Glycogen synthesis by two Ehrlich ascites tumour cell strains in vitro.

Cells of two closely related strains of the Ehrlich mouse ascites tumour synthesize glycogen in vitro. The glycogen is mainly deposited intranuclearly.     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

Heterogeniety of the amino acid pool for protein synthesis in Ehrlich ascites carcinoma cells

Using a new methodological approach based on a step-wise labelling with [14C] and [3H] amino acids, it was demonstrated that the Ehrlich ascite carcinoma cells are capable of utilizing both intracellular and extracellular amino acid pools for protein synthesis. The inhibition of amino acid transport into the cells is accompanied by a more intensive utilization of the exogenous pool. The described procedure permits to calculate the specific radioactivity of the tRNA-bound amino acid and the absolute rate of protein synthesis.     

Protein synthesis in postnuclear supernatants from mengovirus-infected Ehrlich ascites tumor cells.

The effect of mengovirus infection on the protein synthetic capacity of Ehrlich ascites tumor cells cultured in vitro was studied in vivo and in vitro employing postnuclear supernatants prepared at various times post-infection in the absence and in the presence of 1% Triton X-100. The amino acid incorporating activities of extracts obtained in the presence of the detergent were reduced by about 30% compared with the capacities of the corresponding postnuclear supernatants prepared in the absence of Triton X-100; but the course of the activity vs. time curve was not influenced by the detergent. Under the conditions employed, the postnuclear supernatants were unable to reinitiate protein synthesis once elongation of nascent polypeptide chains concomitant with ribosome runoff was completed. After mengovirus infection, a gradual disappearance of polysomes from postnuclear supernatants and a simultaneous accumulation of monosomes was observed. The protein-synthesizing activities of normal and infected cells were inversely proportional to the monosome concentrations of their corresponding extracts. Qualitatively, protein synthesis in intact cells and in postnuclear supernatants responded similarly to mengovirus infection. In both cases an initial reduction of host-specific amino acid incorporation was followed by a burst of viral protein synthesis. However, the two activity vs. time curves showed the following significant differences: 1) The activities of extracts from control cells and from mengovirus-infected cells nearly in the infectious cycle were low compared with the activities observed in vivo. 2) In the middle of the infectious cycle, the peak of viral protein synthesis occurred later and the activity was higher in vitro. 3) Finally, in the late period of the infectious cycle the postnuclear supernatants had considerable protein synthesizing activity, at a time when protein synthesis in vivo was nil.     

Biochemical aspects of cardiac muscle differentiation. Deoxyribonucleic acid synthesis and nuclear and cytoplasmic deoxyribonucleic acid polymerase activity.

DNA synthesis and DNA polymerase activity have been measured in terminally differentiating cardiac muscle of the rat. Incorporation of [3H]thymidine into DNA essentially ceases by the 17th day of postnatal development. Cardiac muscle of neonatal rats contains at least two molecular species of DNA polymerase: a 3.5 S DNA polymerase that can be extracted from nuclei with 0.2 m potassium phosphate and a 6 to 8 S soluble cytoplasmic DNA polymerase. The nuclear DNA polymerase in crude extracts has a pH optimum of 9.0 and is more active with native DNA than with denatured DNA as the primer-template. The cytoplasmic DNA polymerase in crude extracts has a pH optimum of 7.5 and is more active with denatured DNA. The activity of the 6 to 8 S cytoplasmic DNA polymerase decreases 80-fold from day 1 to day 17 after birth, which correlates temporally with the reduced rate of DNA synthesis. The activity of the 3.5 S nuclear DNA polymerase remains relatively constant throughout postnatal development. Mixing experiments (assay of neonatal enzyme extracts with adult enzyme extracts) gave additive results, suggesting that the decline in 6 to 8 S DNA polymerase activity apparently is not due to the presence of absence of soluble activators or inhibitors at different times during development. These studies may provide a system which can be used to investigate the control of DNA synthesis and cellular proliferation during the terminal stages of cardiac muscle differentiation.     

Deoxyribonucleic acid synthesis in mammalian nuclei. Incorporation of deoxyribonucleotides and chain-terminating nucleotide analogues

The properties of a nuclear preparation from rat liver and thymus are described. (1) Nearest-neighbour analysis after incorporation of (32)P-labelled nucleotide residues from dATP, dCTP, dGTP, dTTP and arabinofuranosyl analogues of CTP and ATP shows template-dependent DNA synthesis. (2) Where primer termini are limiting, incorporation of arabinofuranosyl analogues of AMP and CMP residues proceeds to a limit indicating that both of these analogues are DNA chain terminators. (3) No large differences have been found between the priming potentialities or the intrinsic DNA polymerase activities of nuclei from resting or regenerating liver and the relationship of this DNA synthesis in vitro to DNA replication or repair in vivo is briefly discussed.