Kieselguhr Column Chromatography Research Articles

Rapid analysis of propylene glycol in fatty foods.

Rapid analysis of propylene glycol (PG) in fatty foods was developed by using kieselguhr column chromatography. 2-5 g of sample suspended in hot water (70-80°C) was applied to the Extrelut 20 column. After standing for 15 min, 70 ml of hexane was put on the column to remove fat. PG retained in the column was eluted with distilled water. 50 ml of aqueous eluate was collected. PG was quantitated by gas chromatography with trimetylene glycol as an intenal standard. Recoveries of PG added to margarines and mayonnaises were from 98% to 105%. Since proposed purification process is very rapid, many samples can be treated at the same time. Among commercial margarines (25 samples), shortenings (3 samples) and mayonnaises (3 samples), PG was detected in eight margarines (range 37-261 ppm).

A rapid isotope dilution procedure for estimating the relative proportion of mitochondrial DNA in yeast

A method is described for estimating rapidly the relative proportion of total DNA that is of mitochondrial origin in small quantities of the yeast, Saccharomyces cerevisiae. This procedure involves the mechanical disruption of cells followed by the addition of small amounts of radioactively labeled yeast nuclear and mitochondrial DNA to the lysate. Both labeled and unlabeled DNAs are then co-extracted from the mixture and separated into nuclear and mitochondrial DNA components by poly( l-lysine) Kieselguhr column chromatography. The resulting specific radioactivities of each species of DNA, when compared to the amount of labeled DNA initially added, are related to the relative proportion of unlabeled nuclear and mitochondrial DNA in the original cell sample. The isotope dilution procedure reported here is shown to be both reproducible and to reflect the true relative concentrations of each species of DNA in this yeast.

Alterations in molecular size of messenger RNA from ultraviolet-irradiated T-4-phage.

T4-phages were irradiated by UV-light and phage-specific mRNAs were extracted from Escherichia coli BB-1 cells at various periods of infection. The RNAs were fractionated by methylated albumin-coated kieselguhr (MAK) column chromatography into characteristic sizeclasses. With UV-irradiated phages the last-eluted mRNA component of high molecular weight disappeared preferentially after the middle stage of infection but not at the early stage. However, quite the contrasted changes in the last-eluted, phage-specific component in chromatographic profiles were observed in E. coli cells irradiated by UV-light before infection of normal phages. Some unstable proteins might be responsible for the UV-induced, selective inhibition of high molecular weight mRNA synthesis in bacterial cells.

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.

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

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.

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.

Grouping of T4-specific messenger RNA by methylated albumin-coated kieselguhr column chromatography

T4-specific mRNA was fractionated into three characteristic classes by methylated albumin-coated kieselguhr column chromatography and into several discrete components by polyacrylamide gel electrophoresis. From the results of a comparison of these RNAs it is suggested that the methylated albumin-coated kieselguhr column causes grouping of T4-specific RNA. The characteristic peaks revealed on chromatographic profiles are not aggregations of mRNA and rRNA formed during fractionation.

Inhibition of labeling of messenger and nucleoplasmic RNA of HeLa cells by camptothecin

Methyl albumin kieselguhr column chromatography has been employed to follow the action of camptothecin on the labeling of nucleoplasmic and polyribosomal messenger RNA in HeLa cells. The data indicate that both species of RNA are inhibited in a parallel fashion by the drug. The effect cannot be accounted for by a decreased uptake of RNA precursor by the cells.

Fractionation of rapidly-labelled nucleic acids from rhodospirillum rubrum using polylysine kieselguhr column chromatography

Poly- l-lysine kieselguhr column chromatography has been used to fractionate a rapidly labelled RNA fraction from Rhodospirillum rubrum. Labelled nucleic acid mixtures, prepared from R. rubrum grown anaerobically in the light, were fractionated on poly l-lysine kieselguhr columns. Preliminary results of this study are reported in this paper.

