Elliot Volkin

Phosphorus incorporation in Escherichia coli ribonucleic acid after infection with bacteriophage T2

Abstract Incorporation of medium phosphorus into RNA of T2r+-infected Escherichia coli has been demonstrated in a manner that reduces the contribution from uninfected bacteria to an insignificant level, and which unambiguously identifies such phosphorus with RNA phosphorus. In nutrient broth cultures containing P 32 O 4 , accumulation of labeled RNA takes place very shortly after infection with phage and then stops, whereas with P 32 O 4 in synthetic medium accumulation of labeled RNA continues almost linearly for at least 60 minutes after infection. The unequal specific activities of the RNA mononucleotides isolated from alkaline hydrolyzates of RNA requires a heterogeneous uptake of the phosphorus with respect to the total RNA phosphorus. Possible mechanisms to explain the data have been presented.

Properties of ribonucleic acid turnover in T2-infected Escherichia coli

1. 1. The turnover of phosphorus by RNA and phosphorus accumulation by DNA were measured with 32P at varying times after infection of E. coli with bacteriophage T2. The turnover rate of RNA is greatest shortly after infection and decreases at later times. The reverse time dependency is found for phosphorus accumulation by DNA. 2. 2. Comparison of RNA turnover in broth and in synthetic medium reveals that the RNA of infected cells in broth is much more quickly responsive to changes in specific radioactivity of orthophosphate in the medium. 3. 3. When isotope is presented to uninfected cells, the RNA mononucleotides always attain uniform specific radioactivity, whereas within 1 to 4 min after phage infection, the RNA mononucleotides of infected cells have nouniform specific activities in that adenylic and uridylic acids have about twice the activities of guanylic and cytidlic acids. 4. 4. When, after a period of 32P incorporation, infected cells are washed to reduce the acid-soluble 32P pool, further incubation results in a nearly quantitative transfer of radioactivity from RNA to DNA.

Intracellular distribution of labeled ribonucleic acid after phage infection of Escherichia coli

Abstract By rupture of T2r+-infected bacterial cells, and differential centrifugation into component subcellular parts, it can be shown that, during the course of infection, isotopic phosphate is incorporated into the RNA of these constituents in a diverse manner. The RNA of one particulate fraction, though only a small part of the total cell RNA, has the highest specific activity, whereas the major portion of cell RNA, found in another particulate fraction, has extremely low activity. RNA found in the soluble fraction is intermediate in specific activity.

Acid degradation products of deoxyribonucleic acid.

Abstract The acid hydrolysis of calf thymus DNA by normal HCl at 100° results primarily in nucleotides containing one more phosphate than pyrimidine, a number of which have been isolated and identified by ion-exchange chromatography. This is consistent with an elimination mechanism that leaves on the pyrimidine residue those phosphates formerly lying between purine and pyrimidine residues. It also indicates that the loss of purine residues is a necessary prerequisite to the rupture of the polynucleotide chain by mild acidic hydrolysis. The presence of nucleotides containing equal amounts of pyrimidine and phosphate may be caused by the hydrolysis of phosphate from the initial products. About 33% of the phosphate appears as inorganic phosphate after 1 hour of hydrolysis; the bulk of this appears to be phosphate originally lying between purine residues in the original DNA. There is a slow rise to a new plateau of 50% at 3 to 4 hours. The sum of inorganic plus monoesterified phosphate remains constant from 1 hour to 4 hours, indicating a slow hydrolysis of monoester phosphate, presumably from the 3′ positions. The presence of all varieties of mono-, di-, and trinucleotide sequences that are possible from cytidylic and thymidylic acids indicates that all possible sequences of pyrimidine and of purine nucleotides exist in thymus DNA.

Fate of homologous and heterologous DNAs after incorporation into human skin fibroblasts

Abstract Homologous DNA from a human lymphoblastoid cell line is taken up in macromolecular form by human skin fibroblasts in culture. Experiments utilizing the heavy buoyant density and ultraviolet-light sensitivity of DNA containing 5-bromouracil reveal that the homologous DNA is retained in the recipient cells in relatively undegraded form for over 20 h. Similar experiments with heterologous DNA from bacteriophage T7 show that this DNA undergoes extensive degradation after entry into the fibroblasts and that the products of degradation are readily incorporated into DNA synthesized de novo .

The absence of ribonucleic acid in bacteriophage T2r

Abstract The ribonucleic acid (RNA) content of T2r+ bacteriophage was deter mined by ion exchange chromatography of mononucleotides released after alkaline hydrolysis of a mixture containing P 32 -labeled phage nucleic acids and excess carrier RNA. An upper limit of 0.025% of the phage phosphorus was recovered with the RNA mononucleotides, but since the elution of radioactivity was not consistent with the elution of the carrier mononucleotides, it is concluded that RNA is totally absent in T2r+ bacteriophage.

Chromatin-associated RNA: Differential extraction and characterization

Mammalian cells in different states of cytodifferentiation exhibited different RNA-synthesizing and processing patterns that could be used as markers for phenotypic variability. Inherent in these patterns was an RNA class which was differentially extracted from the cellular homogenate by elevating the temperature and pH of the buffer used in the phenol procedure. This class of RNA was initially designated fraction B (chromatin-associated RNA). In the characterization of fraction B, human myeloma cells labeled for 3 and 24 h were fractionated into subcytoplasmic and subnuclear components and the [3H]-RNA was differentially extracted. After 3 and 24 h labeling 84% and 73%, respectively, of the labeled RNA in the chromatin was extracted in fraction B. Only 10-20% of the polysomal RNA was extracted in fraction B with little enrichment in poly(A) RNA. These and other observations suggested that fraction B was a subpopulation of heterogeneous nuclear RNA which was tightly bound to the chromatin complex.

DEAE-Sephadex chromatography of guanylate oligomers using guanidinium chloride

Abstract When a partial alkaline hydrolysate of polyguanylate is chromatographed on DEAE-Sephadex in 7 M urea with a gradient of NaCl, peaks corresponding only to chains of length 1–6 are obtained. The rest remains on the column and can be eluted with 1 M NaCl. We describe a modification of this procedure that overcomes some of the aggregation of oligomers and fractionates chains of up to at least 12 nucleotides in length. The buffer is 0.01 M Tris (pH 7.4)-0.002 M EDTA-7 M urea; a gradient of 0–1 M guanidinium chloride in this buffer is used for elution from DEAE-Sephadex. As the concentration of polyguanylate hydrolysate is increased, the yield of the longer oligomers decreases. The aggregation of guanylate oligomers thus appears to be a function of their concentration on the column.

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Nuclear retention of 18S ribosomal RNA by human myeloma cells.

SummaryNormal quiescent lymphocytes regulate their ribosome content by selectively degrading newly synthesized 18S ribosomal RNA. Unlike actively dividing HeLa cells, lymphocytes retain 18 S ribosomal RNA in the nucleus after synthesis instead of immediately transporting it to the cytoplasm. Subcellular fractionation of the highly differentiated human neoplastic lymphocyte RPMI-8226 reveals that this cell line also retains 18 S ribosomal RNA in the nucleus, a trait not displayed by the less differentiated human lymphoblastoid cell line RPMI-4265. These observations suggest that neoplastic cells can be phenotypically characterized by their ribosomal RNA processing patterns.