Methylated Albumin Kieselguhr Column Research Articles

Fractions of nucleic acids in seedlings and leaves of winter wheat in the vegetative and generative development phases

The composition of nucleic acid preparations isolated from seedlings and leaves of plants grown from vernalized and nonvernalized winter wheat grain was chromatographically investigated on a methylated albumin kieselguhr column (MAK column). The intensity of synthesis of the particular nucleic acid fractions was compared by the <sup>32</sup>P-incorporation technique and the nucleotide composition of the newly synthesized nucleic acid fractions was determined. It was observed that the rate of <sup>32</sup>P incorporation into the sRNA, rRNA+mRNA and TB-RNA fractions of the leaves of vernalized plants was about two times higher as compared with that in the nonvernalized control.

Distribution of Incorporated, Synthetic Cytokinins in Ribosomal RNA Preparations from Tobacco Callus

The distribution of incorporated synthetic cytokinins (N(6)-[8-(14)C]benzyladenine ([8-(14)C]bzl(6)Ade) and N(6)[8-(14)C]furfuryladenine ([8-(14)C]fr(6)Ade) in ribosomal RNA prepared from tobacco callus (Nicotiana tabacum L. var. Wis. No. 38) grown in the presence of one of these for 25 or 26 days has been studied. The rRNA of tissue supplied with [8-(14)C]bzl(6)Ade or [8-(14)C]fr(6)Ade was fractionated by methylated albumin-Kieselguhr column chromatography and preparative gel electrophoresis, respectively. In each case about 80% of the incorporated cytokinin was recovered as the ribonucleoside [8-(14)C]bzl(6)A or [8-(14)C]fr(6)A in the rRNA peak after the fractionations. [8-(14)C]fr(6)A was found associated with both the 18S and 25S rRNA components in quantities roughly proportional to their 260 nm absorbance. This pattern of apparently nonspecific association was not affected by prior denaturation of the RNA with formamide.The distribution of [8-(14)C]fr(6)A moieties in mono- and oligonucleotides derived from combined treatment of [8-(14)C]fr(6)Ade-labeled rRNA with ribonuclease T(1) and pancreatic ribonuclease A was measured by fractionating the digest on a DEAE-cellulose column in the presence of 7 m urea and determining the [8-(14)C]fr(6)A content in each fraction. The [8-(14)C]fr(6)A content in the oligonucleotides varied from 46 to 210 mumol/mol of adenosine (A). The mol ratio of [8-(14)C]fr(6)A to A was three to four times greater for oligonucleotides containing uridine or cytidine ([A](n)Up or [A](n)Cp) than for those containing quanosine ([A](n)Gp).

Mammalian tRNA sulfurtransferase: properties of the enzyme in rat liver.

Transfer RNA sulfurtransferase activity was detected in 105,000 x g supernatant preparations from rat liver and several other rat tissues. Sulfur is transferred from [35S] cysteine to tRNA in a reaction which also requires ATP, Mg2+, and supernatant protein. While [35S] beta-mercaptopyruvate appeared to be a substrate for this enzyme, the reaction product was sensitive to deacylation and the reaction was inhibited by [32S] cysteine. Of the various nucleic acids tested, only tRNAs were effective sulfur acceptors, with rat liver tRNA being the poorest substrate. The [35S] reaction product was sensitive to ribonuclease, cochromatographed with tRNA on methylated-albumin kieselguhr columns, and was converted to nucleotide material after alkaline hydrolysis. DEAE-cellulose chromatography of the neutralized [35S] nucleotide digest revealed a single thionucleotide peak. These studies demonstrate that tRNA sulfurtransferase is present in various rat tissues, and that the requirements of the liver enzyme are similar to those of bacterial enzymes.

Alteration in Two Enzymatically Active Forms of Valyl-tRNA Synthetase during the Sporulation of <italic>Bacillus subtilis</italic>

Two enzymatically active forms of valyl-tRNA synthetase [EC 6.1.1.9] were found in the cells of Bacillus subtilis. The aminoacylation activities of the two forms were altered during the sporulation of B. subtilis. The tRNA'S acylated by these enzymes were analyzed by methylated albumin-Kieselguhr column chromatography.

