino acids (Littauer et al., 1963; Peterkofsky et al. 1964) have been detected. Although this may indicate that the methylated bases do not participate in these functions of t-RNA, it should be recalled that “methionine starved” RNA preparations consist of a mixture of equal amounts of methyl deficient and normal s-RNA. Therefore, small differences in the activities of these RNA species invitro, which could be very critical invivo, might not have been detected with such crude RNA preparations. These considerations prompted us to develop a method for the isolation of methyl deficient t-RNA. Phenylalanyl transfer RNA (phe-t-RNA) from E.coli G-15, a relaxed double mutant which can be starved for methionine or for histidine, was selected for these studies. The present report describes the isolation of methyl deficient phe-t-RNA by chromatography on methylated albumin kieselguhr (MAK) columns.

Chromatography of tobacco mosaic virus, its constituents, and nucleic acids extracted from infected tobacco leaf tissues

Purified tobacco mosaic virus (TMV), TMV-ribonucleic acid (RNA), and aggregated TMV-protein were absorbed and eluted at specific NaCl concentrations by chromatography with methylated albumin kieselguhr columns. The A-protein, an alkaline hydrolyzate of the aggregated TMV-protein, was not absorbed. It is suggested that the specific elution of TMV and the aggregated TMV-protein were due to their quaternary structures. In the chromatography of the nucleic acids extracted from TMV-infected tobacco leaf tissues by the pyrophosphate-phenol method, infectious RNA was eluted later than ribosomal RNA as a single peak. There were no other differences between the chromatographs of nucleic acids from infected tissues and those from healthy tissues. Extraction by pyrophosphate and phenol directly from infected leaves yielded more infectious RNA than extraction by sodium dodecyl sulfate and phenol after homogenization.

Some abnormal properties of chloramphenicol RNA

A detailed analysis of the ribosomal components of chloramphenicol ribonucleic acid has revealed slight but definite differences from normal ribosomal ribonucleic acid. Both the 16 s and the 23 s components of chloramphenicol ribonucleic acid sedimented slightly faster through sucrose gradients, and had slightly less affinity for columns of methylated albumin-coated kieselguhr, than their normal counterparts. These differences are explainable in terms of secondary structure; chloramphenicol ribosomal ribonucleic acid may have a greater degree of ordered helical configuration than normal ribosomal ribonucleic acid.