Initiated complexes of RNA polymerase II are concentrated in the nuclear skeleton associated DNA

Several preparations of nuclear matrices containing varying amounts of DNA were obtained from mouse plasmocytoma P3-X63-Ag8.653 cells and tested for the presence of RNA polymerase II activity. It has been demonstrated that about 25% of RNA polymerase II activity detected in the original nuclei can be recovered in isolated nuclear matrices. Only DNA-bound RNA polymerase II was found in the isolated matrices, while both free and DNA-bound RNA polymerase II activities were detected in the original nuclei. RNA polymerase II activity found in the isolated matrices did not depend on the portion of DNA recovered in the nuclear matrices in a large interval between 91 and 1.5% of DNA content in the original nuclei. The conclusion has been drawn that initiated RNA polymerase II molecules are non-randomly distributed along DNA loops. They are concentrated near the points of DNA attachment to the nuclear skeleton.     

Thermosensitivity of Nuclear RNA Polymerase during the in vitro Senescence of Mouse Fibroblasts

Embryonic mouse fibroblasts divide approximately twelve times in vitro prior to cessation of mitotic activity. During this period of cellular senescence the thermosensitivity of the RNA polymerase activity of isolated nuclei has been examined as a means of detecting the possible accumulation of defective enzyme molecules, as has been found by other workers for several cytoplasmic enzymes during the ageing of human fibroblasts in vitro.
The total RNA polymerase activity of nuclei isolated from old (10th generation) cells is more thermoresistant than that of young (2nd generation) cells. However, the net RNA polymerase activity of nuclei from non-dividing (confluent) cells is more thermoresistant than that of exponentially growing cells of the same age. When allowance is made for the state of growth of the cultures, little difference is seens in the thermosensitivity of the activities of nuclei from old and young cells. Neither is there any difference between the thermosensitivity of the net activity of an established line of murine fibroblasts (L-cells) and cells in primary culture.
Preheating nuclei increases the inhibition of their total RNA polymerase activity by or-α-amanitin, indicating that RNA polymerase II is the most heat resistance species present. There appears to be no difference between the thermosensitivity of the α-amanitin sensitive and resistance species of the enzyme in the nuclei of old and young cells.
It is concluded that old cells resemble non-dividing young cells in containing a higher proportion of RNA polymerase II in their nuclei, resulting in greater thermoresistance of the total RNA polymerase activity over that of exponentially growing cells. However, there appears to be no increase in thermosensitivity of the enzymes with age.     

Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.

In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.