Isolation and characterization of a gene encoding DNA topoisomerase I in Drosophila melanogaster.

We synthesized a DNA probe specific for the gene encoding eucaryotic DNA topoisomerase I by the polymerase chain reaction. The sequences of the primers for this reaction were deduced from the regions with extensive homology among the enzymes from the fission and budding yeasts, and the human. From the clones isolated by screening a Drosophila cDNA library with this DNA probe, two cDNA clones of 3.8 and 5.2 kb were characterized and completely sequenced. Both cDNA sequences contain an identical open reading frame for 972 amino acid residues. The 3.8 kb messenger RNA is likely generated by using a polyadenylation site 5' upstream to that used in generating the 5.2 kb mRNA. The predicted amino acid sequence shows that a segment of 420 amino acid residues at the amino terminus is hydrophilic, similar to the amino terminal 200 residues in the yeast and human enzymes. Furthermore, the Drosophila enzyme is unique in that the amino terminal 200 residues are enriched in serine and histidine residues; most of them are present in clusters. The rest of the Drosophila sequence is highly homologous to those from yeast and human enzymes. The evolutionarily conserved residues are identified and are likely the critical elements for the structure and function of this enzyme. A plasmid vector containing the cloned cDNA was constructed for the expression of Drosophila protein in Escherichia coli. The enzymatic and immunochemical analysis of the polypeptide produced in this heterologous expression system demonstrated that the expressed protein shares similar enzymatic properties and antigenic epitopes with DNA topoisomerase I purified from Drosophila embryos or tissue culture cells, thus establishing the bacterial expression system being useful for the future structure/function analysis of the Drosophila enzyme.     

DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA

In order to study the double-strand DNA passage reaction of eukaryotic type II topoisomerases, a quantitative assay to monitor the enzymic conversion of supercoiled circular DNA to relaxed circular DNA was developed. Under conditions of maximal activity, relaxation catalyzed by the Drosophila melanogaster topoisomerase II was processive and the energy of activation was 14.3 kcal . mol-1. Removal of supercoils was accompanied by the hydrolysis of either ATP or dATP to inorganic phosphate and the corresponding nucleoside diphosphate. Apparent Km values were 200 microM for pBR322 plasmid DNA, 140 microM for SV40 viral DNA, 280 microM for ATP, and 630 microM for dATP. The turnover number for the Drosophila enzyme was at least 200 supercoils of DNA relaxed/min/molecule of topoisomerase II. The enzyme interacts preferentially with negatively supercoiled DNA over relaxed molecules, is capable of removing positive superhelical twists, and was found to be strongly inhibited by single-stranded DNA. Kinetic and inhibition studies indicated that the beta and gamma phosphate groups, the 2'-OH of the ribose sugar, and the C6-NH2 of the adenine ring are important for the interaction of ATP with the enzyme. While the binding of ATP to Drosophila topoisomerase II was sufficient to induce a DNA strand passage event, hydrolysis was required for enzyme turnover. The ATPase activity of the topoisomerase was stimulated 17-fold by the presence of negatively supercoiled DNA and approximately 4 molecules of ATP were hydrolyzed/supercoil removed. Finally, a kinetic model describing the switch from a processive to a distributive relaxation reaction is presented.     

An RNase-sensitive particle containing Drosophila melanogaster DNA topoisomerase II

Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II- containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II- containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II- containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).     

DNA topoisomerase II from Drosophila melanogaster. Purification and physical characterization

A type II DNA topoisomerase has been purified from the nuclei of Drosophila melanogaster 6- to 18-h-old embryos. The enzyme, as assayed by its ability to catenate supercoiled DNA, behaved as a single homogeneous species throughout the procedure and the yield was approximately 0.5 mg of protein/100 g of dechorionated embryos. The final product was entirely ATP-dependent and free of topoisomerase I, endonuclease and protease activities. The purified topoisomerase II had a Stokes radius of 69 A and a sedimentation coefficient (S20,w) of 9.2 S, leading to a calculated native molecular weight of approximately 261,000. The protein consists of a single polypeptide of molecular weight 166,000, as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Taken together with the above hydrodynamic studies, the Drosophila enzyme is probably a homodimer, as has been observed for other eukaryotic type II enzymes. Thus, it appears that during the course of evolution the heterologous subunits which comprise bacterial type II topoisomerases have been combined into a single polypeptide chain in eukaryotes.     

