Autonomous replication in Drosophila melanogaster tissue culture cells

This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and tested them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA fragments were able to replicate autonomously, as were plasmids containing random human and Escherichia coli genomic DNA fragments. Most of the plasmids were detectable 18 days after transfection in the absence of selection, suggesting that transfected DNA is maintained in Drosophila cells without rapid loss or degradation. The finding that all plasmids containing Drosophila, human, or bacterial DNA replicate autonomously in Drosophila cells suggests that the signals that direct autonomous replication in Drosophila contain a low degree of sequence specificity. A two-dimensional gel analysis of initiation on one of the plasmids was consistent with many dispersed initiation sites. Low sequence specificity and dispersed initiation sites also characterize autonomous replication in human cells and Senopus eggs and may be general properties of autonomous replication in animal cells.     

Telomeres and P-element of Drosophila melanogaster contain sequences that replicate autonomously in Saccharomyces cerevisiae

Summary We have isolated restriction fragments from a shotgun collection of Drosophila DNA which function as autonomously replicating sequences (ARS) in Saccharomyces cerevisiae and hybridize with telomeric regions of the 2L, 2R, 4, and X chromosomes. In an independently obtained set of D. melanogaster clones five fragments hybridize in situ with telomeres and a number of internal sites. Two of them also contain ARSs. A Drosophila mobile P-element also possesses ARS activity in yeast.     

Localisation of reiterated nucleotide sequences in Drosophila and mouse by in situ hybridisation of complementary RNA

The location of highly reiterated nucleotide sequences on the chromosomes has been studied by the technique of in situ hybridisation between the DNA of either Drosophila melanogaster salivary gland chromosomes or mouse chromosomes and tritium labelled complementary RNA (c-RNA) transcribed in vitro from appropriate templates with the aid of DNA dependent RNA polymerase extracted from Micrococcus lysodeikticus. The location of the hybrid material was identified by autoradiography after RNase treatment. — When Drosophila c-RNA, transcribed from whole DNA, was annealed with homologous salivary chromosomes in the presence of formamide the well defined labelling was confined to the chromocentre. With heat instead of formamide denaturation there was evidence of discontinuous labelling in various chromosome regions as well, apparently associated with banding. Xenopus ribosomal RNA showed no evidence of annealing to Drosophila chromosomes with the comparatively short exposure times used here. — When mouse satellite DNA was used as template the resulting c-RNA showed no hybridisation to Drosophila chromosomes but, when annealed with mouse chromosomes, the centromeric regions were intensely labelled. The interphase nuclei showed several distinct regions of high activity which suggested aggregation of centromeric regions of both homologous and non-homologous chromosomes. The results of annealing either c-RNA or labelled satellite DNA to homologous chromosomes were virtually indistinguishable. Incubation of Drosophila c-RNA with mouse chromosomes provided no evidence of localisation of grains. — It is inferred that both in mouse and Drosophila the centromeric regions of all chromosomes are enriched in highly reiterated sequences. This may be a general phenomenon and it might be tentatively suggested that the highly reiterated sequences play some role in promoting the close physical approximation of homologous and non-homologous chromosomes or chromosome regions to facilitate regulation of function.     

Structural constraints in expansion segments from a midge 26S rDNA

DNA sequences representing approximately 40% of the large-subunit rRNA gene from the lower dipteran Chironomus thummi were analyzed. Once aligned with their Drosophila counterparts, sequence and base content comparisons were carried out. Sequence identity was found to be high overall, except for six regions that displayed a local bias in nucleotide composition toward AT. These regions were identified as expansion segments D3, D4, D5, D6, D7a, and D12. Besides base sequence divergence, differences in length were observed between the respective variable domains of the two species, particularly for D7a. Prediction of secondary structure showed that the folding of the Chironomus expansion segments analyzed is in agreement with the general patterns proposed for eukaryotic LSU rRNA. The comparison with Drosophila revealed also that the Chironomus secondary structures of the variable domains are supported by multiple compensatory substitutions or even compensatory insertions. Chironomus D7a displayed an unusual structural feature with respect to the insect D7a models that have been inferred up to now. The structural constraint observed in the expansion segments of Diptera so distantly related as midges and Drosophila suggests that these regions contribute to some functional role. Concerning the D7a of insects so far analyzed, there can be, in addition to a conserved secondary structure, a nucleotide composition constraint that might be important for the process giving rise to the alpha and beta halves of the 26S rRNA. Correspondence to: E. Gorab     

In vivo topoisomerase II cleavage of the Drosophila histone and satellite III repeats: DNA sequence and structural characteristics.

We have identified two classes of in vivo topoisomerase II cleavage sites in the Drosophila histone gene repeat. One class co-localizes with DNase I-hypersensitive regions and another novel class maps to a subset of consecutive nucleosome linker sites in the scaffold-associated region (SAR) of the histone gene loop. Prominent topoisomerase II cleavage is also observed in one of the linker regions of the two nucleosomes spanning satellite III, a centromeric SAR-like DNA sequence with a repeat length of 359 bp. At the sequence level, in vivo topoisomerase II cleavage is highly site specific. Comparison of 10 nucleosome linker sites defines an in vivo cleavage sequence whose major characteristic is a prominent GC-rich core. These GC-rich cleavage sites are flanked by extensive arrays of oligo(dA).oligo(dT) tracts characteristic of SAR sequences. Treatment of cells with distamycin selectively enhances cleavage at nucleosome linker sites of the SAR and satellite regions, suggesting that AT-rich sequences flanking cleavage sites may be involved in determining topoisomerase II activity in the cell. These observations provide evidence for the association of topoisomerase II with SARS in vivo.     

