MAK Column Chromatography Research Articles

Effect of IAA on growth, organogenesis and RNA metabolism during the development of Cichorium intybus root explants cultured „in vitro"

The effects of 3-indolylacetic acid (IAA) on growth, organogenesis, RNA content, RNase activity and MAK elution profiles during the development of chicory root explants cultured in vitro were investigated. It was found that the intensive callus growth in the presence of IAA was accompanied by an increase in the RNA content, with simultaneous decrease of RNase activity. Fractionation of RNA by MAK column chromatography showed that the high content of RNA in the callus under the influence of IAA was due to an increased accumulation of the ribosomal fraction of RNA mainly. Experiments with actinomycin D demonstrated that this antibiotic abolished both the auxin-induced callus growth and the inhibitory effect of IAA on bud formation. No significant inhibition of spontaneous bud formation under the influence of actinomycin D was observed. The possible relationship between the IAA-affected morphogenetic processes and RNA metabolism is discussed.

Nucleic acids in the last stages of maturation of two varieties

Using the MAK column chromatography method the process of wheat grain maturation was found to be closely correlated with nucleic acid metabolism. The amount of nucleic acids in the embryos increased until the end of the maturation of the grain. This increase was the result of an intensive synthesis mainly of rRNA fractions and to some extent also of sRNA. The quantitative proportion of various fractions of nucleic acids also changed in the last stages of grain maturation. In the period of wax maturity in wheat embryos a higher content of 4S and 5S RNA was found which partly explains a higher viability of these seeds.

Three Size Classes of Higher Plant DNA-Like RNA Grouped by MAK Column Chromatography IV. Molecular Size of Rapidly Labeled RNA Binding Tightly to MAK Columns

Three Size Classes of Higher Plant DNA-Like RNA Grouped by MAK Column Chromatography IV. Molecular Size of Rapidly Labeled RNA Binding Tightly to MAK Columns

Inosit-Phosphate ausLemna minor L.

Summary Inositol phosphates from Lemna minor L. or technical products of phytic acids show an UV-absorption with a peak at 260 nm. This finding shows another way of indicating inositol phosphates. Inositol phosphates from Lemna minor were extracted together with condensed phosphates and nucleic acids by phenol. During MAK column chromatography most of the inositol phosphates were isolated by elution with 100 ml NaCl-phosphate buffer. In this solution all 6 inositol phosphates could be separated by means of a Dowex 1 X 4 (Cl − ) column according to their charge and by means of a Sephadex G 15 column according to their molecular weight. Inositol phosphates were identified after biosynthetic labeling with 14 C-glucose, 3 H-inositol, and 32 P-orthophosphate. Furthermore extracted inositol phosphates showed an UV-absorption and they could be diminished encymatically by phytase. There was no difference during chromatography on MAK, Dowex 1 X 4, and Sephadex in comparison to the elution of reference compounds. Separated fractions of inositol phosphates always include a portion of condensed phosphates and vice versa, because there are only small differences between inositol phosphates and condensed phosphates regarding charge and molecular weight. For this reason an exact separation and phosphate estimation of each phosphate compound was necessary. The mixture of inositol phosphates and condensed phosphates was dialysed for 20 h to remove orthophosphate. In the following two-dimensional thin layer chromatography on cellulose inositol phosphates appeared beside the poly- and metaoligophosphates exactly in the area of the missing orthophosphate. When inositol phosphates were eluted from combined spots of several chromatograms, one could estimate, by a Sephadex G 15 column chromatography, the phosphate values of all inositol phosphates without any influence of other compounds. All the six differently phosphorylated inositol phosphates could be identified by comparison with commercially available reference compounds. The estimation of the condensed phosphates in Lemna minor is described in previous publications of the authors.