Immunology of DNA. II. The effect of size and structure of the antigen on the farr assay

The Farr assay for the detection of antibodies to double stranded (ds) DNA is influenced by the DNA preparations used as antigen. To elucidate this the molecular weight of the antigen preparation, contamination with proteins and presence or absence of single stranded (ss) regions were studied with the following conclusions: 1) The degree of DNA binding by antibodies is linearly dependent on the molecular weight of the DNA, provided that this does not exceed 10 × 10 6. 2) Deproteinization of E. coli DNA by chromatography on methylated albumin-kieselguhr (MAK) columns results in lower binding by most sera. 3) ds DNA preparations sometimes contain ss regions which bind antibodies to ss DNA. The difference in behaviour of different ds DNA preparations may be ascribed entirely to these factors and not to differences in antigenic determinants. We have standardized the Farr assay and enhanced its specificity by the use of circular DNA isolated from bacteriophage PM 2.

Study of messenger-like RNA extracted from HeLa cells polysomes. II. Identification of the types of RNA eluted from methylated albumin kieselguhr columns.

Together with the elution pattern of pure messenger RNA molecules of various origin, the labelling kinetics of rapidly labelled heterogeneously sedimenting RNA (HSRNA) extracted from polysomes of HeLa cells have been studied by chromatogrphy on columns made of methylated bovine serum albumin adsorbed on kieselguhr. HSRNA is eluted within three peaks-IP, Q2P and TDP-following in that order the increase of NaC1 concentration in the eluting buffer. Besides peak TDP which results from an experimental artefact, our data suggest that the appearance of peaks IP and Q2P reflects the absence and presence respectively of polyadenylic acid stretches in these molecules. Within peak Q2P, the critical factor affecting the order of elution is the size of the polyadenylic acid stretch.

Incorporation of label from radioactive uridine into the stylar nucleic acids of Lilium longiflorum thunb. as affected by heat, 6-methylpurine and actinomycin D

5-(3)H-uridine injected into the stylar canal of detached lily stigma-styles was taken up initially into the rapidly-labeled-RNA of the nucleic acid profile of a methylated albumin kieselguhr (MAK) column but with increasing time was found in all portions of the RNA profile, but not in the DNA. Heat treatment of the style before injection of 5-(3)H-uridine greatly reduced the rate of incorporation of label into and the ultimate amount of label found in the RNA species of the lily style. Translocation of 5-(3)H-uridine through the ovary into heattreated pistils and the injection of 5-(3)H-uridine into styles which had been incubated for 1 or 2 days after heat treatment resulted in stylar nucleic acids more highly labeled than nucleic acids in control styles, with an incorporation pattern different than control styles. Heat treatment of lily pistils resulted in detectable changes in the proportion of stylar RNA species as separated on MAK columns and measured as absorbance units. Actinomycin D and 6-methylpurine treated styles incorporated label from a stylar injection of radioactive uridine in patterns different than each other, different than heat-treated styles and different than non-treated styles. 6-methylpurine and heat treatment of styles only slightly reduced the rate at which 5-(3)H-uridine was removed from the stylar canal into the stylar tissue.

Mechanism of suppressive effect of basic cupric acetate on rat liver carcinogenesis by ethionine.

The rate of ethylation of t-RNA in vivo by L-ethionine[ethyl-1-14C] markedly lowered in the liver of rats fed a diet containing copper than that of control. In corporation of the labeled compound into the t-RNA of liver by a single injection. of L-ethionine[ethyl-1-14C] was inhibited as much as 50% by the preceding concurrent administration of copper and ethionine added in the diet to rats. Direct interaction of copper ion with ethylation by ethione in the specific t-RNA of the rat liver was examined as an important biochemical explanation in molecular level for the inhibitory mechanism of copper on rat liver carcinogenesis by ethionine. Profiles of normal t-RNA on methylated albumin-kieselguhr column chromatography revealed three components for leucine, two of which were found at the tube number 40 to 50 and disappeared in the leucyl t-RNA treated with ethionine. The components for leucine that disappeared were normalized in the liver of rats by the concurrent administration of copper.

Deoxyribonucleic acid strandedness. Partial characterization of the antigenic regions binding antibodies in lupus erythematosus serum.

