Biomedical subjects

I Grummt

Publications and source records attributed to I Grummt.

108 records · Page 6Linked to original sources

PubMed1979

Characterization of a cloned ribosomal fragment from mouse which contains the 18S coding region and adjacent spacer sequences.

The large EcoRI fragment of mouse ribosomal genes containing parts of the non-transcribed spacer, the external transcribed spacer located at the 5' end of the precursor molecule and about two thirds of the 18S sequence has been cloned in bacteriophage lambda gtWES. A physical map of the DNA was constructed by cleavage with several restriction endonucleases and hybridization of the restriction fragments of the recombinant DNA with labelled 18S and 45S rRNA. The orientation of the inserted fragment as well as the length of the 18S sequence was determined by electron microscopy of R-loop containing molecules. The absence of hybridization of the cloned fragment to other fragments in the genome shows that the non-transcribed spacer does not have a significant length of sequences in common with other sequences in the genome.

Animals↗

PubMed1979

Localisation of an endonuclease specific for double-stranded RNA within the nucleolus and its implication in processing ribosomal transcripts.

Nucleoli of both chick embryos and mouse Ehrlich ascites cells contain an enzymatic activity that is very similar to RNase DII, an enzyme isolated from total chick embryos for its ability to degrade double-stranded RNA. The enzyme can be extracted by low salt/EDTA from nucleoli and is associated with pre-ribosomal 80-S and 55-S particles. Under ionic conditions which are inhibitory for the nucleolytic activity the transcript in vitro of nucleoli is not processed and sediments around 45 S. Under salt conditions which are optimal for the nucleolar enzyme the nucleolar transcripts are cleaved to distinct intermediate-sized molecules. Addition of the chicken RNase DII or RNase III to the nucleolar transcription system results in a similar shift of the chain length of the RNA molecules. It is concluded that a nucleolar RNase recognizing double-stranded regions in the pre-ribosomal RNA is involved in the maturation of ribosomal RNA.

Animals↗

PubMed1979

Ribosome biosynthesis is not necessary for initiation of DNA replication.

Synthesis of mature 28-S ribosomal RNA and 60-S ribosomal subunits is inhibited in baby hamster kidney (BHK) cell line ts 422E at non-permissive temperature (39 degrees C). This leads to a 66% decrease of total ribosomes per cell, a marked imbalance between the large and small ribosomal subunits in the cytoplasm and a decrease of cells per dish after prolonged culture at 30 degrees C. However, inhibition of ribosome synthesis does not affect progression of cells through the G1 period of the cell division cycle, the length of the pre-replicative period, and the rate of entry of cells into S phase. In contrast to culture at non-permissive temperature, culture of BHK ts 422E cells in the presence of 0.04 micrograms/ml actinomycin D at 33 degrees C inhibits markedly the entry into S period. It is concluded that low doses of actinomycin D exert their inhibitory effect on cell growth by preventing maturation and transport of mRNA rather than by interfering with ribosome synthesis. Microfluorometric analysis revealed only slight differences in the distribution of BHK ts 422E cells in G1, S and G2 phases of the cycle either when cultured at 33 degrees C or at 39 degrees C. When too few ribosomes per cell are produced in BHK ts 422E cells at 39 degrees C, cells do not seem to be arrested reversibly at a specific point of the cell cycle but rather to die at random.

Animals↗

PubMed1977

The effect of cyclic nucleotides on cellular ATP levels and ribosomal RNA synthesis in Ehrlich ascites cells.

Amino acid starvation of Ehrlich ascites cells leads to a significant decrease of the intracellular ATP concentration concomitant with a marked decrease in nucleolar RNA polymerase activity. Addition of 8-bromoguanosine 3':5'-monophosphate (br8cGMP) to the amino-acid-deficient culture medium increased the cellular ATP levels and restored the rRNA synthesis capacity of nucleoli to control levels. Exogenous br8cAMP overcame the effects of br8cGMP. Administration of br8cAMP to exponentially growing ascites cells resulted in a shrinkage of ATP levels and in an inhibition of nucleolar RNA synthesis similar to that observed under shift-down conditions. These effects of br8cAMP could be antagonized by exogenous br8cGMP or hypoxanthine. Since the br8cGMP-induced increase in the total adenine nucleotides was abolished in the presence of azaserine (an inhibitor of the amidation of formylglycineamide ribonucleotide) it is concluded that cyclic nucleotides exert at least a part of their regulatory effects on cell proliferation by regulating nucleotide biosynthesis de novo.

Adenosine Triphosphate↗

PubMed1977

The effects of histidine starvation on the methylation of ribosomal RNA.

The effect of amino acid starvation on the control of ribosome biosynthesis at the post-transcriptional level has been studied in Ehrlich ascites cells. A comparison of the turnover rates of ribosomal precursor RNA (pre-rRNA) and the degree of methylation of ribosomal RNA after histidine deprivation revealed that the slow down of ribosome formation is accompanied by a significant inhibition of rRNA methylation. Analysis of nucleolar and cytoplasmic RNA double-labelled with L-[Me-3H]methionine and [14C]uridine, as well as a quantitative determination of alkali-stable dinucleotides on DEAE-Sephadex, showed that methylation of rRNA species was inhibited by about 50% under shift-down conditions. This decrease in RNA methylation does not reflect an inhibition of rRNA methylases caused by amino acid starvation but is rather brought about by a shrinkage in the pool size of S-adenosylmethionine, the donor of methyl groups. It is suggested that amino acid starvation might exert its blocking effect on proper ribosome maturation by affecting the methylation of 45-S RNA.