Three size classes of higher plant DNA-like RNA grouped by MAK column chromatography II. Species-specific grouping of DNA-like RNAs from various kinds of organisms

Three size classes of higher plant DNA-like RNA grouped by MAK column chromatography II. Species-specific grouping of DNA-like RNAs from various kinds of organisms

Evidence for ribosomal RNA synthesis in pollen tubes in culture

The evidence is presented that pollen tubes ofNicotiana tabacum L. cultivated in shaken suspension do synthesize 5S, 18S and 28S RNA. Following incubation of pollen tubes in the presence of radioactive uracil or uridine, RNA was isolated from total pollen tube material after the removal of 4S RNA, from polysomes and from 80S ribosomal particles, and fractionated by density gradient centrifugation and MAK column chromatography. The results obtained further suggest a higher rate of 5S RNA synthesis with respect to 18S+28S RNA.

Three Size Classes of Higher Plant DNA-like RNA Grouped by MAK Column Chromatography I. Fractionation and Characterization

Summary Rapidly labeled DNA-like RNA from broad bean shoots was separated with salt concentration gradients into three distinct size classes utilizing MAK 1 ) column chromatography. It was found that two minor classes were eluted just before light and heavy components of rRNAs, respectively, and a major one after them. Proportions of these three newly-formed RNA classes observed in the chromatographic profiles depend significantly upon the tissues used. The two minor classes of DNA-like RNA sedimented rather monodispersedly at about 13 S and 20 S but another of higher molecular weight was largely heterogeneous and sedimented more slowly than the heavy component of rRNA. These DNA-like RNA components were distinguishable from each other by SDS-polyacrylamide gel electrophoresis, and showed high hybridization with single-stranded homologous DNA. Hybridization of the 13 S RNA was effectively inhibited by the high-molecular-weight DNA-like RNA. In contrast, hybridization of the high-molecular-weight RNA class was only incompletely inhibited by the 13 S RNA. The poly(A) content in various classes of DNA-like RNA was found to be rather low. These observations suggest that the three classes of DNA-like RNA exist in the cell as entities of different molecular weights and that the MAK fractionation profiles result from grouping of heterogeneous DNA-like RNAs. The 13 S and 20 S RNA classes are most likely fractions of mRNA , most of which are deficient in sufficiently long poly(A)-segments. The grouping of DNA-like RNA into three size classes by MAK column chromatography is a general phenomenon in various kinds of organisms.

Immunogenicity of rice dwarf virus-ribonucleic acid.

Using rice dwarf virus (RDV)-RNA which was extracted from RDV and further purified by MAK-column chromatography, anti-RNA antibodies were produced in rabbits immunized with RDV-RNA antibodies were prods immunized with RDV-RNA-methylated bovine serum albumin complexes. The antisera, as analzyed by complement fixation, cross-reacted with synthetic double stranded RNAs (poly (A)-poly (U), poly (I)-poly (C)), but not with native or denatured DNA, rRNA, tRNA, 5 S RNA and nucleic acids from rice plants. RDV-RNA treated with heat or dimethylsulfoxide was markedly reduced in reactivity to the antisera. When RDV-RNA was digested with RNase A at low salt concentration, its complement fixation activity was abolished. In double diffusion tests, two different precipitation lines were demonstrated between the antiserum and RDV-RNA. One of the precipitation lines connected with those was formed between the antisera, poly (A)-poly (U) and poly (I)-poly (C). As regards immunoglobulin classes of the antibodies, one of the two rabbits employed had antibody activity only in IgG.

Three size classes of higher plant DNA-like RNA grouped by MAK column chromatography HI. Selective extractions and distributions in cytoplasm

Three size classes of higher plant DNA-like RNA grouped by MAK column chromatography HI. Selective extractions and distributions in cytoplasm

Further investigations on the property of chromatins from apical and axial parts of cauliflower inflorescence

To evaluate the importance of histone F1 for RNA synthesis in cauliflower in relation to the differentiation between apical and axial tissues, the F1 fraction from apical chromatin was removed and the property of the residual (F1-depleted) chromatin was investigated compared to that of axial chromatin. It was found that the derivative melting profile of the residual chromatin was quite similar to that of the axial chromatin, and the nucleotide composition of RNA synthesized with the residual chromatin as template was also like that of the axial chromatin. The phosphate content of the histone F1 fraction isolated from apical chromatin was markedly lower than that of the F1 fraction from axial chromatin. Further, it was ascertained from MAK-column chromatography of [ 14C]uridine incorporation into RNA synthesized in excised tissues that axial chromatin in situ has a much higher template activity for RNA synthesis than does apical chromatin. It was postulated that the higher RNA synthesizing activity in the axial part of the cauliflower head can be attributed mainly to the presence of active chromatin in which the histone F1-depleted region is more prevalent than it is in apical chromatin.