This study shows that tritiated thymidine labeled DNA prepared from mammalian cells by the Marmur technique is a pure preparation of nucleic acid that is composed essentially of two populations of molecules. One molecular population consists of primarily double-standed nucleic acid, while the other population is of double-stranded nucleic acid with significant single-stranded regions. The double-stranded DNA with single-stranded regions can, depending upon the length of the single strand, behave as "native" DNA or "denatured" DNA on methylated albumin kieselguhr (MAK) column chromatography, Using MAK chromatography we have separated the DNA into a saltelutable fraction composed of primarily double-stranded molecules and an alkaline-elutable fraction containing double-stranded nucleic acid with variable length, single-stranded regions. Endonuclease enzyme removal of the single-stranded regions from the alkaline fraction DNA yield nucleic acid that behaves identically to the salt elutable DNA. Exonuclease removal of the single-stranded regions suggests they are located primarily at the ends of the molecules. Our data show that the alkaline-elutable DNA differs from salt-elutable DNA only in that the former has significant single-stranded regions. Sera of patients with systemic lupus erythematosus (SLE) selected for anti-DNA by hemagglutination bind significantly less to the alkaline fraction DNA than the sale fraction DNA. This difference in binding clearly does not represent simply an affinity for double-stranded vs. single-stranded nucleic acid since the alkaline fraction DNA contains predominately double-stranded nucleic acid. A model for antibody-DNA binding is suggested from the present data and information contained in the literature.

CHANGES IN LABELLING OF RIBONUCLEIC ACID SPECIES FRACTIONATED FROM THE RAT VENTRAL PROSTATE IN RESPONSE TO TESTOSTERONE

ABSTRACT The total RNA isolated at various times up to 24 h after testosterone administration from the ventral prostate of castrated rats, was labelled either by injecting 3H-orotic acid directly into the ventral prostate 6 h before the animals were killed, or by incubating prostatic tissue in vitro with 3H-uridine for 20 to 60 min. The isolated RNA was separated into tRNA, ribosomal RNA (Q1 RNA) and two DNA-like RNA fractions (Q2) and TD RNA) by chromatography on methylated albumin kieselguhr (MAK) columns, and the fractions were further analysed by sucrose gradient centrifugation. Testosterone given into castrated animals for 12 h, stimulated the labelling of all main fractions. The radioactivity of TD RNA after a 60-min incubation period in vitro with 3H-uridine was approximately twice that seen in the castrated rat, while there was a 3.1- and 2.2-fold increase in the radioactivity of the Q1 and Q2 RNA fractions respectively. Kinetics of incorporation of 3H-uridine into different RNA fractions revealed that the hormone facilitated the labelling of the TD RNA fraction relatively more than that of the Q2 fraction. The injection of 3H-orotic acid into the ventral prostate labelled the Q1 RNA preferentially. More than 60 % of the recovered radioactivity was found in Q1 RNA (as 18 and 28 S). Testosterone increased markedly (9.4-fold) the labelling of this fraction. It was concluded that testosterone has an activatory effect on the production of ribosomal RNA, and the bulk of the testosterone effect on the total RNA labelling is to be found in this fraction. Furthermore, it seems likely that testosterone also stimulates both the synthesis and processing of DNA-like RNA. When actinomycin D was given 2 h before the hormone administration in a dose of 25 μg per 100 g of body weight, there was no noticeable increase in the labelling of any fraction above the level seen in the untreated castrated rat. There is evidence that testosterone exerts some effects on the labelling of proteins with radioactive amino acids and 14C-glucose metabolism in the absence of that fraction of the total RNA synthesis which is sensitive to a low dose (25 μg per 100 g of body weight) of actinomycin D (Isotalo & Santti 1972). In this way it may be concluded that the major changes of the RNA synthesis after testosterone administration are likely to be secondary to the protein synthesis and glucose metabolism, or the hormone exerts its anabolic effect on prostatic cells at different sites and by different modes of action, each of which can be operated independently.

Heterogeneity of the DNA preparation from light-anaerobically grown Rhodopseudomonas spheroides cells as revealed by methylated albumin kieselguhr column chromatography

Heterogeneity of the DNA preparation from light-anaerobically grown Rhodopseudomonas spheroides cells as revealed by methylated albumin kieselguhr column chromatography

Human leukemic intermediate DNA components.

