Control of telomere elongation and telomeric silencing in Drosophila melanogaster.

Chromosome length in Drosophila is maintained by the targeted transposition of two families of non-LTR retrotransposons, HeT-A and TART. Although the rate of transposition to telomeres is sufficient to counterbalance loss from the chromosome ends due to incomplete DNA replication, transposition as a mechanism for elongating chromosome ends raises the possibility of damaged or deleted telomeres, because of its stochastic nature. Recent evidence suggests that HeT-A transposition is controlled at the levels of transcription and reverse transcription. HeT-A transcription is found primarily in mitotically active cells, and transcription of a w+ reporter gene inserted into the 2L telomere increases when the homologous telomere is partially or completely deleted. The terminal HeT-A array may be important as a positive regulator of this activity in cis, and the subterminal satellite appears to be an important negative regulator in cis. A third chromosome modifier has been identified that increases the level of reverse transcriptase activity on a HeT-A RNA template and greatly increases the transposition of HeT-A. Thus, the host appears to play a role in transposition of these elements. Taken together, these results suggest that control of HeT-A transposition is more complex than previously thought.

Directional gene silencing induced by a complex subtelomeric satellite from Drosophila.

The telomeric regions in Drosophila cause transcriptional silencing of integrated transgenes. A complex satellite has recently been identified in the subterminal region of the left arm of chromosome 2 that is a good candidate for the source of the observed telomeric silencing, because genetically marked transposable elements that have inserted into this subtelomeric array show repression and variegation of the reporter gene. We asked whether this satellite can also cause transcriptional repression in ectopic chromosomal positions by placing it upstream of a mini-white reporter gene in P element constructs used for germ line transformation. The transgenes are shielded from external influences at the integration site using SU(HW) binding sites at either end. It was found that the satellite represses transcription of the reporter gene in an orientation dependent and an array length dependent manner. The satellite does not, however, induce variegation under the conditions used. The repressed transgenes do not respond to typical modifiers of centromeric position effect variegation, such as Su(var)2055, Su(var)2-11, Su(var)3-11, and Su(var)3-61, or to the addition of a Y chromosome. However, as with the original variegating telomeric insertion, suppression in the transgenes is relieved by Su(z)25, suggesting that suppression induced by the subtelomeric satellite retains aspects of telomeric silencing in ectopic positions.

Telomere functions. A review.

Telomeres are structurally and functionally complex. They consist of an array of simple DNA repeats at the extreme end of the chromosome with a more complex array of repeats adjacent to it. A large number of proteins have been identified that bind to the telomeric DNA repeats or to the protein complexes that are built at the chromosome end. Telomeres tend to form associations with each other. These associations have been implicated in the formation of nuclear domains that may be important for transcriptional regulation, for sister chromatid pairing at mitosis, and for homologous meiotic synapsis. Telomeric chromosome ends do not cause delays in cell cycle progression, nor are they subject to DNA repair as are broken chromosome ends. Telomeres also provide a separate mechanism for adding additional copies of the telomeric DNA to chromosome ends. This is needed to counterbalance the loss of DNA sequences from chromosome ends due to incomplete DNA replication. The components that participate in the latter mechanism and this process have been characterized in detail; the other functions of telomeres are less well understood but are the subjects of active investigation.

The Y chromosome of Drosophila melanogaster contains a distinctive subclass of Het-A-related repeats.

The HeT-A element is a transposable element with an apparent role in the structure of the telomeres of Drosophila melanogaster chromosomes. HeT-A transposition is the earliest event detected in healing of broken ends; HeT-A is also found on telomeres of unbroken chromosomes. Sequences with homology to HeT-A are never detected in euchromatic regions; however, clusters of HeT-A-related sequences occur in nontelomeric regions of the heterochromatic Y chromosome. Analysis of two of these Y-associated clusters shows them to be significantly different in structure from telomeric HeT-A elements, although the regions of shared sequence have > 80% sequence identity in all cases. Telomeric HeT-A elements occur in chains, with the elements in the same orientation but variably truncated at their external ends and irregularly interspersed with unrelated sequences. In contrast, the nontelomeric Y elements are regular tandem repeats of parts of the HeT-A sequence joined to unrelated sequences which are not the same in the two clusters studied. The sequence structures suggest that the nontelomeric clusters on the Y chromosome do not arise by the same transposition mechanism that forms the telomeric clusters; instead the clusters on the Y may arise by a mechanism that is used more generally in the evolution of Y chromosomes. Although the telomeric and nontelomeric clusters appear to be formed differently, both are enriched in parts of the HeT-A sequence which may be important in the structure of heterochromatin.

A repetitive DNA element, associated with telomeric sequences in Drosophila melanogaster, contains open reading frames.

He-T sequences are a complex repetitive family of DNA sequences in Drosophila that are associated with telomeric regions, pericentromeric heterochromatin, and the Y chromosome. A component of the He-T family containing open reading frames (ORFs) is described. These ORF-containing elements within the He-T family are designated T-elements, since hybridization in situ with the polytene salivary gland chromosomes results in detectable signal exclusively at the chromosome tips. One T-element that has been sequenced includes ORFs of 1,428 and 1,614 bp. The ORFs are overlapping but one nucleotide out of frame with respect to each other. The longer ORF contains cysteine-histidine motifs strongly resembling nucleic acid binding domains of gag-like proteins, and the overall organization of the T-element ORFs is reminiscent of LINE elements. The T-elements are transcribed and appear to be conserved in Drosophila species related to D. melanogaster. The results suggest that T-elements may play a role in the structure and/or function of telomeres.

