DNA sequence adjacent to and specific for the 1.672 g/cm3 satellite DNA in the Drosophila genome.

The Drosophila genome contains nearly 2.8 X 10(4) kilobases of satellite DNA. This simple sequence satellite DNA is contained within transcriptionally inactive heterochromatin that is distributed among all chromosomes with a concentration at the centromeres and along the length of the Y chromosome. To investigate the relationship of the satellite DNA with the surrounding sequences, we have isolated a satellite junction sequence that is repetitive and specifically adjacent to the 1.672 g/cm3 satellite DNA. It is conserved between strains of Drosophila melanogaster and localized to the chromocenter of polytene chromosomes. The characteristics of this sequence suggest a functional role involving the specific organization of large regions of chromosomes.

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.

Genetic modulation of RNA metabolism in Drosophila. III. Requirement for an rDNA-deficient X chromosome in YbbSuVar-3-mediated increases in RNA synthesis.

Males of the genotype car bb/YbbSuVar-5 have an approximately two-fold increase in the rate of accumulation of 4S, 5S, 18S plus 28S and poly-A+ RNA molecules when compared to males of the genotype car bb/Ybb- (CLARK, STRAUSBAUGH AND KIEFER 1977; CLARK and KIEFER 1977). Experiments were designed to determine if the modulation of RNA metabolism by YbbSuVar-5 requires an rDNA deficiency on the X chromosome. Synthetic rates for 4S, 5S, and 18S plus 28S RNA's were measured in the genotypes Sam + iso/Ybb- and Sam + iso/YbbSuVar-5 by injecting adult males with 3H-uridine, allowing them to incorporate label for two hours, fractionating extracted RNA by polyacrylamide gel electrophoresis and determining the specific activities of RNA's (dpm/microgram RNA). The results of these determination showed that the synthetic rates for 4S, 5S, and 18S plus 28S RNA's are the same in these two genotypes, demonstrating that the YbbSuVar-5 chromosome does not increase transcription when paired with an X chromosome that is wild type for rDNA. Saturation hybridization was used to measure the rDNA content in several genotypes. These results showed that Sam + iso/YbbSuVar-5 males and Sam + iso/Ybb- males have equivalent amounts of rDNA.--These results are consistent with the suggestion that the Ybb- and YbbSuVar-5 chromosomes have similar amounts of rDNA. The nature of compensatory strategies in rDNA-deficient genotypes is discussed.

Genetic modulation of RNA metabolism in Drosophilia. I. Increased rate of ribosomal RNA synthesis.

It has been suggested that a particular Y chromosome which is rDNA-deficient (YbbSuVar-5) may be associated with an increased utilization of rDNA template in adult testes (Shermoen and Kiefer 1975). To extend the observations on this chromosome, experiments were designed to determine if the chromosome has an effect on rRNA synthesis in bobbed adults and on classic bobbed phenotypes (shortened and thinner scutellar bristles and delayed development). Specific activity measurements were made on rRNA extracted from adult males of the genotypes car bb/YbbSuVar-5, which are rDNA-deficient to the same extent, and from Samarkand+ isogenic (Sam+ iso), which is a wild-type stock. The resulting data demonstrated that the presence of the YbbSuVar-5 chromosome increases the rate of ribosomal RNA synthesis in adult flies. In addition, it was found that the presence of this particular Y chromosome restores wild-type bristle phenotype and development time. Appropriate genetic crosses indicate that the observed effects (increased rRNA synthesis, restoration of wild-type phenotype) are a function of this particular Y chromosome, and are not due to autosomal factors. The results of these experiments suggest that the rate of rRNA accumulation is under genetic control.

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.

Regulation in rDNA-deficient Drosophila melanogaster.

Bobbed mutants of Drosophila melanogaster were used to determine whether there is any functional compensation for deficiency in rDNA content. The rate of rRNA accumulation was measured in the testes of five bb mutants of different phenotypic severity and in a wild type strain. The different rates of rRNA accumulation were compared to the phenotype (macroscutellar bristle length) and found to have a direct correlation (as in Weinman, 1972). However, there was not a direct relationship between the rate of rRNA accumulation and rDNA content. It is concluded that there is regulation of rRNA accumulation in some mutants, but that the regulation cannot be considered to be a compensation for the lack of rDNA. These results are discussed relative to other observations on the regulation of RNA synthesis in Drosophila.

Mitotic abnormalities in sea urchin embryos exposed to Dactinomycin.

Dactinomycin at concentrations of 10, 20, 25, and 50 mug/ml causes mitotic abnormalities in sea urchin embryos. Sister chromosome separation is impaired and anaphase bridges are frequently formed. The result is an unequal distribution of the chromosome complement to daughter cells. Possible explanations are discussed.