rRNA Gene Expression Research Articles

Expression of the ribosomal DNA insertions in bobbed mutants of Drosophila melanogaster.

We have shown earlier that interrupted rRNA genes in Drosophila melanogaster do not contribute significantly to rRNA production by a splicing mechanism (Long and Dawid 1979). In the work reported here the expression of interrupted rRNA genes was tested in several stocks that carry bobbed mutations, i.e., have partial deletions of their rRNA gene clusters. Transcripts of the major 5 kb type 1 insertion are very rare in bobbed flies as they are in the wild type, occurring at a concentration in embryos of less than one copy per nucleus. Transcripts of short type 1 insertions are more abundant in certain bobbed stocks, especially those carrying the car bb chromosome. However, other severely bobbed flies have no increase in these insertion transcripts over the wild-type levels. Type 2 insertions are transcribed into very rare RNA molecules in the wild type and in the bobbed genotypes that were studied. From these results we conclude that interrupted rRNA genes are not expressed through a splicing mechanism into mature rRNA in mutant or wild-type flies. Since even severely bobbed flies fail to utilize their interrupted rRNA genes, we suggest that these genes cannot be transcribed productively in D. melanogaster.

Cloning and characterization of ribosomal RNA gene of Physarum polycephalum.

As a first step in studies on the structure and expression of rRNA gene of Physarum polycephalum, the cloning of the gene in Escherichia coli with plasmid vector DNAs was performed and 8 different kinds of clones containing 26S, 19S, and 5.85 rRNA genes were obtained. Using these cloned fragments, the location of these rRNA genes was determined by the Southern hybridization and S1 mapping method. Cloning of rDNA fragments made it possible to analyze the fine structure of the rDNA.

Reactivation of ribosomal RNA genes in human-mouse hybrid cells by 12-O-tetradecanoylphorbol 13-acetate.

The effect of the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on the expression of rRNA genes was studied in human greater than mouse hybrid cells (55-54 cells). These hybrids retain the rRNA genes of both parent species but express only human rRNA. 55-54 cells were treated with either TPA (1-100 nM), phorbol (0.01-1 micro M) or phorbol 13,20-diacetate (0.01-1 micro M). After 24 hr the cells were harvested and the extracted rRNA was analyzed by agarose/2.4% polyacrylamide gel electrophoresis. The results show that only the TPA-treated hybrid cells synthesized both human and mouse 28S rRNA. Untreated cells and cells treated with phorbol or phorbol 13,20-diacetate synthesized only human 28S rRNA. The reactivation of silent rRNA genes by TPA was not restricted to mouse rRNA genes. In mouse-human hybrids in which both human and mouse rRNA genes are present but only the mouse genes are expressed, TPA treatment led to reactivation of the silent human rRNA genes. Although tumor promoters are generally believed to alter gene expression, we have found a specific gene whose transcription is modified by treatment with TPA.

Ribosomal RNA synthesis in imbibing radish (Raphanus sativus) embryo axes

The first hours of seed germination are characterized by an increase in the rate of RNA synthesis. Although this change is most easily accounted for by changes in the ribonucleotide pool sizes, we investigated two other aspects of rRNA synthesis which are likely to contribute to the phenomenon. Using isolated radish embryo axes, we demonstrate that processing of rRNA gene transcripts is much slower during early germination than during the growth of the seedling. We also provide evidence that rRNA gene expression is sequentially reactivated in different tissues, starting in the provascular tissue and apex cells and only later in the cortical cells of the rootlet.

Reactivation of silent rRNA genes by simian virus 40 in human-mouse hybrid cells.

Mouse-human hybrid cells were used to study the ability of simian virus 40 to regulate the expression of rRNA genes in vivo. In these hybrid cells, only the rRNA genes of the dominant species are expressed; the genes for the rRNA of the recessive species are silent. Simian virus 40 infection of these hybrids led to the production of two distinct 28S rRNA species as analyzed by agarose/2.4% polyacrylamide gel electrophoresis. These species were identified as human and mouse rRNAs. This result was confirmed by histochemical studies which indicated that the nucleolus organizer regions of both mouse and human chromosomes were actively synthesizing rRNA in the virus-infected hybrid cells. These results indicate that simian virus 40 infection can induce the expression of otherwise silent rRNA genes.

Deletion analysis of the expression of rRNA genes and associated tRNA genes carried by a lambda transducing bacteriophage

Transducing phage lambdailv5 carries genes for rRNA's, spacer tRNA's (tRNA1 Ile and tRNA1B Ala), and two other tRNA's (TRNA1 Asp and tRNA Trp). We have isolated a mutant of lambdailv5, lambdailv5su7, which carries an amber suppressor mutation in the tRNA Trp gene. A series of deletion mutants were isolated from the lambdailv5su7 phage. Genetic and biochemical analyses of these deletion mutants have confirmed our previous conclusion (E. A. Morgan, T. Ikemura, L. Lindahl, A. M. Fallon, and M. Nomura, Cell 13:335--344, 1978) that the genes for tRNA1 Asp and tRNA Trp located at the distal end of the rRNA operon (rrnC) are cotranscribed with other rRNA genes in that operon. In addition, these deletions were used to define roughly the physical location of the promoter(s) of the rRNA operon carried by the lambdailv5su7 transducing phage.

Regulation of rRNA gene expression in a human familial 14p+ marker chromosome.

Chromosome studies were carried out on normal individuals from three generations of one family with a 14p+ chromosome. The short arm of the 14p+ chromosome stained well using Giemsa but poorly using quinacrine or trypsin-Giemsa methods; in each case there was an unstained secondary constriction near the distal end of the short arm. Two Ag bands of average size were present on the 14p+ short arm, indicating that there were two active nucleolus organizer regions; the Ag band near the distal end of the short arm was slightly larger than that near the centromere. Each of the two Ag bands was seen associated with the short arm of one or more of the other acrocentric chromosomes, with a combined frequency of association no greater than that of other chromosomes with an Ag band of the same size. In one individual, hybridization in situ with radioactive 18S and 28S ribosomal RNA showed six times as many autoradiographic silver grains over the short arm of the 14p+ chromosome as over that of any other acrocentric chromosome. The results obtained using in situ labeling indicated that the 14p+ chromosome had a large number of rRNA genes compared with the other acrocentric chromosomes, whereas the results obtained using Ag-staining and association frequency indicated that the 14p+ chromosome had no greater nucleolus organizer activity than did the other acrocentrics. The difference in these findings suggests that not all the rRNA genes on the 14p+ chromosome were active.

Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.