Kieselguhr Column Chromatography Research Articles

Presence and function of sulur-containing transfer ribonucleic acid of Bacillus subtilis.

Bacillus subtilis transfer ribonucleic acid (tRNA) was analyzed for the occurrence of thionucleotides by in vivo labeling with (35)S and fractionation by methylated albumin kieselguhr column chromatography. Alkaline hydrolysates of tRNA were also examined by column chromatography and paper electrophoresis, and the amino acid-accepting ability of thionucleotide-containing tRNA was tested after iodine oxidation. The results showed that B. subtilis tRNA contains 4-thiouridylate, a second nucleotide with properties similar to 2-thiopyrimidine, and a third unidentified thionucleotide. The amino acid-accepting ability for serine, tyrosine, lysine, and glutamic acid was markedly inhibited after oxidation of the tRNA with iodine, suggesting the presence of thionucleotides in these tRNA species. This inhibition could be reversed by thiosulfate reduction. The iodine treatment totally inactivated all lysine tRNA species, partially inactivated the serine tRNA species, and did not affect the accepting ability for valine. A comparison of tRNA from cells in the log and stationary phases and from spores revealed similar iodine inactivation patterns in all cases. The thionucleotide content in B. subtilis tRNA differed from that in Escherichia coli, both in extent and in distribution. A possible function of the thionucleotides in tRNA is discussed.

Pattern of Valine Transfer Ribonucleic Acid of Bacillus subtilis under Different Growth Conditions

Abstract The relative amounts of two valyl-tRNA species from Bacillus subtilis W23 were compared by methylated albumin Kieselguhr column chromatography and by diethylaminoethyl cellulose column chromatography after T1 ribonuclease digestion. The ratio of the two valyl-tRNAs changes significantly during sporulation, during step-up and step-down growth transitions, during the stationary phase of an asporogenous mutant, and during sporulation of B. subtilis 168 Marburg. The change in ratio could not be induced in vitro by treating the tRNA preparations with several chemicals and was not the result of differential hydrolysis of the two valyl-tRNA species during the experimental procedures. The change in ratio was due to the increased valine-accepting ability of one of the two valine tRNAs. These results suggest that the valyl-tRNA ratio changes may be a function of the growth condition and that a regulatory mechanism exists which controls the amino acid accepting capacity of the two valine accepting tRNAs.

Ribonucleic Acid Synthesis in Isolated Mitochondria from Tetrahymena

Abstract Some conditions of RNA synthesis in vitro in isolated mitochondria of Tetrahymena pyriformis were investigated. Sucrose gradient centrifugation analyses and methylated albumin-coated Kieselguhr column chromatography of the RNA synthesized in vitro demonstrate that RNA ranging from 14 S to 18 S, not transfer RNA, was synthesized. Furthermore, the RNA is hybridizable with mitochondrial DNA, and the hybridization is not inhibited by the addition of RNA isolated from the material sedimenting at 105,000 x g after centrifugation of mitochondrial lysate for 1½ to 2 hours. Although net transfer RNA synthesis does not occur, the transfer RNA fraction was labeled if 3H-ATP was used for incorporation, suggesting that the incorporation is due to the addition of 3H-ATP at the terminus of the transfer RNA molecule.

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.

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.

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.

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.

Placental transmission and mammary gland secretion of thyroxin in the rat.

When large doses of thyroxin are administered to pregnant rats, propylthiouracil goitrogenesis in the fetuses as well as in the mother is prevented. One hour following the intravenous injection of thyroxin-I131 into pregnant rats a small fraction of TCA-precipitable, butanol-extractable radioactivity was recovered from homogenates of their fetuses. Part of this was shown by means of Kieselguhr column chromatography to represent thyroxin. Large amounts of thyroxin administered to lactating rats over a 10-day period failed to prevent propylthiouracil-induced goiter in their suckling offspring. It is concluded that the rat placenta is slightly permeable to thyroxin but the rat mammary gland does not secrete thyroxin in a physiologically active form.