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.     

Developmental regulation of Drosophila DNA topoisomerase II

Affinity-purified polyclonal antibodies were used to quantitate steady-state levels of DNA topoisomerase II (topo II) throughout Drosophila development. Although wide fluctuations were recorded at different stages, these fluctuations were paralleled by changes in levels of the nuclear lamin, a nuclear structural protein used as an internal standard. The exception to this was adult males where lamin levels were significantly elevated relative to topo II. Northern blot analyses of topo II and lamin mRNA, performed in conjunction with immunoblot analyses of protein revealed fluctuations in levels of the two different messages that paralleled changes in each other and in their respective translation products. Biochemical and immunochemical analyses were complemented by indirect immunofluorescence and immunoperoxidase experiments performed in situ. topo II was found distributed throughout nuclei in most but not all cell types examined. These results for Drosophila topo II are apparently at odds with those obtained by others working in vertebrate systems (see, for example, Heck, M. M. S. and W. C. Earnshaw. 1986. J. Cell Biol. 103:2569-2581; Heck, M. M. S., W. N. Hittelman, and W. C. Earnshaw. 1988. Proc. Natl. Acad. Sci. USA. 85:1086-1090) and suggest that in Drosophila, topo II may not be a useful marker for the proliferative state.     

Essential genes in proximal 3L heterochromatin of Drosophila melanogaster

We have further characterized essential loci within the centric heterochromatin of the left arm of chromosome 3 (3L) of Drosophila melanogaster, using EMS, radiation and P element mutagenesis. We failed to find any new essential genes, a result that suggests a lower-than-average gene density in this region. Mutations affecting expression of the most proximal gene [lethal 1, l1 or l(3)80Fj] act as dominant suppressors of Polycomb (Pc), behavior which is consistent with a putative trithorax group (trx-G) gene. The third gene to the left of the centromere [lethal 3, l3 or l(3)80Fh] is likely to correspond to verthandi (vtd), a known trx-G gene that plays a role in the regulation of hedgehog (hh) expression and signalling. The intervening gene [lethal 2, l2 or l(3)80Fi] is required throughout development, and mutant alleles have interesting phenotypes; in various allelic combinations that survive, we observe fertility, bristle, wing, eye and cuticle defects.     

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.     

Enzymatic studies of DNA repair in Drosophila melanogaster

Thus far, our studies in Drosophila have concentrated primarily on the various enzymes involved in the in vitro repair of modified or nonconventional DNA substrates. In some cases, our findings have led us to investigate events that may not have a bearing on DNA repair, but rather may be associated with developmental signals important to the maturation of the organism. As appealing as some of these models seem, however, they must await confirmation through detailed genetic studies before any substantial conclusions can be drawn. This combination of genetic and biochemical knowledge makes Drosophila an exciting organism for an eventual detailed understanding of the developmental expression and cellular location of DNA-repair systems.     

Initial phases of DNA synthesis in Drosophila melanogaster

Replication patterns of the X chromosomes and autosomes in D. melanogaster male and female larvae during the discontinuously labeled initial and end phases of DNA synthesis were compared. In female larvae X and autosomes behaved correspondingly during all the replication stages. In males, however, the X chromosome shows a differential replication behavior from that of the autosomes already during the discontinuously labeled initial stage.—In those nuclei of both sexes, in which the autosomes correspond in their initial replication patterns, significantly more labeled regions are to be found over the male X than over the female X. The complementary behavior during the end phases (Berendes, 1966), i.e. the reverse of that above, leads to an earlier completion of the replication cycle in most of the labeled regions of the male X chromosome. The differential replication revealed in the autoradiograms is interpreted as a consequence of the polytene structure in giant chromosomes.     