Effects of chemical animalization and cell dissociation on RNA synthesis in sea urchin embryos

Newly synthesized RNAs from chemically animalized embryos and from dissociated embryonic cells were analysed by means of MAK column chromatography in order to study whether temporal transition of transcriptional pattern during development of sea urchin embryos is closely relevant to the morphological change and the proper cell-to-cell interaction. The results show that both Evans Blue animalization and cell dissociation failed to alter the normal temporal transition of transcriptional pattern. Therefore, it seems unlikely that the temporal progression of stagedependent transcription is closely related to intercellular contact and morphogenetic events.

DNA eluted at unusual regions on mak column chromatography during metamorphosis in Bombyx mori

Nucleic acids from whole insects and isolated pupal abdomens of Bombyx mori, at successive stages of pupal-adult development were analyzed on MAK column chromatography. Four major peaks corresponding to tRNA, 5S RNA, DNA, and rRNA were eluted at all stages examined. Besides the four peaks, two new peaks X 1 and X 2 were found between the peaks of 5S RNA and DNA on the chromatograms in both whole insects and isolated abdomens. The occurrence of the new peaks was stage specific, being found up to the half-way stage in pupal-adult development in whole insects. Both the peaks were DNAse sensitive and RNAse resistant and were strongly positive to the diphenylamine reaction. Experiments on tissue distribution showed that the new peaks were detectable particularly in the midgut contents, the degenerating silkgland, and fat body of pupae and pharate adults: they were completely absent from the ovary, testis, Malpighian tubules, and from the carcass of the same insects. They were also absent from the larval midgut and silkgland. It is suggested that the new peaks may be composed of partly-degraded DNAs accompanying the histolysis during metamorphosis.

Biochemical studies during the development of the chick embryo. VI. On a deoxyribosyl compound in the ribonucleic acid preparation.

Ribonucleic acid prepared according to modified Kirby’s phenol method from 14 day old chick embryos was fractionated into three peaks by MAK column chromatography. To the peak eluted in the range from 0.65 to 0.7 m NaCl, the name “Peak II” was given tentatively. Purified Peak II was homogeneous upon ultracentrifugation, polyacrylamide gel electrophoresis and elution patterns of chromatographies.The peak gave a positive result with the orcinol reaction. Furthermore, the hydrolyzate by DNase and snake venom was active in promoting the growth of L. leichmannii 7830. The peak was not disrupted by treatment with RNase, but was with DNase. Peak II treated with heat gave the same fractionation profile as the native peak by gel filtration on Sephadex G–200.By treatment with 0.3 n KOH at 37°C for 18 hr, Peak II was resolved into two peaks, Peak II–1 and Peak II–2. It was found that Peak II consisted of the susceptible and the non-susceptible components to the alkali. From the susceptible component (Peak II–2) the ...

