Sequence arrangement of tRNA genes on a fragment of Drosophila melanogaster DNA cloned in E. coli.

A plasmid with the vector Col E1 attached to an insert of Drosophila melanogaster DNA carrying four tRNA genes has been cloned in E. coli. Some features of the sequence arrangement and the positions of the tRNA genes have been determined by electron microscopic methods and by restriction endonuclease mapping. tRNA genes were mapped at 1.4, 4.7, 5.9 and 8.6 kb from one of the Drosophila/Col E1 junctions in the Drosophila insert of total length 9.34 kb. There are several secondary structure features consisting of inverted repeat sequences of length about 70-100 nucleotide pairs, some with and some without intervening loops, irregularly distributed on the insert. Cross-hybridization of tRNAs isolated by hybridization to separated restriction fragments indicate that the tRNA genes at 4.7, 5.9 and 8.6 kb are identical and differ from the one at 1.4 kb. Thus the positions of the genes, of the secondary structure features and of the restriction endonuclease sites all indicate that the spacers between the genes are not identical tandem repeats. In situ hybridization with cRNA transcribed from the plasmid showed localization at region 42A of chromosome 2R.     

A novel arrangement of the 18S and 28S sequences in a repeating unit of Drosophila melanogaster rDNA.

The sequences corresponding to the 18S and 28S rRNAs have been mapped within a cloned 17 kilobase (kb) fragment formed by Eco R1 cleavage of Drosophila melanogaster rDNA. This fragment, Dm103, represents the longer of two major types of repeating units that are present in the rDNA of this fly, and was cloned as a hybrid plasmid, pDm103, consisting of Dm103 inserted at the Eco R1 site of the pSC101 vector (Glover et al., 1975). Mapping of the 18S and 28S rDNA in Dm103 was accomplished by quantitative determination of the amount of these rDNAs in each member of an ordered set of restriction fragments obtained by Hind III and Eco R1 ccleavage of pDm103. The amounts of 18S and 28S rDNAs were determined by hybridization of the rRNAs to fragments that were purified by cloning, and an unambiguous order of the fragments within pDm103 was established by heteroduplex mapping and from the stoichiometry of the fragment lengths. The resulting map revealed that the 4 kb of 28S rDNA within the long repeating unit represented by Dm103 is divided into two blocks that are separated by 5.4 kb of DNA of unknown function. It is this unusual arrangement of the 28S rDNA that distinguishes the long repeating units (17 kb) from the short units (11.5) kb), whose 4 kb of 28S rDna is confined to a single block, as is shown in the accompanying paper (White and Hogness, 1977). The remainder of the DNA in this long unit appears to be typically arranged, with the 2 kb of 18S rDNA confined to a single block that is separated by about 1 kb from the closest block of 28S rDNA.     

Evidence for an excess of rDNA in the testis of Drosophila melanogaster during rDNA magnification.

Summary Hybridization of rRNA with DNA extracted from different tissues of different genotypes have been performed. The results show that: 1) in DNA extracted from the testis of premagnified males there exists an excess of rDNA, which is consistent with the model proposed by Ritossa (1972) and by us (1973) to explain the phenomenon of magnification. 2) in DNA extracted from diploid tissues of different genotypes the percent of rDNA is directly proportional to the number of ribosomal genes. 3) in polytene cells the percent of rDNA for all genotypes so far studied is less than that in diploid cells and is not significantly dependant on the genotype. This last result is consistent with those of Spear and Gall (1973).     

Bromodeoxyuridine inhibits the rDNA compensatory response of Drosophila melanogaster

The compensatory response is a regulatory event influencing the redundancy of the ribosomal RNA cistrons (rDNAs) of Drosophila melanogaster. In this report we attempt to demonstrate that the compensatory event and the thymidine analogue bromodeoxyuridine (BrdU) specifically interact. We conclude that the drug inhibits the compensatory response of Drosophila melanogaster XO males and argue that the compensatory event is not the passive consequence of replicational dominance known to occur in Drosophila polytene tissues.     

Sequence of histone 2B of Drosophila melanogaster.

