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.     

Proportion of HeLa cell genome complementary to transfer RNA and 5 s RNA

The proportion of the HeLa cell genome complementary to tRNA and to 5 s RNA has been investigated by using the technique of RNA-DNA hybridization. The specificity of hybrids formed between HeLa DNA and these highly purified RNA classes has been assessed by analysis of the size distribution and nucleotide composition of the RNA recovered from the ribonuclease-treated hybrids.     

The Synthesis of 5s RNA and Its Relationship to 18s and 28s Ribosomal RNA in the Bobbed Mutants of DROSOPHILA MELANOGASTER

Ribosomes contain one molecule each of 5S, 18S and 28S RNA. In Drosophila melanogaster although the genes for 18S+28S are physically separated from the 5S RNA genes, the multiplicity of various ribosomal RNA genes is roughly the same. Thus a coordinate synthesis of these three molecules might seem feasible. This problem has been approached by determining the molar ratios of various RNA's in ovaries and in adult flies. In ovaries there is a slight excess of 5S RNA molecules over other rRNA's, but in adult flies no such differences exist. Bobbed mutants also have the same molar ratios as wild-type flies. Results on 5S RNA synthesis in both in vitro and in vivo studies show that it is reduced in coordination with 18S+28S rRNA in the bobbed mutants of Drosophila melanogaster. Various possibilities are discussed in considering the implications of these results.     

Localization of some 5s RNA cistrons on Escherichia coli chromosome

Summary 3 5S RNA cistrons, characterized by specific nucleotides sequences, are shown not to be linked.     

Genetic Analysis of the 5s RNA Genes in DROSOPHILA MELANOGASTER

The 5S RNA genes of Drosophila melanogaster in either an isogenic wild-type or a multiply inverted (SM1) chromosome 2 increase their multiplicity when opposite a deficiency for the 5S gene site. This is analogous to the compensation phenomenon previously described for the 18S and 28S ribosomal RNA genes of the X chromosome nucleolus organizer region. Molecular hybridization of 5S RNA to DNA containing various doses of the 56F1-9 region of chromosome 2 demonstrates that most, if not all, of the 5S genes reside in or near this region. Also, a deficiency missing approximately one-half of the wild-type number of 5S genes was isolated and genetically localized. This mutant has a phenotype like that of bobbed, a mutant known to be partially deficient in 18S and 28S ribosomal RNA genes. Finally, we report the existence of a chromosomal rearrangement which splits the second chromosome into two segments, each containing 5S DNA.     

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.     

Study of the maturation of 5 s RNA precursors in Escherichia coli

The existence of three precursors to 5 s RNA's (p5 s RNA's) has been confirmed. p5 s RNA's and mature 5 s RNA have different 5′-terminal sequences and produce the following 5′-terminal oligonucleotides: p5 s RNA-I:pAUUUG; p5 s RNA-II:pUUUG; p5 s RNA-III:pUUG; 5 s RNA:pUG. The results of experiments on pulse-labelled cells treated with actinomycin D, on chloramphenicol-inhibited cells, on various ribosome assembly-defective mutants and on the state of 5 s RNA in polysomes after a short labelling period, support the following conclusions. (1) p5 s RNA-I is the first identifiable precursor which appears during a pulse. (2) The amount of p5 s RNA-II, relative to those of the other p5 s RNA's, is very low at all times during pulse-chase experiments. On the contrary, it becomes significant during chloramphenicol inhibition and in one assembly-defective mutant under non-permissive conditions. (3) The maturation steps which lead to p5 s RNA-III and 5 s RNA normally occur after binding to 50 s subunit precursor particles and are, consequently, dependent upon protein synthesis. (4) The transition from p5 s RNA-III to 5 s RNA is a slow process, which is neither dependent upon nor required for the proper functioning of 50 s subunits in protein synthesis.