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Telomeric regions in Drosophila are composed of three subdomains. A chromosome cap distinguishes the chromosome end from a DNA double-strand break; an array of retrotransposons, HeT-A, TART, and TAHRE (HTT), maintains telomere length by targeted transposition to chromosome ends; and telomere-associated sequence (TAS), which consists of a mosaic of complex repeated sequences, has been identified as a source of gene silencing. Heterochromatin protein 1 (HP1) and HP1-ORC-associated protein (HOAP) are major protein components of the telomere cap in Drosophila and are required for telomere stability. Besides the chromosome cap, HP1 is also localized along the HTT array and in TAS. Mutants for Su(var)205, the gene encoding HP1, have decreased the HP1 level in the HTT array and increased transcription of individual HeT-A elements. This suggests that HP1 levels directly affect HeT-A activity along the HTT array, although they have little or no effect on transcription of a white reporter gene in the HTT. Chromatin immunoprecipitation to identify other heterochromatic proteins indicates that TAS and the HTT array may be distinct from either heterochromatin or euchromatin.

Telomeres are generally considered heterochromatic. On the basis of DNA composition, the telomeric region of Drosophila melanogaster contains two distinct subdomains: a subtelomeric region of repetitive DNA, termed TAS, and a terminal array of retrotransposons, which perform the elongation function instead of telomerase. We have identified several P-element insertions into this retrotransposon array and compared expression levels of transgenes with similar integrations into TAS and euchromatic regions. In contrast to insertions in TAS, which are silenced, reporter genes in the terminal HeT-A, TAHRE, or TART retroelements did not exhibit repressed expression in comparison with the same transgene construct in euchromatin. These data, in combination with cytological studies, provide evidence that the subtelomeric TAS region exhibits features resembling heterochromatin, while the terminal retrotransposon array exhibits euchromatic characteristics.

Heterochromatin is a specialized chromatin structure in chromosomal regions associated with repeated DNA sequences and low concentrations of genes. Formation of heterochromatin is determined in large part by enzymes that modify histones and structural proteins that bind to these modified histones in a cooperative fashion. In Drosophila, mutations in genes that encode heterochromatic proteins are often dominant and increase expression of genes placed into heterochromatic positions. To find components of telomeric heterochromatin in Drosophila, we screened a collection of autosomal deficiencies for dominant suppressors of silencing of a transgene at the telomere of chromosome 2L. While many deficiency chromosomes are associated with dominant suppressors, in the cases tested on chromosome 2 the suppressor mapped to the 2L telomere, rather than the deficiency. We infer that background effects may hamper the search for genes that play a role in telomeric heterochromatin formation and that either very few genes participate in this pathway or mutations in these genes are not dominant suppressors of telomeric position effect. The data also suggest that the 2L telomere region plays a major role in telomeric silencing.

One model of telomeric position effect (TPE) in Drosophila melanogaster proposes that reporter genes in the vicinity of telomeres are repressed by subterminal telomere-associated sequences (TAS) and that variegation of these genes is the result of competition between the repressive effects of TAS and the stimulating effects of promoters in the terminal HeT-A transposon array. The data presented here support this model, but also suggest that TPE is more complex. Activity of a telomeric white reporter gene increases in response to deletion of some or all of the TAS on the homolog. Only transgenes next to fairly long HeT-A arrays respond to this trans-interaction. HeT-A arrays of 6-18 kb respond by increasing the number of dark spots on the eye, while longer arrays increase the background eye color or increase the number of spots sufficiently to cause them to merge. Thus, expression of a subtelomeric reporter gene is influenced by the telomere structure in cis and trans. We propose that the forces involved in telomere length regulation in Drosophila are the underlying forces that manifest themselves as TPE. In the wild-type telomere TAS may play an important role in controlling telomere elongation by repressing HeT-A promoter activity. Modulation of this repression by the homolog may thus regulate telomere elongation.