The DNA extracted from human leukemic leucocytes has been fractionated on a methylated albumin kieselguhr (MAK) column. The different fractions obtained have been reannealed to a Cot value of 20 mol times sec/1 to study the distribution of the intermediate DNA on the MAK column. Intermediate DNA contains two components, one (CsCl density after reannealing, 1.703 g/ml) obtained by reannealing high molecular weight DNA, the other (CsCl density after reannealing, 1.707 g/ml) obtained only by reannealing sonicated low molecular weight DNA. High molecular weight intermediate DNA (1.703 component) is eluted early from the MAK column in the fractions corresponding to the main DNA peak, while low molecular weight intermediate DNA (1.707 component) is more widespread on the MAK column, but appears to be enriched in the fractions eluted later. The possibility is discussed that the latter component is interspersed in that part of the genome which is apparently more homogeneous in density in an analytical CsCl gradient, and is absent on the skewed, more heterogeneous, heavy side of the main DNA in CsCl.

Incorporation of radioactive label into nucleic acids of compatible and incompatible pollen tubes of Lilium longiflorum Thunb.

Stylar canal cells of Lilium longiflorum were labeled before pollination with 5-(3)H-uridine or 5-(3)H-orotic acid dissolved in water, or the stylar canal was filled 6 hr after pollination with stigmatic exudate supplemented with 5-(3)H-uridine, 2-(14)C-uridine or H3 (32)PO4. Nucleic acids extracted from compatible and incompatible pollen tubes grown in these styles were separated by methylated albumin kieselguhr column chromatography. Label occurred in all portions of the RNA profile, but no label appeared in the DNA peak. Using 5-(3)H-uridine, pollen tubes of different genotype had different labeling patterns.

Biochemical properties of fat body DNA of the silkworm, Bombyx mori

Some properties of DNA, especially of pupal fat body of the silkworm, Bombyx mori, were studied. Pupal fat body DNA was separated into at least three components called α-DNA, β 1-DNA, and β 2-DNA on methylated albumin kieselguhr (MAK) column chromatography. All of these classes of DNA were demonstrated to be pure DNA, neither contaminated nor hybridized with RNA, by their being positive to the diphenylamine reaction, sensitive to DNase, resistant to RNase, and incorporating thymidine-6- 3H but not uridine-5- 3H. The GC contents calculated from T m values were around 38 per cent for all of these three components, almost coinciding with that of bulk DNA. But the molecular weight of α-DNA, roughly calculated from the sedimentation coefficient on a sucrose density gradient centrifugation was several-fold larger than that of β 1-DNA. In the pupal stage, fat body DNA was mainly composed of β 1- and β 2-DNA with a minor amount of α-DNA, while in larval stage, it consisted only of α-DNA. Larval fat body type DNA was observed in the larval silk gland, and in pupal and/or pharate adult tissues like the integument, muscles, and gonads. On the other hand, pupal fat body type DNA was detected in the tissues destined to degenerate or in the process of degeneration, such as pupal silk gland and midgut. These facts indicate that β 1- and β 2-DNA may be the degradation products of α-DNA.

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.

Metabolism of Adenine in Healthy and Blighted Potato Leaves

Analysis of the distribution of 14C in extracts prepared from leaf tissue which had been exposed to labelled adenine by petiolar uptake revealed that this purine is extensively metabolized in both healthy and Phytophthora-infected potato leaves. Incorporation of labelled adenine into the major ribonucleic acid species of the leaf was also extensive as determined by radioactive assays performed on individual fractions which were separated on columns of methylated albumin kieselguhr. Examination of individual nucleotides released by alkaline hydrolysis showed that both the adenylic and guanylic acid moieties were labelled. Although the labelling patterns were similar for RNA from healthy and infected leaf tissue, the specific activity of the latter was consistently higher than the former. When partially purified leaf extracts were assayed for phosphoribosyltransferase, they exhibited relatively high levels of activity with adenine as substrate, but were virtually devoid of activity with hypoxanthine and guanine. However, direct petiolar uptake of labelled hypoxanthine resulted in highly labelled RNA. A comparison of adenine phosphoribosyltransferase activity in extracts from healthy and blighted leaves failed to reveal measurable differences. Therefore, it was concluded that the differential incorporation of labelled adenine into the RNA of healthy and infected leaves was due neither to increased activity of this enzyme in response to infection nor to its differential activation. Apart from its role in the recovery of preformed purines for nucleic acid synthesis, adenine phosphoribosyltransferase may function as part of a mechanism for regulating levels of adenine in the potato leaf.