The kinetics of labelling and intracellular distribution of mammalian DNA-like RNA isolated by methylated albumin kieselguhr column chromatography

1. 1. Methylated albumin kieselguhr column chromatography has been used to follow the kinetics of incorporation of labelled precursors into total and cytoplasmic DNA-like RNA. 2. 2. After a 5 min labelling period 81 % of the radioactivity is incorporated into two main species of DNA-like RNA. 43 % of this label is present as the larger DNA-like RNA which elutes towards the end of a NaCl concentration gradient (previously termed Q2), and the remaining 38 % of the label is present as DNA-like RNA which is tenaciously bound to the column after elution with salt (termed TD RNA). 3. 3. The results of longer labelling periods show that both species of DNA-like RNA are turning over more rapidly than rRNA and that Q2 is turning over more rapidly than TD RNA. 4. 4. No Q2 RNA was found in the cytoplasm of either liver or cells in culture. The percentage of TD RNA in labelled cytoplasmic RNA is less than the percentage of TD RNA in labelled total RNA. These results illustrate the danger of equating rapidly labelled DNA-like RNA with mRNA. 5. 5. A small amount of another DNA-like RNA species was also found in the cytoplasm. This DNA-like RNA has yet to be characterised, but it originates from either Q2 or TD RNA. 6. 6. Low concentrations of actinomycin D were used to follow the kinetics of labelling of the DNA-like RNA species without interference from labelled rRNA. The results of chasing labelled DNA-like RNA (taken with similarities of base composition and structure) indicate that Q2 is a precursor of at least part of TD RNA, which is then eventually broken down to acid-soluble products. 7. 7. These results are in agreement with some of the current theories on the role of DNA-like RNA in the regulation of gene expression.

A procedure for gene purification: The purification of the ribosomal RNA genes of Bacillus subtilis as DNA-RNA hybrids

A procedure for the purification of specific genes as DNA-RNA hybrids is described. The ribosomal RNA genes of Bacillus subtilis were selected as a model system for this study, since the ribosomal RNA from this species can be readily obtained in pure form and since much biochemical and genetic background information about the genes was already available. B. subtilis DNA was mechanically sheared and the complementary strands were separated by methylated albumin kieselguhr column chromatography. The ribosomal RNA gene fraction was then identified by hybridization with a mixture of 16 s and 23 s ribosomal RNA. Passage through a hydroxyapatite column eliminated any renatured DNA. The DNA-RNA hybrid was then separated from the bulk, nonhybridized, single-stranded DNA by cesium sulfate density-gradient centrifugation in the presence of mercuric ions. The over-all purification was 80-fold, as measured by the increase in hybridizing activity with 16 s or 23 s ribosomal RNA. This result indicated that at least 30% of the purified DNA was composed of ribosomal RNA genes. The techniques used for the purification are discussed in detail and future applications are considered.

Separation of 5S RNA from other nucleic acids by polyamino acid kieselguhr column chromatography

Separation of 5S RNA from other nucleic acids by polyamino acid kieselguhr column chromatography

A study of aminoacyl transfer RNA synthetases by methylated albumin kieselguhr column chromatography in Paracentrotus lividus

A study of aminoacyl transfer RNA synthetases by methylated albumin kieselguhr column chromatography in Paracentrotus lividus

Uracil incorporation and photopigment synthesis in Rhodospirillum rubrum

The relationships between bacteriochlorophyll and RNA synthesis in non-growing cells of Rhodospirillum rubrum were studied during transition from dark aerobic to light anaerobic metabolism. 1. 1. At high cell densities, uracil incorporation and bacteriochlorophyll synthesis were greatly stimulated by illumination. The light-stimulation of uracil incorporation occurred earlier. Cell mass and protein content did not increase significantly. 2. 2. The incorporated uracil was distributed in a slowly sedimenting (“slow”) RNA fraction during 1 h of incubation. The ribosomal and soluble RNA's were gradually labeled at later times. The rate of synthesis of the slow RNA was greater than those of the other RNA fractions particularly in the light. 3. 3. Sucrose-gradient centrifugation and methylated albumen kieselguhr column chromatography produced the same profiles for the pulse-labeled (3 min) and 60-min labeled RNA's. 4. 4. Mitomycin, chloramphenicol, antimycin A, 2,4-dinitrophenol, carbonylcyanide m- chlorophenylhydrazone , 2-n- nonylhydroxyquinoline-N- oxide and 5-fluorouracil inhibited both RNA and bacteriochlorophyll synthesis. Puromycin inhibited bacteriochlorophyll synthesis, but not RNA synthesis. 5. 5. It is concluded that under non-growing conditions during transition, the slow RNA fraction produced could contain a messenger RNA and that the possibility it includes a specific light messenger component requires further investigation of this fraction.

