English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Tools annual

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Presents the access of premium content as premium feature

Premium Content

Global: Zendy Plus annual

Global: Zendy Free Plan

A recent study from this laboratory1 gave a preliminary account of experiments aiming at the isolation of chromosome preparations from synchronized HeLa cells in metaphase arrest. It appeared of interest to examine the template activity of these preparations in directing the incorporation of ribonucleotides by RNA polymerase, with respect to the amount and, more importantly, to the composition of the polymeric product formed; and to do this in comparison with the template behavior of the total free DNA isolated from the same HeLa cell cultures. The RNA normally present in the preparations of metaphase chromosomes is, similarly to cytoplasmic RNA, of a high GC-type, quite unlike the DNA of HeLa cells. It was one of the purposes of this study to ascertain whether the enzymic product formed with chromosomal preparations as templates resembled in any way the RNA component of the chromosomes. This proved not to be the case. Materials and Procedures.-HeLa cells in suspension culture were grown in phosphate medium containing 0.3% (w/v) Methocel HG (Standard Grade, 4000 cP, Dow Chemical Co.) and 5% (v/v) whole horse serum.2 The cells were synchronized and arrested at metaphase as described previously for stationary cultures.1 Three cultures, each containing 1.2-1.5 X 10' cells of which about 40 c were in metaphase, were fractionated so as to yield the preparations rich in chromosomes and free of nuclei and soluble DNA described previously as fraction C.' Two cycles of resuspenision in 0.16 A/IT NaCl solution and centrifugation (1100 X g, 15 mim, 40) resulted in the recovery of 95%0 of the chromosomes, as judged from DNA assay. In the incorporation experimenits the washed chromosomes were tested as a suspension in 0.16 M NaCl solution. Four preparations of DNA were used. Pireparations 1-3 were isolated from HeLa cells with the aid of Duponol' and purified in the usual manner (compare p. 327 of ref. 4). The fibers obtained by precipitation with ethanol were dissolved in, and dialyzed against, physiological saline, and the solution was stored in the frozen state at -35?. For the isolation of DNA preparation 4, another procedure described recently1 was adapted. The preparation from HeLa cells comprising intact nuclei and aggregated chromosomes, designated as fraction P in a recent paper,' was washed twice by centrifugation (1100 X g, 15 min, 40) with 0.15 M NaCI-0.015 M sodium citrate (pH 7) and then treated with trypsin (0.3 mg/ml) in the same saline-citrate solution for 4 hr at 37?. After cenitrifugation the entire DNA was found in the supernatanlt layer which was then subjected to ilncubation with pronase and ribonuclease anid to extractioni with phenol, as described in the papers mentioned before.' The final preparation was essentially free of protein and RNA. Two preparations of RNA were isolated with the use of a procedure applied previously to the preparatioln of RNA from HeLa cells.' The starting material for the specimen designated as cytoplasmic RNA was the cell lysate freed by centrifugation of n-uclei and chromosomal particles.' The chromosomal RNA preparation was made from fraction C.' The base composition of DNA was determined in the usual manner.7 RNA was hydrolyzed to the mononucleotides and analyzed by the two-dimensional chromatography of the latter (isopropanol-NH3, buffered isobutyrate) in the arrangement described before.8 RNA polymerase was prepared from frozen cells of Escherichia coli, strain W (Grain Processing Co., Muscatine, Iowa) by a procedure patterned after one described previously,9 with the modifications menitioned in a recent paper." Other enzymes used were commercial preparations.

proceedings of the national academy of sciences of the united states of america

Template functions in the enzymic formation of polyribonucleotides, II. Metaphase chromosomes as templates in the enzymic synthesis of ribonucleic acid.