Ribosomal DNA organization before and after magnification in Drosophila melanogaster

In all eukaryotes, the ribosomal RNA genes are stably inherited redundant elements. In Drosophila melanogaster, the presence of a Ybb(-) chromosome in males, or the maternal presence of the Ribosomal exchange (Rex) element, induces magnification: a heritable increase of rDNA copy number. To date, several alternative classes of mechanisms have been proposed for magnification: in situ replication or extra-chromosomal replication, either of which might act on short or extended strings of rDNA units, or unequal sister chromatid exchange. To eliminate some of these hypotheses, none of which has been clearly proven, we examined molecular-variant composition and compared genetic maps of the rDNA in the bb(2) mutant and in some magnified bb(+) alleles. The genetic markers used are molecular-length variants of IGS sequences and of R1 and R2 mobile elements present in many 28S sequences. Direct comparison of PCR products does not reveal any particularly intensified electrophoretic bands in magnified alleles compared to the nonmagnified bb(2) allele. Hence, the increase of rDNA copy number is diluted among multiple variants. We can therefore reject mechanisms of magnification based on multiple rounds of replication of short strings. Moreover, we find no changes of marker order when pre- and postmagnification maps are compared. Thus, we can further restrict the possible mechanisms to two: replication in situ of an extended string of rDNA units or unequal exchange between sister chromatids.     

Replication of chromosomal DNA in diploid Drosophila melanogaster cells cultured in vitro

Replication rate and replicon sizes in chromosomal DNA of in vitro cultured diploid D. melanogaster cells were determined using autoradiography of ³H-thymidine labeled DNA. Synthesis of DNA in euchromatic and heterochromatic regions of Drosophila diploid cells occurs at different periods of the S phase which lasts 10 h. During the first 4 h the synthesis is observed only in euchromatic regions. The heterochromatic synthesis starts shortly before the synthesis in euchromatic regions is completed and lasts for 6 h until the end of the S phase. The cells were synchronized by 5fluorodeoxyuridine which blocked the diploid cell DNA synthesis. Synthesis was found to start simultaneously in most euchromatic replicons. In the majority of the replicons the synthesis started at a single point and proceeded bidirectionally. The average rate of DNA synthesis per fork was 12.5 m/h (38 kb). The mean distance between the middle points of adjacent labeled regions was 70 m (210 kb). The size of most replicons ranged from 40 to 120 m. — These estimates do not apply to the heterochromatic portions of the D. melanogaster genome since the measurements have been carried out on DNA preparations obtained during the first 2 h of the S phase. — On the average, a replicon can consist of 7 chromomeres since the size of a replicon in diploid cell chromosomal DNA and DNA length of a polytene chromomere average 210 and 30 kb, respectively.     

Ribosomal DNA in Drosophila melanogaster. I. Isolation and characterization of cloned fragments.

Fragments of rDNA3 from Drosophila melanogaster produced by the restriction endonuclease EcoRI were cloned in the form of recombinant plasmids in Escheriehia coli. Maps were prepared showing the location of the coding regions and of several restriction endonuclease sites. Most rDNA repeats have a single EcoRI site in the 18 S gene region. Thus, 19 of 24 recombinant clones contained a full repeat of rDNA. Ten repeats with continuous 28 S genes and repeats containing insertions in the 28 S gene of 0.5, 1 and 5 kb were isolated. The 0.5 and 1 kb insertion sequences are homologous to segments of the 5 kb insertions; because of this homology they are grouped together and identified as type 1 insertions. Four recombinant clones contain an rDNA fragment that corresponds to only a portion of a repeating unit. In these fragments the 28 S gene is interrupted by a sequence which had been cleaved by EcoRI. The interrupting sequences in these clones are not homologous to any portion of type 1 insertions and are therefore classified as type 2. In one of the above clones the 28 S gene is interrupted at an unusual position; such a structure is rare or absent in genomic rDNA from the fly. Another unusual rDNA fragment was isolated as a recombinant molecule. In this fragment the entire 18 S gene and portions of the spacer regions surrounding it are missing from one repeat. A molecule with the same structure has been found in uncloned genomic rDNA by electron microscopic examination of RNA/DNA hybrids.     

Localization of chromosomal DNA sequences homologous to ribosomal gene type I insertion DNA in Drosophila melanogaster

Summary Chromosomal sites which have DNA homology to the 1 kb (kilobase pair) BamHI restrictable fragment of the 5 kb type I insertion present in many ribosomal genes in Drosophila melanogaster, were identified by using in situ hybridization and autoradiography. XX and XY complements of polytene chromosomes showed the nucleolus and chromocenter to be heavily labeled. Of the light label over euchromatic regions, the 102C band of chromosome 4 labeled particularly intensely. In mitotic XX and XY complements, the NORs (nucleolus organizer regions) of both sex chromosomes labeled as did the centromeric heterochromatin of autosomes. Label also appeared less frequently over telomeric and euchromatic regions.     

Chromosomal rearrangements which affect the chromosomal integration of the ribosomal genes in Drosophila melanogaster.

Sucrose gradient analysis of DNA from detergent-pronase lysates of whole adult flies has been used to examine a variety of genotypes for the presence of ribosomal genes not integrated into the DNA of the chromosome. Such genes were found in females in which one X chromosome carries an inversion, having one of its breakpoints between the nucleolus organizer and the centromere. These inversions move the nucleolus organizer to the distal end of the X chromosome. Other inversions which do not move the nucleolus organizer, as well as a series of bobbed deficiencies, did not induce unintegrated genes. The same inversions which induce unintegrated genes in adults also produce them in the diploid brain and imaginal discs of larvae. On the other hand, in the polytene salivary glands, unintegrated genes were found in every genotype examined.     