The response of mammalian cells to alkylating agents: II. On the mechanism of the removal of sulfur-mustard-induced cross-links

1. The ability of L-cells to remove alkylation products produced by sulfur mustard di-(2-chloroethyl)-sulfide from their DNA has been studied. 2. The loss of total alkylation products as well as the loss of the diguaninyl mustard product, di-(guanin-7-yl)-ethyl sulfide, were followed as a function of time using 35S-labelled mustard. Both processes were exponential and occurred with half-lives of 18 h. 3. Parallel measurements of the amount of renaturable cross-linked DNA present were made using methylated albumin kieselguhr column chromatography or ultracentrifugation in CsCl gradients to separate renaturable, i.e., bihelical, DNA from non-renaturable, i.e., denatured, DNA. These studies showed that (a) the loss of renaturable DNA occurred with a half-life of 2 h; (b) the loss of renaturable DNA occurred more rapidly than did the loss of either the total or the diguaninyl alkylation products; (c) this loss of renaturable DNA occurred in the absence of preferential excision of the diguaninyl alkylation product. 4. The following mechanism is proposed to account for these findings. It appears that L-cells are able to excise both monoguaninyl (7-hydroxyethylthioethylguanine) and diguaninyl alkylation products from their DNA and further, that the latter product is removed by a two-step process that involves first, the ‘unhooking’ of one arm of the cross-link and second, excision at some later time of the remaining product.

Kinetics of cellular and viral DNA synthesis in equine abortion (herpes) virus infection of L-M cells

Deoxyribonucleic acid (DNA) synthesis in control and equine abortion virus (EAV)-infected L-M cell suspension cultures was examined by measuring the incorporation of labeled thymidine (TdR-2- 14C) and deoxycytidine (CdR-2- 14C) into the DNA fraction. Employment of methylated-albumin kieselguhr (MAK) column chromatography to separate viral and cellular DNA revealed the relative amounts being synthesized during infection. These studies showed that there was a marked and progressive inhibition in cellular DNA synthesis beginning as early as 2–4 hours post infection (PI) and continuing throughout the cycle. Viral DNA synthesis was initiated by 4–6 hour PI and the rate increased rapidly; by 12–14 hours (the period of maximal EAV DNA synthesis), it was approximately 15 times that observed for cellular DNA. The kinetics of biosynthesis of both species of DNA throughout the infection cycle are described and possible mechanisms responsible for inhibition of cellular DNA synthesis are discussed.

X-irradiation Effects on Protein Synthesis and Synthesis of Messenger Ribonucleic Acid from Peanut Cotyledons

Abstract Exposure of peanut seed to 500 kr of x-rays followed by 6 weeks of storage before planting resulted in a severe reduction of 14C-amino acid incorporation by the ribosomal material. Ribosomes from irradiated tissue were just as effective as those from control tissue in amino acid incorporation directed by exogenous messenger ribonucleic acid and polyuridylic acid. Polyribosomes were nearly absent from the ribosomal material obtained from irradiated tissue, and the synthesis of RNA and protein in vivo was much less than that for control cotyledons. The 105,000 x g supernatant from irradiated tissue was superior to the control supernatant when control ribosomes were used, but gave the same activity with ribosomes from irradiated tissue. Assay of leucyl and phenylalanyl transfer RNA synthetases showed that x-irradiation does not affect their activities. The supernatant fraction from irradiated tissue contains, however, only one-third as much ribonuclease activity as that of the control. The high level of RNase activity in the control 105,000 x g supernatant possibly leads to degradation of the template and ribosomal RNAs, thereby reducing amino acid incorporation. Other experiments showed that mRNA, purified by methylated albumin Kieselguhr column chromatography from irradiated tissue, hybridized with control DNA about 20% as well as that from control tissue. It is concluded from our results that x-irradiation reduces the capacity of cells to produce biologically functional mRNA.

Further studies on the heterogeneity of lens ribonucleic acid

Ribonucleic acid (RNA) from separated parts of cattle lenses, i.e. epithelial cells, cortical and nuclear fibres was prepared by phenol treatment. The heterogeneity of ribonucleic acid was studied by sucrose density-gradient ultracentrifugation and methylated albumin kieselguhr column chromatography. Due to the differentiation of epithelial cells to lens fibres, marked decrease in relative amounts of high molecular weight RNA and an increase of low molecular weight RNA were observed by both methods. Similar distribution was shown when RNA heterogeneity of chick lenses during ontogenesis, starting from the embryonic stage, was studied. The method of methylated albumin chromatography proved to be more advantageous when the features of low molecular weight RNA, present in large quantity in lens, are studied.