In most eukaryotes telomeres are extended by telomerase. Drosophila melanogaster, however, lacks telomerase, and telomere-specific non-LTR retrotransposons, HeT-A and TART, transpose specifically to chromosome ends. A Drosophila strain, Gaiano, that has long telomeres has been identified. We extracted the major Gaiano chromosomes into an Oregon-R genetic background and examined the resulting stocks after 60 generations. In situ hybridization using HeT-A and TART sequences showed that, in stocks carrying either the X or the second chromosome from Gaiano, only the Gaiano-derived chromosomes display long telomeres. However, in stocks carrying the Gaiano third chromosome, all telomeres are substantially elongated, indicating that the Gaiano chromosome 3 carries a factor that increases HeT-A and TART addition to the telomeres. We show that this factor, termed Telomere elongation (Tel), is dominant and localizes as a single unit to 69 on the genetic map. The long telomeres tend to associate with each other in both polytene and mitotic cells. These associations depend on telomere length rather than the presence of Tel. Associations between metaphase chromosomes are resolved during anaphase, suggesting that they are mediated by either proteinaceous links or DNA hydrogen bonding, rather than covalent DNA-DNA bonds.

A mutator, [mu2.sup.a], in Drosophila melanogaster potentiates terminal deficiencies. In the female germ line the y mutant frequency induced by irradiation of mature oocytes with 5 Gy increases approximately twofold in heterozygotes and 20-fold in homozygotes compared with wild type. The recovery of terminal deficiencies is not limited to breaks close to chromosome ends; high frequencies of deficiencies can be recovered with breakpoints located in centric heterochromatin or near the middle of a chromosome arm. Lesions induced by [Gamma]-rays are repaired slowly in [mu2.sup.a] oocytes, but become 'fixed' as terminal deficiencies upon fertilization. A few lesions induced in wild-type females also produce terminal deficiencies. Mutator males do not exhibit an increase in terminal deletions, regardless of the germ cell stage irradiated. In addition, there is no increase in the mutant frequency when mature sperm are irradiated and fertilize eggs produced by [mu2.sup.a] females. The data are consistent with the hypothesis that lesions induced in sperm chromosomes are repaired after fertilization, while lesions induced in oocyte chromosomes are shunted instead to a mechanism that stabilizes broken chromosome ends. We propose that mu2 affects chromosomal structure during oogenesis, thereby modulating DNA repair.

Genetically marked P elements inserted into the subtelomeric satellites of Drosophila show repression and variegation of the reporter gene. One such [white.sup.+] reporter, inserted between the subtelomeric satellite and the terminal HeT-A array in the left arm of chromosome 2 (2L), is sensitive to its context; changes in the structure of the telomere region can be identified by changes in eye color. Addition of HeT-A or TART elements to the 2L terminus increases [w.sup.+] expression, and loss of sequence from the end decreases expression. This indicates that the telomeric retrotransposons in Drosophila have an activating influence on the repressed subterminal reporter gene. Changes in eye color due to altered expression of the transgene also allow the detection of interactions between homologous telomeres. The 2L arms that terminate in long HeT-A/TART arrays showed increased expression of the subterminal [w.sup.+] transgene when the terminal repeats on the homologue are absent or markedly shorter. We propose that the chromatin structure of the terminal HeT-A/TART array and the activity of a putative promoter/enhancer element on HeT-A are affected by telomeric interactions. Such trans-activation may reflect control over HeT-A transcription and, thus, transposition activity.

We present here the molecular cloning and characterization of the mutator2 (mu2) gene of Drosophila melanogaster together with further genetic analyses of its mutant phenotype, mu2 functions in oogenesis during meiotic recombination, during repair of radiation damage in mature oocytes, and in proliferating somatic cells, where mu2 mutations cause an increase in somatic recombination. Our data show that mu2 represents a novel component in the processing of double strand breaks (DSBs) in female meiosis, mu2 does not code for a DNA repair enzyme because mu2 mutants are not hypersensitive to DSB-inducing agents. We have mapped and cloned the mu2gene and rescued the mu2phenotype by germ-line transformation with genomic DNA fragments containing the mu2 gene. Sequencing its cDNA demonstrates that mu2 encodes a novel 139-kD protein, which is highly basic in the carboxy half and carries three nuclear localization signals and a helix-loop-helix domain. Consistent with the sex-specific mutant phenotype, the gene is expressed in ovaries but not in testes. During oogenesis its RNA is rapidly transported from the nurse cells into the oocyte where it accumulates specifically at the anterior margin. Expression is also prominent in diploid proliferating cells of larval somatic tissues. Our genetic and molecular data are consistent with the model that mu2 encodes a structural component of the oocyte nucleus. The MU2 protein may be involved in controlling chromatin structure and thus may influence the processing of DNA DSBs.