Small molecular weight DNA of the silkworm, Bombyx mori

DNA from 6-day-old isolated pupal abdomens of silkworm, Bombyx mori, was clearly separated into three components designated as X 1, X 2 and normal DNA on MAK column chromatography. These DNA components were characterized by means of some biochemical techniques. All DNA components exhibited a hyperchromic effect when they were treated with either heat or alkali, whereas treatment with formaldehyde caused no increase in u.v. absorbance of those components. Thermal denaturation curves were also typical compared to those from double-stranded nucleic acids. Base compositions of the three different components were very similar to each other having G + C contents around 39 to 40%. The ratios of both A/T and G/C were nearly unity. Centrifugation in an ultracentrifuge showed that X 1, X 2, and normal DNA all migrated as a single peak with S 20,w of 4.6, 7.3, and 17.6, respectively. When mol. wt was estimated from the S 20,w, X 1, and X 2-DNA were found to decrease in size approximately to 1 60 and 1 15 respectively, of the normal DNA. In polyacrylamide gel electrophoresis, X 2-DNA showed a polydisperse migration pattern consisting of at least four broad bands, while X 1 and normal DNA were both detected as a single band, the former being a rather broad one. The mol. wt of four DNA components given from X 2-DNA on polyacrylamide gel were integral multiples of the mol. wt of the smallest DNA component, X 1-DNA. These facts strongly indicate that X 1 and X 2-DNA are degradation products of normal DNA, and suggest that subunit structure of chromatin may contribute to produce such DNA components.

Methylation of RNA in Dormant and After-ripened Embryos of Agrostemma githago

The correlation between the physiological stage of Agrostemma githago seeds and the methylation of RNA have been studied. Dormant and after-ripened embryos were incubated with 3 H-uracil and 14 C-D,L-methionine (methyl-labelled). The 3 H/ 14 C-ratio was used as a measure for the degree of methylation (related to synthesis) of the RNA prepared from the embryos. The precipitable RNA compounds labelled with 3 H and 14 C, respectively, were hydrolyzed by pancreatic RNase to 95 and 70 %. It is concluded that some label of the [ 14 C-methyl]-group was incorporated into other TCA-insoluble substances, probably into pectins. In order to characterize the degree of methylation of the major classes of RNA in dormant and after-ripened embryos RNA was separated either by gel chromatography on Sephadex G 150 columns, by MAK-column chromatography, by polyacrylamidegel electrophoresis or by affinity chromatography on oligo (dT)-cellulose columns. The [ 14 C-methyl]-group of methionine was found predominantly in tRNA, 25S- and 18S RNA. The degree of methylation proved 3fold higher in tRNA than in rRNA, related to the rate of synthesis. 5S-RNA, heterogeneous RNA, and the poly(A)-RNA, prepared by affinity chromatography, were methylated not at all or only weakly. Methylation of DNA during the period of investigation could not be detected. Despite of their different physiological stages dormant (blocked) and after-ripened (activated) embryos of Agrostemma githago are not differentiated in the activity of methylation as well as synthesis of tRNA and rRNA. Those and other findings support our hypothesis that the dormancy of Agrostemma githago seeds is caused by a specific developmental block.

Untersuchungen über die DNA-Synthese während der Frühphase der Kallusbildung bei Zea mays-Wurzeln

The formation of callus tissues from root segments of Zea mays seedlings was generally induced on synthetic media by 2, 4-D at concentrations ranging from 2 to 20 mg/1; the subcultures of this callus tissue, however, were successfully cultivated at lower auxin concentrations (1 — 4 mg/1). Studies on the uptake and incorporation of (3H-methyl)-thymidine by root segments grown on a medium containing 5 mg/1 2,4-D have shown that the thymidine incorporation increased after a lag phase of 6 h and then reached two maxima: The first one after 24 h and the second one after 120 h. By treatment with specific inhibitors, actinomycin D (10−5 and 10−6 M), puromycin (10−4 and 10−5 M) or cycloheximide (10−4 and 10−5 M) the thymidine incorporation during the first period can be inhibited either partially or completely. The total nucleic acids prepared by phenol extraction were separated by MAK-column chromatography. In order to demonstrate the incorporation of 3H-thymidine into DNA the nucleic acids from all fractions of the resulting DNA-RNA-peak were again precipitated and furtherly characterized. The nucleic acids from the DNA-RNA-peak show a very high specific activity and are the only one labelled with 3H-thymidine. They are stable against RNase but not against pancreatic DNase I. Preparations from callusing roots show a higher 3H-thymidine incorporation and a higher specific activity in comparison to normal roots. From these findings it was concluded that an additional DNA-synthesis is necessary in the early period of callus formation.

