Genetic Modulation of RNA Metabolism in Drosophila. II. Coordinate Rate Change in 4s, 5s, and Poly-a Associated RNA Synthesis

It has been demonstrated that a particular rDNA-deficient Y chromosome (Y(bbSuVar-5)) increases the rate of ribosomal RNA synthesis in adult testes (Shermoen and Kiefer 1975) and in whole flies (Clark, Strausbaugh and Kiefer 1977). As an initial attempt to explore the molecular basis of this phenomenon, experiments were designed to determine if the rate increase was specific for rRNA as opposed to the other species of RNA. The genotypes used in these studies were car bb/Y(bb-), car bb/Y( bbSuVar-5), and Sam(+) iso. car bb/Y(bbSuVar-5 ) and car bb/Y(bb-) are deficient to the same extent in rDNA and Sam(+) iso is a wild-type stock. Following isotope incorporation, total RNA was extracted by a phenol:chloroform method and separated by polyacrylamide gel electrophoresis. The various RNA species were quantified by UV absorption and their radioactivity determined by gel fractionation and liquid scintillation counting. The resulting data permitted the calculation of a specific activity (i.e., dpm/microg RNA) which was defined as synthetic rate. Polyadenylated RNA was isolated using a poly-U sepharose column and similar rate calculations were made. The data from these studies indicate that the rate of synthesis of all species of RNA examined (28S + 18S, 5S, 4S transfer RNA and polyadenylated RNA) is increased by the presence of the Y(bbSuVar-5) chromosome. Genetic and molecular mechanisms are discussed.     

Replication of Sindbis virus. I. Relative size and genetic content of 26 s and 49 s RNA

The genome of Sindbis virus, 49 s RNA, is a single, intact polynucleotide chain having a molecular weight of 4.3±0.3 × 10⁶ daltons. This has been determined using a variety of methods including polyacrylamide gel electrophoresis, sedimentation after reaction with formaldehyde, determination of the molecular weights of the double-stranded forms of Sindbis-specific RNA and hybridization competition. The second major species of single-stranded RNA made after infection with Sindbis, 26 s RNA, has been found to have a molecular weight of 1.6 × 10⁶ daltons as determined by sedimentation in dimethylsulfoxide. Hybridizationcompetition experiments carried out between these two species of RNA, using double-stranded forms of Sindbis RNA isolated from infected cells, showed that 26 s RNA contains only one-third of the base sequences in 49 s RNA and thus represents a unique fraction of the viral genome.     

Regulation of Ribosomal RNA and 5s RNA Synthesis in DROSOPHILA MELANOGASTER: I. Bobbed Mutants

Analysis of the rates and amounts of rRNA and 5s RNA synthesized in Drosophila melanogaster bobbed mutants was done by using acrylamide-gel electrophoresis. The results show that the amounts of rRNA synthesized are constant, although the rates of rRNA synthesis in bb's are reduced to 30% of the wild-type level. The rates of synthesis of 5s RNA were constant. The rate of synthesis of the two kinds of molecules that enter in equimolar amounts into the mature ribosome is non-coordinated.-The rates of rRNA synthesis were shown to be proportional to the length of the scutellar bristles, supporting the notion that in trichogen cells there is no developmental delay, but the size of the bristle depends directly on the rate of rRNA synthesis.     

Studies of fractionated HeLa cell metaphase chromosomes. II. chromosomal distribution of sites for transfer RNA and 5 s RNA

DNA extracted from HeLa cell metaphase chromosomes fractionated on the basis of sedimentation velocity in glycerol-sucrose gradients has been tested for the capacity to hybridize with highly purified tRNA and 5 s RNA. The results obtained indicate that the sites for these two RNA classes, in contrast to those for the high molecular weight rRNA, are distributed among chromosomes of all size ranges.     

Release of template restriction in chromatin by nuclear 4.5s RNA.

The stimulatory mechanism of RNA synthesis of calf-thymus chromatin by nuclear 4.5 S RNA from the homologous tissue was investigated by using exogenously added Escherichia coli RNA polymerase. The RNA synthesis was initiated at low concentration of salt, and then the chain elongation was achieved at high concentration of ammonium sulfate in the presence of polyvinyl sulfate. Under these conditions the number of binding sites of RNA polymerase on chromatin which were capable of initiating RNA chain was increased by the addition of the 4.5 S RNA. This stimulation was presumed to result from the release of template restriction in chromatin. The polyvinyl salt minimized ribonuclease activity without changing the RNA polymerase activity bound to the template. Neither rearrangement nor release of chromatin proteins affected the amount or size of RNA produced. Preliminary analysis suggested that the molecular species of RNA produced upon the addition of the 4.5 S RNA from various tissues seemed to be heterologous.