The complete sequence of histone 2B of Drosophila has been determined by using an improved Beckman sequenator. Comparing these data with those previously published by other investigators on the histone 2B of calf [Iwai, K., Hayashi, H., & Ishikawa, K. (1972) J. Biochem. (Tokyo) 72, 357--367], trout [Koostra, A., & Bailey, G. S. (1978) Biochemistry 17, 2504--2510], and Patella (a limpet) [van Helden, P. D., Strickland, W. N., Brandt, W. F., & von Holt, C. (1979) Eur. J. Biochem. 93, 71--78], it is possible to assess the evolutionary stability of this protein. There is little conservation of sequence in the N-terminal portion of the molecule (residues 1--26 numbering according to calf H2B), while the remainder of the protein, which we designate the C-terminal portion, is highly conserved. In the region of 27--125 residues, there are 9 substitutions in the composite data among the 98 positions, 8 of them conservative. These data indicate that very different selective pressures operate on the two different portions of the H2B molecule, implying the existence of two well-defined regions. Studies on the structure of the nucleosome by others have suggested that the C-terminal portion of H2B is involved in histone-histone interactions while the N-terminal portion is a relatively free "tail" binding to DNA. The sequence data indicate that the function of the C-terminal region of H2B requires considerable sequence specificity while that of the N-terminal region does not.     

Differential elimination of rDNA genes in bobbed mutants of Drosophila melanogaster.

In Drosophila melanogaster, the multiply repeated genes encoding 18S and 28S rRNA are located on the X and Y chromosomes. A large percentage of these repeats are interrupted in the 28S region by insertions of two types. We compared the restriction patterns from a subcloned wild-type Oregon R strain to those of spontaneous and ethyl methanesulfonate-induced bobbed mutants. Bobbed mutations were found to be deficiencies that modified the organization of the rDNA locus. Genes without insertions were deleted about twice as often as genes with type I insertions. Type II insertion genes were not decreased in number, except in the mutant having the most bobbed phenotype. Reversion to wild type was associated with an increase in gene copy number, affecting exclusively genes without insertions. One hypothesis which explains these results is the partial clustering of genes by type. The initial deletion could then be due either to an unequal crossover or to loss of material without exchange. Some of our findings indicated that deletion may be associated with an amplification phenomenon, the magnitude of which would be dependent on the amount of clustering of specific gene types at the locus.     

Duplicated rDNA sequences of variable lengths flanking the short type I insertions in the rDNA of Drosophila melanogaster.

We describe cloned segments of rDNA that contain short type I insertions of differing lengths. These insertions represent a coterminal subset of sequences from the right hand side of the major 5kb type I insertion. Three of these shorter insertions are flanked on both sides by a short sequence present as a single copy in uninterrupted rDNA units. The duplicated segment is 7, 14 and 15 nucleotides in the different clones. In this respect, the insertions differ from the 5kb type I insertion, where the corresponding sequence is found only at the right hand junction and where at the left hand side there is a deletion of 9 nucleotides of rDNA (Roiha et al.,1981). One clone is unusual in that it contains two type I insertions, one of which is flanked by a 14 nucleotide repeat. The left hand junction of the second insertion occurs 380 nucleotides downstream in the rDNA unit from the first. It has an identical right hand junction to the other elements and the 380 nucleotide rDNA sequence is repeated on both sides of the insertion. We discuss the variety of sequence rearrangements of the rDNA which flank type I insertions.     

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 sequence arrangement of Drosophila melanogaster 5s DNA cloned in recombinant plasmids.

Segments of Drosophila melanogaster DNA containing 5S rRNA genes have been propagated in recombinant plasmids using E. coli as a host and Col E1 as a vector. Electron microscope partial denaturation mapping, mapping by ferritin labeling and restriction enzyme-gel electrophoresis analysis all indicate that the Drosophila DNA inserts of these plasmids consist of tandem repeats of 5S genes and spacer regions. The repeat length is approximately 380 nucleotide pairs (ntp), corresponding to a gene of length 120 ntp and a spacer of length 260 ntp. The insert in one plasmid (pCIT9) consists of 32 contiguous repeats. Restriction enzyme-gel electrophoresis analysis shows that all these repeats have the same length within ± 5 nucleotides. This repeat length is estimated as 370 ± 20 ntp by gel electrophoresis and 390 ± 40 ntp by partial denaturation mapping. A second plasmid (pCIT19) contains three complete genes, two complete spacers and incomplete flanking spacer sequences. The two complete repeat units released by suitable restriction endonuclease digestions differ in length by 20 ± 5 ntp, with estimated lengths of 370 and 390 ntp. The positions and spacings of the genes on this plasmid have been observed directly by ferritin labeling and by partial denaturation mapping. The A+T content of the 5S DNA spacer region is calculated to be 68%. By in situ hybridization, cRNA transcribed from one plasmid hybridizes to polytene chromosomes only at band 56F, the known locus of the 5S rRNA genes. Spontaneous excision of some of the tandem repeat units from the recombinant plasmids occurs during growth in E. coli; the frequency of excision does not depend upon the recA character of the host, but is greatly increased by chloramphenicol treatment.