Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.     

The genes for ribosomal RNA in diploid and polytene chromosomes of Drosophila melanogaster

The proportion of the Drosophila genome coding for ribosomal RNA was examined in DNA from both diploid and polytene tissues of Drosophila melanogaster by rRNA-DNA hybridization. Measurements were made on larvae with one, two, three and four nucleolus organizer regions per genome. In DNA from diploid tissues the percent rDNA (coding for 28S and 18S ribosomal DNA) was found to be in proportion to the number of nucleolus organizers present. The number of rRNA genes within a nucleolus organizer therefore does not vary in response to changes in the number of nucleolus organizers. On the other hand, in DNA from cells with polytene chromosomes the percent rDNA remained at a level of about 0.1% (two to six times lower than the diploid values), regardless of either the number of nucleolus organizers per genome or whether the nucleolus organizers were carried by the X or Y chromosomes. This independence of polytene rDNA content from the number of nucleolus organizers is presumably due to the autonomous polytenization of this region of the chromosome. When the rDNA content of DNA from whole flies is examined, both the rDNA additivity of the diploid cells and the rDNA independence of polytene cells will affect the results. This is a possible explanation for the relative rDNA increase known to occur in X0 flies, but probably not for the phenomenon of rDNA magnification. — In further studies on DNA from larval diploid tissues, the following findings were made: 1) the Ybb-chromosome carries no rDNA; 2) flies carrying four nucleolus organizers do not tend to lose rDNA, even after eleven generations, and 3) the nucleolus organizer on the wild type Y chromosome may have significantly less rDNA than does that on the corresponding X chromosome.     

Transcriptional organization of the Drosophila melanogaster ribosomal RNA genes.

Five distinct DNA replicating intermediates have been separated from lysates of bacteriophage G4-infected cells pulse-labelled during the period of replicative form synthesis using propidium diiodide/caesium chloride gradients. These are a partially single-stranded theta structure that is labelled in both the viral and complementary DNA strands; partially single-stranded circles, some with an unfinished viral DNA strand (25%) and some with an unfinished complementary DNA strand (75%); replicative form II(RFII) and replicative form I(RFI) DNA labelled only in the complementary DNA strand. To explain the pulse-label data a model is proposed in which G4 replicative form replication takes place by a displacement mechanism in which synthesis of the new viral DNA strand displaces the old viral DNA strand as a single-stranded DNA loop (D-loop) and when the displacement reaches half way round the molecule (the origin of synthesis of the G4 viral and complementary DNA strands are on opposite sides of the genome, Martin &; Godson 1977) synthesis of the complementary DNA strand starts, but in the opposite direction. Strand separation of the parent helix runs ahead of DNA synthesis, releasing two partially single-stranded circles from the replicating structure which then complete their replication as free single-stranded DNA circles. No evidence was found to support a rolling circle displacement mechanism of G4 replicative form synthesis.     

Unintegrated ribosomal genes in diploid and polytene tissues of Drosophila melanogaster

We have examined DNA from polytene salivary glands and diploid brains and imaginal discs of male and female larvae having one or two nucleolus organizers. DNA having an estimated molecular weight of 5×10⁹ or greater was obtained by sucrose gradient sedimentation of gently prepared lysates. Hybridization of the gradient fractions with ³H-ribosomal RNA reveals that 42% of the ribosomal genes are found in DNA of lower molecular weight (approximately 3×10⁸ daltons) in the salivary glands of every genotype examined. In the brains and imaginai discs, by contrast, all of the ribosomal genes are found in the high molecular weight peak except in females with one nucleolus organizer where 42% are found in lower molecular weight DNA, as in the salivary gland. Thus unintegrated genes are not an exclusive feature of polytene tissue, but can occur in diploid tissue as well in at least one genotype.     

The Drosophila melanogaster RPS17 gene encoding ribosomal protein S17

A human ribosomal protein S17 cDNA [Chen et al., Proc. Natl. Acad. Sci. USA 83 (1986) 6907-6911] was used as heterologous probe to isolate S17 clones from Drosophila genomic and cDNA recombinant libraries. Five S17 genomic clones were recognized; all contained overlapping regions of a single chromosomal site. Subsequently the Drosophila RPS17 gene was mapped by in situ hybridization to chromosome 3L, band 67B1-5. The locus spans approximately 1000 bp of DNA and includes four exons. It is preceded by conventional CAAT and TATA RNA polymerase II promoter motifs. The 131 amino acid protein encoded within Drosophila RPS17 is similar to ribosomal proteins from several other eukaryotes. Comparison of eukaryotic S17 proteins' primary structures as well as the number and location of their genes' intervening sequences suggest that S17 is a relatively recent addition to the ribosomal protein family, probably post-dating divergence of eukaryotes and prokaryotes.     

Dosage of 5 S and ribosomal genes during oogenesis of Drosophila melanogaster

During growth, the Drosophila egg chamber increases its DNA content over a thousandfold, mainly by polyploidization of the nurse cell nuclei. We wanted to determine if 5 S and ribosomal genes are replicated to the same extent as the remaining DNA. Egg chambers were mass fractionated to represent different size classes and, therefore, different stages of oogenesis. Nucleic acids were extracted from each class of egg chambers, and after removal and quantitation of the RNA, the content of 5 S and ribosomal genes in the different DNA fractions was assayed by filter hybridization. Diploid DNA and DNA from polytene salivary gland cells served as references. It was concluded that: (1) Ribosomal genes become underreplicated as oogenesis proceeds, but to a much lower extent than in polytene chromosomes of salivary glands of the same organism. (2) By contrast, 5 S genes are equally replicated in egg chambers of all stages of oogenesis. (3) Notwithstanding the large increase in DNA content of egg chambers during oogenesis, the increase in total RNA content (mostly ribosomal RNA) is over 15 times as large.     

Cardiomyopathy is associated with ribosomal protein gene haplo-insufficiency in Drosophila melanogaster

The Minute syndrome in Drosophila melanogaster is characterized by delayed development, poor fertility, and short slender bristles. Many Minute loci correspond to disruptions of genes for cytoplasmic ribosomal proteins, and therefore the phenotype has been attributed to alterations in translational processes. Although protein translation is crucial for all cells in an organism, it is unclear why Minute mutations cause effects in specific tissues. To determine whether the heart is sensitive to haplo-insufficiency of genes encoding ribosomal proteins, we measured heart function of Minute mutants using optical coherence tomography. We found that cardiomyopathy is associated with the Minute syndrome caused by haplo-insufficiency of genes encoding cytoplasmic ribosomal proteins. While mutations of genes encoding non-Minute cytoplasmic ribosomal proteins are homozygous lethal, heterozygous deficiencies spanning these non-Minute genes did not cause a change in cardiac function. Deficiencies of genes for non-Minute mitochondrial ribosomal proteins also did not show abnormal cardiac function, with the exception of a heterozygous disruption of mRpS33. We demonstrate that cardiomyopathy is a common trait of the Minute syndrome caused by haplo-insufficiency of genes encoding cytoplasmic ribosomal proteins. In contrast, most cases of heterozygous deficiencies of genes encoding non-Minute ribosomal proteins have normal heart function in adult Drosophila.