The genomic organization of HeT-A retroposons inDrosophila melanogaster

Members of theDrosophila HeT-A family of transposable elements are LINE-like retroposons that are found at telomeres and in centric heterochromatin. We recently characterized an active HeT-A element that had transposed to a broken chromosome end fewer than mine generations before it was isolated. The sequence arerangement of this element, called 9D4, most likely represents the organization of an actively transposing member of the HeT-A family. Here we assess the degree of divergence among members of the HeT-A family and test a model of telomere length maintenance based on HeT-A transposition. The region containing the single open reading frame of this element appears to be more highly conserved than the non-coding regions. The HeT-A element has been implicated in theDrosophila telomere elongation process, because frequent transpositions to chromosome ends are sufficient to counter-balance nucleotide loss due to incomplete DNA replication. The proposed elongation model and the hypothetical mechanism of HeT-A transposition predict a predominant orientation of HeT-A elements with their oligo (A) tails facing proximally at chromosome ends, as well as the existence of irregular tandem arrays of HeT-A elements at chromosome ends resulting from transposition of new HeT-A elements onto chromosome ends with existing elements. Twenty-nine different HeT-A fragments were isolated from directional libraries that were enriched in terminal DNA fragments. Sequence analyses of these fragments and comparisons with the organization of the HeT-A element, 9D4, fit these two predictions and support the model ofDrosophila telomere elongation by transposition of HeT-A elements.     

DNA from Drosophila melanogaster Interband 61C7/C8 Evolves at a High Rate

The results of a comparative study of cloned DNA fragments ofDrosophila simulans, D. mauritiana, D. teissieri, and D. erecta are presented. The fragments were amplified in PCR with primers specified to the region of D. melanogaster interband 61C7/C8. The uniqueness of all cloned fragments in the genomes of these species was confirmed. A comparative analysis of nucleotide sequences revealed that the rate of evolution of DNA from D. melanogaster interband 61C7/C8 is close to the rate of neutral evolution in the genusDrosophila.     

Developmental puffing patterns in salivary gland chromosomes of Rhynchosciara hollaenderi

An analysis of puff formation and regression has been carried out in 3 morphologically distinct regions of the Rhynchosciara hollaenderi salivary gland during mid-larval through pupal development. Puffing differences among these 3 regions have been found and analysed for both RNA and DNA puffs. The presence of such differences suggests that the gland regions may also be functionally differentiated. — Developmentally specific sequences of puffs have been distinguished and correlated with morphological and physiological events which occur during the development of Rhynchosciara. The DNA puffs as well as the RNA puffs enlarge and regress at predictably specific developmental stages. The presence of particular puffing sequences in the late larval to pupal period has been compared with the occurrence of known changes in the developmental ecdysone titre for Rhynchosciara. Certain aspects of this developmental picture appear to fit the ecdysone-stimulated puffing model for Drosophila, but other aspects indicate that the Drosophila-based model may not be completely applicable to Rhynchosciara.     

DNA organization and polymorphism of a wild-type Drosophila telomere region

Telomeres at the ends of linear chromosomes of eukaryotes protect the chromosome termini from degradation and fusion. While telomeric replication/elongation mechanisms have been studied extensively, the functions of subterminal sequences are less well understood. In general, subterminal regions can be quite polymorphic, varying in size from organism to organism, and differing among chromosomes within an organism. The subterminal regions of Drosophila melanogaster are not well characterized today, and it is not known which and how many different components they contain. Here we present the molecular characterization of DNA components and their organization in the subterminal region of the left arm of chromosome 2 of the Oregon RC wildtype strain of D. melanogaster, including a minisatellite with a 457 bp repeat length. Two distinct polymorphic arrangements at 2L were found and analyzed, supporting the Drosophila telomere elongation model by retrotransposition. The high incidence of terminal chromosome deficiencies occurring in natural Drosophila populations is discussed in view of the telomere structure at 2L.     