He-T family DNA sequences in the Y chromosome of Drosophila melanogaster share homology with the X-linked stellate genes.

The genome of Drosophila melanogaster contains a class of repetitive DNA sequences called the He-T family, which is unusual in being confined to telomeric and heterochromatic regions. The specific He-T fragment designated Dm665 was cloned in yeast by selection for an autonomously replicating sequence (ARS). Dm665 contains a restriction fragment length polymorphism (RFLP) that is specific to males and thus derives from the Y chromosome. Deletion mapping using X-Y translocations indicates that sequences homologous to Dm665 occur in at least one major cluster in each arm of the Y chromosome. Among 20 yeast artificial chromosome (YAC) clones containing Drosophila sequences homologous with Dm665, four clones derive from defined regions of the long arm of the Y and two from the short arm. The sequence of Dm665 is 2443 bp long, consists of 59% A + T, and contains no significant open reading frames or direct or inverted repeats. However, Dm665 contains a region of 650 bp that shares homology with portions of the X-linked locus Stellate.

[Hoppel-family of mobile elements of Drosophila melanogaster, flanked by short inverted repeats and having preferential localization in the heterochromatin regions of the genome]. / Hoppel-semeistvo mobil'nykh élementov Drosophila melanogaster, flankirovannoe korotkimi invertirovannymi povtorami i imeiushchee aredpochtitel'nuiu lokalizatsiiu v geterokhromatinovykh oblastiakh genoma.

A mobile element (ME) having 91% homology with Dm1360 (Kholodilov et al., 1987) has been cloned from the Drosophila melanogaster genome and sequenced. The family of ME was designated hoppel. The members of this family are flanked by short inverted repeats likewise P, hobo and HB. The hoppel is hybridized with 10-30 euchromatic sites of polytene chromosomes of different Drosophila stocks. Abundant hybridization with heterochromatic regions of chromosomes-chromocenter, pericentric heterochromatin, the 4 chromosome and telomeres was observed in all stocks of D. melanogaster examined and in D. simulans. At least six genomic variants of ME differing in length of the central part were revealed. Hoppel possesses ARS activity similar to the P element. Two ME hoppel were shown to be arranged as a direct repeat in the recombinant phage.

[A family of repeated sequences in Drosophila genome with telomeric localization: properties and polymorphism]. / Semeistvo povtoriaiushchikhsia posledovatel'nostei genoma drozofily, imeiushchee telomernuiu lokalizatsiiu: svoistva i polimorfizm.

The previously cloned Drosophila genome fragment Dm665 (2.4 kb) hybridizing with telomers on polytene chromosomes is a representative of the family of repeats, a part of which being organized in tandem clusters. The repeats are not transcribed in cell culture, are species-specific and represented in 200-250 copies per haploid genome. In natural and laboratory Drosophila lines polymorphism has been revealed with regard to homology with Dm665 in the telomeres.

[The variability of the telomeric and of a number of internal regions of Drosophila chromosomes according to the intensity of hybridization in situ with labelled probes]. / Variabel'nost' telomernykh i riada vnutrennikh raionov khromosom drozofily po intensivnosti gibridizatsii in situ s mechenymi zondami.

Cloned Drosophila DNA fragments containing telomere-specific sequences have been hybridized in situ. It is found that intensity of their hybridization with telomeres greatly varies in different nuclei of the same salivary gland. This phenomenon is also observed for internal sites where some mobile elements included in several DNA fragments under investigation are located. Within each nucleus different regions are hybridized non-uniformly as well. It is suggested that these phenomena can be explained by varying polytenization in telomeres and some internal chromosomal regions.

[Localization in the P-element of Drosophila melanogaster of sequences ensuring the autonomic replication of plasmids in yeasts]. / Lokalizatsiia v P-élemente Drosophila melanogaster posledovatel'nostei, obespechivaiushchikh avtonomnuiu replikatsiiu plazmid v drozhzhakh.

Two sequences (ARS) capable of maintaining the autonomous replication of plasmids in yeast cells were localized in the right part of Drosophila melanogaster P-element subcloned from the pi 25.1 plasmid. An ARS was found in the DNA region of genome adjacent to P-element. ARS sequences contain imperfect (10 out of 11) consensus typical of yeast ARS and have a complicated domain structure.

[Cloning of the promoter of the RNA polymerase operon located before the structural gene of the beta-subunit]. / Klonirovanie promotora RNK-polimeraznogo operona, raspolozhennogo pered strukturnym genom beta-sub"edinitsy.

An EcoRI-fragment of the rpoBC operon carrying the end of the rplL gene, the intercistron region and the beginning of the rpoB gene cloned on the pBRH4 plasmid makes it tetracyclin-resistant, i. e. possesses the properties of a promoter. There is no appreciable difference in the degree of antibiotic resistance of recombinant plasmids carrying the main PJ promoter and the additional promoter.