Essential Loci in Centromeric Heterochromatin of Drosophila melanogaster. I: The Right Arm of Chromosome 2

With the most recent releases of the Drosophila melanogaster genome sequences, much of the previously absent heterochromatic sequences have now been annotated. We undertook an extensive genetic analysis of existing lethal mutations, as well as molecular mapping and sequence analysis (using a candidate gene approach) to identify as many essential genes as possible in the centromeric heterochromatin on the right arm of the second chromosome (2Rh) of D. melanogaster. We also utilized available RNA interference lines to knock down the expression of genes in 2Rh as another approach to identifying essential genes. In total, we verified the existence of eight novel essential loci in 2Rh: CG17665, CG17683, CG17684, CG17883, CG40127, CG41265, CG42595, and Atf6. Two of these essential loci, CG41265 and CG42595, are synonymous with the previously characterized loci l(2)41Ab and unextended, respectively. The genetic and molecular analysis of the previously reported locus, l(2)41Ae, revealed that this is not a single locus, but rather it is a large region of 2Rh that extends from unextended (CG42595) to CG17665 and includes four of the novel loci uncovered here.THE term “heterochromatin” was introduced by Heitz (1928) to describe regions of mitotic chromosomes that remain condensed throughout the cell cycle, in contrast to regions of euchromatin, which condense only during cell division. Heterochromatin was later divided into two classes: constitutive and facultative heterochromatin (Brown 1966). Constitutive heterochromatin is found in large blocks near centromeres and telomeres, while facultative heterochromatin can be described as silenced euchromatin that undergoes heterochromatization at specific developmental stages. Other properties of constitutive heterochromatin include late replication in S phase, low gene density, strikingly reduced level of meiotic recombination, enrichment in transposable element sequences and highly repetitive satellite DNA sequences, and the ability to silence euchromatic gene expression in a phenomenon called position effect variegation.Approximately 30% of the Drosophila melanogaster genome consists of constitutive heterochromatin (Gatti and Pimpinelli 1992). Centromeric heterochromatin in D. melanogaster is composed of mainly middle-repeat satellite DNA sequences and clusters of transposable element sequences (Lohe et al. 1993; Pimpinelli et al. 1995). Genes that reside in the heterochromatin are scattered like islands between the satellites and clusters of transposable elements. On average, heterochromatic genes are larger than euchromatic genes, primarily due to the prevalent accumulation of transposable element sequences in their introns (Devlin et al. 1990; Biggs et al. 1994; Dimitri et al. 2003a,b; Hoskins et al. 2007). Heterochromatic genes also tend to be AT-rich compared to their euchromatic counterparts; there is some evidence suggesting that the coding sequences of heterochromatic genes evolve toward AT richness in response to being located in heterochromatin (Yasuhara et al. 2005; Díaz-Castillo and Golic 2007).Drosophila heterochromatin is vastly under-replicated in polytene chromosomes, so heterochromatic genes cannot easily be mapped through polytene analysis. However, by using Hoechst 33258 and N-chromosome banding techniques, Dimitri (1991) was successful in dividing heterochromatin in mitotic chromosomes into distinct cytological bands; this was an important step in mapping the precise location of heterochromatic genes because before this time heterochromatic genes could be mapped only relative to one another. Here we focus on further refining the previous mapping work on essential genes in the proximal heterochromatin of the right arm of the second chromosome (2Rh) in cytological region h41–h46 of D. melanogaster (Hilliker 1976; Hilliker et al. 1980; Coulthard et al. 2003; Myster et al. 2004).Early mapping studies in D. melanogaster putatively placed the light (lt) and rolled (rl) genes in, or near, chromosome 2 heterochromatin (Schultz 1936; Hannah 1951; Hessler 1958). The first