The integrity of human lymphoblastoid RNA after incorporation by human skin fibroblasts

RNA from a human lymphoblastoid cell line is taken up at the macromolecular level by human skin fibroblasts in culture. Most of the donor RNA is found in the nuclei of recipient cells, apparently associated there with deoxynucleoprotein. Two techniques were designed to determine whether the RNA undergoes gross degradation intracellularly, with resynthesis of the degradation products into newly synthesized fibroblast RNa. The first method involves a comparison of the chromatographic profiles from methylated albumin Kieselguhr columns of 3H-labeled donor RNA with 14C-labeled RNA formed de novo from the simultaneous incorporation of nucleosides. The other method utilizes the ability of 5-fluorouridine (FUrd) to substitute for uridine in RNA. A nearest-neighbor analysis was made of the RNA in the recipient cells after the uptake of [ 3H]FUrd together with donor 32P-labeled RNA or of 32PO 4 together with donor [ 3H]FUrd-labeled RNA. The results of these experiments indicate that the RNA taken up retains its polymeric form for some hours and is not present in significant amounts as a result of breakdown-resynthesis inside the cell.

Two isoaccepting species of tryptophan transfer ribonucleic acid from Chlamydomonas reinhardii.

Two isoaccepting species of tryptophanyl-tRNA have been isolated from the green alga Chlamydomonas reinhardii One species, the first to be eluted from a methylated albumin–kieselguhr column, resembles the tryptophanyl-tRNA from Escherichia coli described by Gartland and Sueoka (Proc. Natl. Acad. Sci. Wash. 55, 948 (1966)) and Ishida and Sueoka (J, Mol. Biol 37, 313 (1968)), in that it can be interconverted to and from form I and form II. The second species does not interconvert and appears to be chemically different from the first species.

Fractionation of denatured bacterial DNA on methylated albumin kieselguhr columns. Is it strand separation?

Fragmented denatured total DNA of Salmonella typhimurium can be separated on methylated albumin kieselguhr columns into two fractions by the intermittent gradient elution technique, or into several fractions by increasing the salt concentration of the eluting buffer stepwise. The fractionation is based mainly on differences of nucleotide composition, fragments with high Pu/Py ratio are eluted at lower salt concentrations. In contrast to the similar fractionation of Bacillus subtilis DNA, this procedure does not lead to the separation of all the complementary DNA strands. Only those fragments, which exhibit higher than average asymmetry in the Pu/Py ratio, such as the DNA sequences coding for ribosomal RNA, can be separated into complementary strands by either of the two procedures.

Induction of Deoxyribonucleic Acid Synthesis in Potato Tuber Tissue by Cutting

Incorporation of (32)P-orthophosphate was found in the DNA fraction of aerobically incubated potato discs when examined by methylated albumin kieselguhr column chromatography. The estimation of DNA content of the discs was by a method developed for starchy tissues and showed that the incorporation of (32)P was due to net synthesis of DNA. The DNA content of a disc rapidly increased after a lag period of about 12 hours. The increase continued during the entire test period although at a lower rate during the later period of aging. DNA synthesis was further examined by measuring the rate of incorporation of (3)H-thymidine. The striking similarity which was found between changes in the rate of DNA accumulation and in the rate of (3)H-thymidine incorporation indicates that the incorporation of (3)H-thymidine actually represents the net synthesis of DNA. Although the experiments with microautoradiography revealed that DNA synthesis occurred exclusively in nuclei, no signs of cell division were detected by microscopic observation. DNA synthesis in potato discs was further examined by using inhibitors of protein and RNA synthesis and was sensitive to those inhibitors. The significance of the present results is discussed in relation to the role of wounding in the induction of DNA synthesis.