Ribosomal DNA in Drosophila melanogaster. II. Heteroduplex mapping of cloned and uncloned rDNA.

Sequences in the cloned Drosophila melanogaster rDNA fragments described by Dawid et al. (1978) were compared by heteroduplex mapping. The nontranscribed spacer regions in all fragments are homologous but vary in length. Deletion loops were observed at variable positions in the spacer region suggesting that spacers are internally repetitious.Many rDNA repeats in D. melanogaster have a 28 S gene interrupted by a region named the ribosomal insertion. Insertions of 0.5, 1 and 5 kb were found in repeat-length EcoRI fragments. These DNA regions, named type 1 insertions, are homologous at their right ends. Although 1 kb insertions are quite precisely twice as large as 0.5 kb insertions they do not represent a duplication of the shorter sequence. Some insertions have at least one EcoRI site and therefore yield EcoRI fragments which are only part of a repeat. The sequences in two cloned right-hand partial insertion sequences are homologous, but the sequences in two lefthand partial insertions are not. None of the EcoRI-restrictable insertion sequences has any homology to any part of type 1 insertions; they are thus grouped together as type 2. Evidence for insertion sequences of at least two types in uncloned rDNA was obtained by annealing a cloned fragment with a 1 kb insertion to genomic rDNA. About 15% of the rDNA repeats show substitution type loops between the 1 kb type 1 insertion derived from the cloned fragment and type 2 insertions in the rDNA.     

The Drosophila virilis dopa decarboxylase gene is developmentally regulated when integrated into Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) has been isolated from Drosophila virilis and introduced into the germ-line of Drosophila melanogaster by P-element mediated transformation. The integrated gene is induced at the correct stages during development with apparently normal tissue specificity, indicating that cis-acting elements required for regulation are functionally conserved between the two species. A comparison of the DNA sequences from the 5' flanking regions reveals a cluster of small (8-16 bp) conserved sequence elements within 150 bp upstream of the RNA startpoint, a region required for normal expression of the D. melanogaster Ddc gene.     

Small circular DNA in Drosophila melanogaster

Covalently closed small circular DNA isolated from Drosophila melanogaster is described. The small circular DNA is found in blastema stage eggs and in Schneider's cell culture line 2 and a cloned subline of line 2. It is heterogeneous in size, although the size distributions and mean sizes differ for each source. The small circular DNA from Schneider's line 2 cells ranges from 0.09-7.3 μm, with a mean contour length of 1.1 μm. This DNA has a buoyant density of 1.703 g/cc and appears to be present predominantly in the nuclear fraction of detergent-disrupted cells. The restriction enzyme EcoRI cleaves approximately 40% of the small circular DNA with a bias toward the larger size classes.Both logarithmic and stationary phase cells contain approximately 3–40 average sized small circular DNA molecules per cell, representing a maximum of 0.03% of the total cellular DNA. Exposure to cycloheximide or puromycin for 14 hr results in a 30 fold increase in the number of small circles per cell, but reduces the mean length of the circular DNA to 0.3 μm. The drug-amplified DNA has a buoyant density in the range of 1.698-1.703 g/cc. No amplification was seen in cells treated with either inhibitor for 3.5 hr. Ethidium bromide, cytosine arabinoside, β-ecdysone, and insulin all had no significant effect on the amount per cell of either small circular DNA or mitochondrial DNA.     

Two new Y-linked genes in Drosophila melanogaster

The Y chromosome and other heterochromatic regions present special challenges for genome sequencing and for the annotation of genes. Here we describe two new genes (ARY and WDY) on the Drosophila melanogaster Y, bringing its number of known single-copy genes to 12. WDY may correspond to the fertility factor kl-1.     

Monoclonal antibodies against chromosomal proteins of Drosophila melanogaster

Total nuclear protein from the embryonic D. melanogaster cell line Kc and crude hydroxyapatite fractions thereof were used for immunization of mice. From the spleen cells of these mice we established 755 permanent lymphoid cell lines using the hybridoma technique originally developed by Köhler and Milstein (1975). Radioimmunoassay showed 455 of these cell lines secreted antibodies which bound to component(s) contained in the antigen mixtures used for immunization. Screening of 311 cell lines using indirect immunofluorescence revealed 58 lines whose antibodies showed a highly selective staining pattern on polytene chromosomes from the salivary glands of D. melanogaster third instar larvae. Eight of these cell lines were cloned and further characterized. We were able to order the staining patterns into three distinct classes based on the staining behaviour of the monoclonal antibodies: staining of active regions, staining of phase dark bands or staining of most interbands. The molecular weight of those antigens against which the monoclonal antibodies were directed was determined in SDS polyacrylamide gels.     

Drosophila melanogaster U1 snRNA genes

We have isolated and characterized a recombinant which contains a Drosophila melanogaster U1 small nuclear RNA (snRNA) gene colinear with the published snRNA sequence. Southern hybridizations of the fly genomic DNA, using as probe a plasmid containing only the coding region of the gene, shows that the fly contains at most three or four genes and very few related sequences for the small nuclear U1 RNA. These genes were localized by in situ hybridization at different chromosomal loci and show no spatial relationship to the U2 snRNA genes.