RNA from callus cultures and leaves of Nicotiana tabacum

MAK column chromatography has been used to analyse RNA from normal and crown gall callus cultures and leaves of Nicotiana tabacum. To determine the elution behaviour of well-defined DNA-like RNAs with different GC content, complementary RNAs ( c-RNA) synthesized on Agrobacterium tumefaciens DNA and crown gall DNA were used. The elution profile of the RNA from all three tissues followed a similar pattern. By salt gradient elution the RNA in the tRNA region showed a remarkably high CMP content which was significantly higher for the normal tissues than for crown gall tissue. RNA from the callus cultures contained more DNA-like RNA (D-RNA) with a higher turnover rate than RNA from leaves. Because of its relatively low poly A content, measured as RNase A + T 1 resistance, as well as its high turnover rate, the salt-eluted D-RNA is thought to be heterogeneous nuclear RNA (Hn-RNA) and not mRNA. RNA molecules that might represent the mRNA population, having intramolecular poly A tracts, were subsequently eluted by a salt gradient, a low salt buffer and with the chaotropic agent guanidine thiocyanate, which removed tenaciously bound (TB-RNA) in two fractions, α and β. Crown gall RNA showed both a different labelling behaviour and a higher poly A content in the α and β fractions compared to the normal tissues. c-RNAs may be eluted at different salt concentrations because of their different GC content. They give rise to a considerable fraction of TB-RNA which in the presence of tobacco leaf RNA was split into fractions similar to α and β. No fraction was found amongst these RNAs which did have intramolecular poly A tracts.

Biochemical evidence of DNA transport from the silk gland to the fat body of the silkworm, Bombyx mori

β-DNA, a component of DNA found in the pupal fat body of the silkworm, Bombyx mori, has the same GC content but a smaller molecular weight than typical silkworm DNA (α-DNA). Its origin and time of synthesis were studied by MAK column chromatography of phenol extracts after labelling with radioactive precursors. The DNA components of the fat body changed greatly during the early pupal stage, the β-DNA showing a striking increase relative to α-DNA. Thymidine-6- 3H and phosphoric acid- 32P injected into the animals 1 day before analysis caused labelling of α-DNA, but not of β-DNA of the fat body, indicating that β-DNA was not synthesized during the stage of its appearance in the fat body. On the other hand, injection of thymidine-6- 3H into 2-day-old fifth instar larvae, when DNA of the silk gland was being actively synthesized, gave high incorporation of the isotope into β-DNA of the pupal fat body. The sudden appearance of highly labelled β-DNA in the fat body during the early pupal stage as well as the occurrence of β-DNA in both the silk gland and fat body suggested that DNA might move from the silk gland to the fat body. It is possible that the fat body stores DNA as a nutrient from the degenerating silk gland.

RNA Synthesis and Polyribosome Metabolism in X-Irradiated Synchronized Tetrahymena

MAK column chromatography of RNA extracted from synchronized Tetrahymena after X-irradiation revealed age-dependent depressions in the cellular content of the various species of RNA and in the synthesis of Qi, Q2, and TD RNA. Sucrose densitygradient analysis of po]yribosomes extracted from irradiated cells revealed age-dependent depressions of cellular polyribosomes, of newly synthesized messenger RNA, and of nascent polypeptide synthesis. The results are interpreted as supporting the hypothesis that the radiation-induced delay of cell division may be due primarily to the radiationinduced depression of division-essential messenger RNA synthesis.

Increased aminoacylation of tRNA as a function of the Abnormal abdomen genotype of Drosophila melanogaster

Post-microsomal supernatant enzymes isolated from adults of the Abnormal Abdomen (A53g) mutant strain of Drosophila melanogaster stimulate the in vitro aminoacylation of tRNA. This stimulation is due to an altered enzyme system in the mutant which exhibits an increased level of aminoacylation of isoaccepting tRNA molecules. Observations of in vitro aminoacylation studies, as well as the results of MAK and BD-cellulose column chromatography of tRNAs aminoacylated by mutant and wild-type Drosophila supernatant enzymes, support this hypothesis.