Replication analysis of plasmid DNAs injected into Drosophila embryos

From a shotgun collection of DNA fragments, isolated from Drosophila melanogaster, we selected sequences that function as autonomously replicating sequences (ARS) in the yeast Saccharomyces cerevisiae. To investigate the replicative potential of such sequences in Drosophila, five of these ARS elements and also the Adh gene of D. melanogaster, which has been described earlier to have ARS function in yeast, were microinjected into developing Drosophila eggs and analysed after reisolation from first instar larvae. As an assay for DNA replication, we determined the sensitivity of recovered plasmid DNA to restriction enzymes that discriminate between adenine methylation and nonmethylation. within the limits of detection our results show that none of the plasmids replicated two or more rounds. However, a fraction of all injected plasmid DNAs, including vector DNA, seems to replicate once. The same result was obtained for a DNA sequence from mouse that had been reported to have replication origin function in mouse tissue culture cells. We excluded the possibility that methylation of the plasmids is the reason for their inability to replicate. These results demonstrate that homologous and heterologous DNA sequences that drive replication of plasmids in cells of other species are not sufficient to fulfil this function in Drosophila embryos.by J.A. Huberman     

Drosophila genome project: One-hit coverage in yeast artificial chromosomes

We present a strategy for assembling a physical map of the genome of Drosophila melanogaster based on yeast artificial chromosomes (YACs). In this paper we report 500 YACs containing inserts of Drosophila DNA averaging 200 kb that have been assigned positions on the physical map by means of in situ hybridization with salivary gland chromosomes. The cloned DNA fragments have randomly sheared ends (DY clones) or ends generated by partial digestion with either NotI (N clones) or EcoRI (E clones). Relative to the euchromatic portion of the genome, the size distribution and genomic positions of the clones reveal no significant bias in the completeness or randomness of genome coverage. The 500 mapped euchromatic clones contain an aggregate of approximately 100 million base pairs of DNA, which is approximately one genome equivalent of Drosophila euchromatin.by W. Hennig     

Non-reciprocal chromosomal bridge-induced translocation (BIT) by targeted DNA integration in yeast

Several experimental in vivo systems exist that generate reciprocal translocations between engineered chromosomal loci of yeast or Drosophila, but not without previous genome modifications. Here we report the successful induction of chromosome translocations in unmodified yeast cells via targeted DNA integration of the KAN^R selectable marker flanked by sequences homologous to two chromosomal loci randomly chosen on the genome. Using this bridge-induced translocation system, 2% of the integrants showed targeted translocations between chromosomes V-VIII and VIII-XV in two wild-type Saccharomyces cerevisiae strains. All the translocation events studied were found to be non-reciprocal and the fate of their chromosomal fragments that were not included in the translocated chromosome was followed. The recovery of discrete-sized fragments suggested multiple pathway repair of their free DNA ends. We propose that centromere-distal chromosome fragments may be processed by a break-induced replication mechanism ensuing in partial trisomy. The experimental feasibility of inducing chromosomal translocations between any two desired genetic loci in a eukaryotic model system will be instrumental in elucidating the molecular mechanism underlying genome rearrangements generated by DNA integration and the gross chromosomal rearrangements characteristic of many types of cancer.Electronic Supplementary Material Supplementary material is available for this article at     

Cloning and characterization of a tomato GTPase-like gene related to yeast and Arabidopsis genes involved in vesicular transport

The reduced translation product of a tomato cDNA derived from a gene expressed in a number of tomato tissues of different developmental stages contained sequence motifs characteristic of the GTPase superfamily of proteins. The sequence was closely related to the Sar1 protein of Saccharomyces cerevisiae, a protein essential for the formation of protein transport vesicles at the endoplasmic reticulum (ER) (A. Nakano and M. Muramatsu, Cell Biol 109 (1989): 2677–2691). From analysis of the GTPase superfamily gene sequences, including the tomato SAR-like gene, it is proposed that the SAR genes comprise a distinct GTPase subfamily, presumably with a common, essential function in vesicular transport.     

Return of the H-word (heterochromatin)

Recent advances in studies of yeast, Drosophila and humans have renewed interest in heterochromatin. These recent studies have demonstrated the interspersion and rapid spread of transposable elements into Drosophila heterochromatin; documented the requirement of heterochromatic genes for heterochromatin; identified heterochromatin-like regions in yeast chromosomes; confirmed an important role for satellite DNA in human centromere function; and suggested potential functions for heterochromatin-associated proteins.     

Construction of pFRT,a Convenient Drosophila Transformation Vector with Functional FRT Sites

P-element transformation vector pCaSpeR3 was modified to obtain pFRT. The new vector contains two tandem FRT sites flanked by several unique restriction sites and separated by a polylinker harboring five convenient restriction sites, and allows easy cloning of DNA fragments between or close to the FRT sites. FLP-mediated excision of DNA sequences cloned between the FRT sites was demonstrated in vivo. The vector was proposed for molecular genetic studies of the position effect variegation, structural and molecular organization of Drosophila polytene chromosomes, etc.     

Telomeric sequences derived from laser-microdissected polytene chromosomes

Telomeric fragments from salivary gland squashes of Drosophila melanogaster Oregon R. were produced by a new microdissection technique, UV laser microbeam dissection. Microdissection, an essential step in microcloning procedures, is usually performed using micromanipulators and microneedles. Recently it has been shown that microdissection can be improved to very high precision if a laser coupled into a microscope is used. A laser microbeam, generated by an excimer pumped dye laser, allows chromosomes to be cut into slices of less than 0.5 m. Here it is shown, that single copy DNA probes prepared from Drosophila chromosomes by laser microdissection and microcloning relocalize to the chromosomal regions from which they are derived. The combination of laser technique and microcloning provides an advantageous approach for rapid genetic analysis with potential for the study of genetic diseases and genome mapping.