Animals↗

PubMed1977

Regulation of ATP pools, rRNA and DNA synthesis in 3T3 cells in response to serum or hypoxanthine.

The serum-induced transition of 3T3 fibroblasts from resting to growing state is characterized by a marked increase in cellular ATP content, the maximal level of which is reached at the onset of DNA replication. This increase in cellular ATP during the G 1 period of the cell cycle is correlated with about 3-fold stimulation of transcription of rRNA measured in permeabilized cell in vitro. Addition of hypoxanthine to serum-depleted quiescent 3T3 cells gives rise to an increase in both the ATP pool and the rate of rRNA synthesis. The expansion of cellular ATP pools after growth induction by serum seems to be a prerequisite for initiation of DNA synthesis since inhibition of purine de novo biosynthesis by azaserine inhibits both ATP pool expansion and DNA replication. This effect of azaserine can be abolished by addition of hypoxanthine to the culture medium. It is concluded that (a) the increase of the rate of rRNA synthesis in 3T3 cells in response to growth factors or serum is controlled by the cellular purine nucleoside triphosphate concentration and (b) an increased ATP level is necessary for initiation of DNA synthesis but is not sufficient to trigger the events that lead to DNA replication.

Adenosine Triphosphate↗

PubMed1976

Studies on the role of uncharged tRNA in pleiotypic response of animal cells.

Experiments were carried out to assess the physiological significance of the charging level of tRNA. Histidinol, a competitve inhibitor of charging of tRNAHis, was used to induce uncharged tRNA in mammalian cells. It is demonstrated that both in the presence of histidinol and under histidine depletion about 40% of the tRNAHis is uncharged. Concomitant with this appearance of uncharged tRNA(a) the pools of GTP and ATP are decreased rapidly by 25--30%; (b) the synthesis of both protein and ribosomal RNA is inhibited, whereas that of nucleoplasmic RNA is not affected; (c) the uptake of 2-deoxyglucose, phosphate, Ca2+; uridine and adenosine is inhibited; and (d) the growth of 3T6 fibroblasts is arrested. It is suggested that the appearence of uncharged tRNA is one of the earliest events occurring under conditions of amino acid starvation, which in turn causes the various metabolic changes observed.

Animals↗

PubMed1976

Amino acid starvation affects the initiation frequency of nucleolar RNA polymerase.

The synthesis of ribosomal precursor RNA in mouse ascites nucleoli derived from cells starved for amino acids is compared with the activity of nucleoli from control cells cultivated in the presence of all amino acids. It is shown that deprivation of a single essential amino acid from the culture medium results in a drastic decrease of the RNA-forming capacity of the isolated nucleoli by a factor of 2-3. This switchoff in rRNA synthesis is a very fast process. Half-maximal inactivation occurs after only 30 min. Addition of amino acids to starved cells leads to a rapid recovery, which is reflected by a sharp increase in the RNA polymerase activity of the isolated nucleoli. Studies on the molecular mechanism of this amino acid-mediated control of rRNA synthesis indicate that this effect is not caused by different growth rates of the RNA chains, but rather by an altered initiation frequency of the RNA polymerase in vivo. Whereas in nucleoli derived from cells grown in full medium almost all the polymerase is tightly bound in a transcriptional complex, a high amount of "free" polymerase which becomes active after addition of exogenous template is present in nucleoli from starved cells.

Cell Division↗

PubMed1976

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.

Adenosine Triphosphate↗

PubMed1975

Methylation of ribosomal-precursor RNA, synthesized in vitro, by isolated rat-liver nucleoli.

Nucleoli isolated from rat liver were incubated for synthesis of RNA in vitro in the presence or absence of S-adenosyl [3H] methionine. The results obtained indicate that neither the rate of RNA synthesis not the processing of pre-ribosomal RNA was changed if methylation was allowed to take place. The methylation process acts on the RNA most recently synthesized, rather than on the bulk of the RNA already present in the nucleoli. The reaction seems to occur faithfully both quantitatively and qualitatively. It is calculated that 104 mol methyl groups were incorporated per mol of newly synthesized 45-S RNA. Methylation of the ribose rather than the bases predominated. The pattern of alkali-stable oligonucleotides of RNA methylated in vitro was analyzed and found to correspond closely to that of ribosomal RNA labelled in vivo.

Animals↗

PubMed1975

Synthesis of RNA molecules larger than 45 S by isolated rat-liver nucleoli.

Nucleoli, isolated from rat liver, synthesize in vitro high-molecular-weight RNA, the base composition and sedimentation pattern of which resembles that of ribosomal precursor RNA. In addition, RNA molecules larger than 45 S have been found. In this paper experiments are described which indicate that these large RNA molecules represent geniune transcription products and are not aggregates arising under the experimental conditions employed. This was established by comparing different extraction methods, by sedimentation analysis of the RNA after denaturation with formamide and by pulse-chase experiments. Hybridisation-competition studies showed that 45-S RNA competes with those rapidly molecules to about 80-90%, thus providing evidence for the presence of ribosomal precursor RNA sequences in those long transcription products. Intact nuclei are able to synthesize in the presence of Mg2+ and alpha-amanitin RNA molecules larger than 45 S too, provided that the RNAase activity is suppressed effectively by the addition of cytoplasmic RNAase inhibitor. The significance of these results is discussed with respect to the initial transcript of the rDNA genes in rat liver nucleoli.

Amanitins↗

PubMed1974