Friedrich Grummt

Control of nucleolar RNA synthesis by the intracellular pool sizes of ATP and GTP

The influence of amino acid starvation on both the pool sizes of nucleoside triphosphates and the rRNA synthetic capacity of Ehrlich ascites cells was studied. The results indicate that under shiftdown conditions, an immediate shrinkage of the cellular ATP and GTP levels occurs. Concomitant with this, protein and rRNA syntheisis are markedly inhibited. If the pool sizes of purine nucleaside triphosphates are expanded by adding adenosine or guanosine to cells cultured in histidine-free medium, the nucleolar RNA synthesis is fully restored, while protein synthesis remains inhibited. The results suggest that the rate of pre-rRNA transcription may be controlled by the actual nucleoside triphosphate levels of the cells rather than by short-lived protein(s), as has been previously postulated.

Diadenosine tetraphosphate (Ap4A) is compartmentalized in nuclei of mammalian cells

The intracellular compartmentation of Ap4A in various growth and cell-cycle stages in mammalian cells was studied by applying a non-aqueous extraction procedure for cell nuclei. In both slowly and in exponentially growing Ehrlich ascites tumour cells from random cultures, more than 75% of the whole cellular Ap4A content is localized in the nuclei. In G1 and early S-phase cells of synchronized baby hamster kidney (BHK) fibroblast cultures, approx. 90% of the intracellular Ap4A pool is confined to the nuclear compartment. In contrast, Ap4A is distributed to nearly equal amounts between cytoplasm and nuclei during mid-S phase. After transition through the S-phase, increasing proportions of Ap4A (78% 18 h and 96% 22 h after serum replenishing, respectively) are again localized in the nuclear compartment.

Effects of TψCG on the enzymatic binding of eukaryotic and prokaryotic initiator tRNAs to rat liver ribosomes

In 1964 Rosset et al. discovered ribosomal5 S RNA [ 11, which was subsequently sequenced from a number of prokaryotic [2-41 and eukaryotic organisms [S-7]. Comparison of the sequence of KB cell 5 S RNA [5] with that of E. coli [2] lead Forget and Weissman [ 51 to propose that the common 5 S RNA sequence CGAAC would interact with the common GT\kCPu (loop IV) sequence of tRNA [8,9]. More recent data has shown that all 5 S RNAs contain the oligonucleotide or one similar to CGAAC [2-7, lo] . In contrast recent tRNA sequencing experiments have shown that not all tRNAs contain the G’NrCPu oligonucleotide. The exceptions being: 1) eukaryotic initiator tRNA [ 1 l-131 and 2)

Studies on the phosphorylation of nucleolar proteins. Comparison of the phosphorylation patterns of nuclear and nucleolar proteins labeled in vivo and in vitro

Evidence has been accumulated which suggests that the control of transcription may involve the phosphorylation of nuclear proteins, in particular nonhistone proteins [l-3]. It was therefore interesting to compare the phosphoproteins of nuclear and nucleolar chromatins which show remarkable differences in template activity. In the preceding paper [4] we demonstrated the presence of a protein kinase activity in purified rat liver nucleoli. In this study the proteins of whole nuclei and isolated nucleoli were fractionated into saline-soluble and chromosomal proteins and the distribution of phosphoproteins was analyzed. Since up to now very little is known about the specificity of the phosphorylation reactions in vitro we compared the distribution of nuclear and nucleolar proteins labelled both in vivo and in vitro. [ 101.

Studies on free and 5 S RNA-bound ribosomal GTPase and ATPase

A complex consisting of one molecule of 5 S RNA and two molecules of ribosomal protein was recently reconstituted in bacterial systems [1 ]. This complex was shown to be associated with both a GTP and an ATP hydrolyzing activity [2,3]. A 5 S RNA-protein complex was isolated previously by Blobel [4] by treatment of rat liver ribosomes with EDTA. Using a similar procedure to isolate this mammalian 5 S RNAprotein complex, we looked for GTPase and ATPase activities like these in the reconstituted bacterial 5 S RNA-protein complex. As reported previously [5], wo found the proteins associated with 5 S RNA active both in GTP and ATP hydrolysis. In this paper we describe the purification and the chemical composition of this complex. Furthermore, the kinetic behaviour and the thermal stability of the GTPase and ATPase associated with 5 S RNA are compared with those of GTPase and ATPase not bound to RNA.

DIADENOSINE TETRAPHOSPHATE (A P4 A) — A LIGAND OF DNA POLYMERASE α AND TRIGGER OF REPLICATION

Diadenosine tetraphosphate (A P4 A) Induces replicative DNA synthesis in quiescent mammalian cells. By equilibrium dialysis an A P4 A binding activity is shown to be present in mammalian cells. The A P4 A binding activity co-purifies with DNA polymerase α during the isolation procedure including chromatography on phospho-, DEAE- and DNA-cellulose, gel filtration, sucrose gradient centrifugation and electrophoresis in non-denaturing polyacrylamide gels. After these purification steps DNA polymerase α appears as a homogeneous protein complex with an apparent M r of 404,000, consisting of 7 subunits with apparent M r of 64,000, 63,000, 62,000, 60,000, 57,000, 55,000 and 52,000. By affinity labeling the protein with M r of 57,000 has been shown to be the A P4 A binding constituent of DNA polymerase α. The A P4 A binding site is lost in neuronal cells during maturation of rat brains concomitantly with the loss of DNA polymerase α and mitotic activity in those cells. From these results DNA polymerase α seems to be the intracellular target of A P4 A. For the elucidation of the mechanism of A P4 A action during DNA synthesis a structural analog has been synthesized in this laboratory. This analog, methylene-bis-ADP, has been shown to act as an antqgonist to A P4 A during binding to DNA polymerase α as well as in DNA replication in vivo and in vitro, and inhibits cell proliferation.