large-scale mutagenesis specifically directed at finding vital loci in second chromosome heterochromatin was conducted by Hilliker (1976). Using heterochromatic deletions created by Hilliker and Holm (1975), Hilliker (1976) set out to map vital loci using the mutagen ethyl methanesulfonate (EMS). He identified seven individual lethal complementation groups in 2Rh that were interpreted as representing seven vital loci. One of these heterochromatic loci was identified as the previously described rl gene. Two of the remaining vital loci have since been identified: Nipped-A is synonymous with the l(2) 41Ah complementation group (Rollins et al. 1999) and RpL38 is synonymous with Minute(2)41A and Hilliker''s (1976) l(2)41Af complementation group (Marygold et al. 2005; also referred to as l(2)Ag in FlyBase). In addition, Rollins et al. (1999) found the Nipped-B gene to be located in 2Rh, but how this locus fit into the data from Hilliker (1976) was unclear.With the limited release of some of the more distal heterochromatic sequences (Hoskins et al. 2002), a more recent mutagenesis screen focusing on distal 2Rh was conducted by Myster et al. (2004). In the region defined by the overlap between Df(2R)41A8 and Df(2R)41A10 (the latter was previously shown to be deficient for most of 2Rh; Hilliker and Holm 1975), Myster et al. (2004) reported the existence of 15 vital loci, considerably more than the 4 essential loci predicted by Hilliker (1976). The discrepancy between these two studies was the catalyst for this current work. Each group used the same mutagen, EMS, yet each group came up with very different interpretations of the number of vital loci.Hilliker''s interpretation relied on earlier evidence that EMS preferentially produced point mutations and not large-scale aberrations (Lim and Snyder 1974). Assuming that the mutants isolated in his study were point mutations, or small aberrations limited to one locus, Hilliker found that some of the loci that he identified exhibited complex interallelic complementation; the most complex complementation pattern was observed with locus l(2)41Ae. On the other hand, the interpretation of Myster et al. (2004) was that heterochromatin was more sensitive to EMS and that EMS could produce large heterochromatic deletions; they proposed that the complex interallelic complementation in l(2)41Ae was due to the presence of deletions and that l(2)41Ae represented a region of 2Rh containing many genes, rather than being a single locus.To resolve these different interpretations of the genomic segment containing l(2)41Ae (i.e., is it a single locus or a region of 2Rh), we set out to map l(2)41Ae and the region surrounding the presumed location of l(2)41Ae (as in Myster et al. 2004) by performing a large-scale inter se complementation analysis between all available mutant lines that were previously mapped to l(2)41Ae (including Nipped-B). In addition, we undertook a molecular mapping and sequence analysis, using a candidate gene approach with the most recent annotation of 2Rh (Hoskins et al. 2007), to characterize the region and identify as many essential genes as possible. We also used these approaches to map l(2)41Ab and unextended [two of the more proximal complementation groups identified by Hilliker (1976)]. Finally, we also utilized available RNA interference (RNAi) lines to knock down the expression of 12 genes in 2Rh in an attempt to identify essential genes.     

DNA of Drosophila melanogaster contains 5-methylcytosine

It is commonly accepted that the DNA of Drosophila melanogaster does not contain 5-methylcytosine, which is essential in the development of most eukaryotes. We have developed a new, highly specific and sensitive assay to detect the presence of 5-methylcytosine in genomic DNA. The DNA is degraded to nucleosides, 5-methylcytosine purified by HPLC and, for detection by 1D- and 2D-TLC, radiolabeled using deoxynucleoside kinase and [gamma-(32)P]ATP. Using this assay, we show here that 5-methylcytosine occurs in the DNA of D. melanogaster at a level of approximately 1 in 1000-2000 cytosine residues in adult flies. DNA methylation is detectable in all stages of D.melanogaster development.