Fragments from section 3 of the salivary gland X chromosome of D. melanogaster were dissected with a micromanipulator. The DNA was extracted, cut and ligated to a lambda vector in a volume of a few nanoliters in an oil chamber monitored through a microscope. From about 10 pg of DNA we obtained 80 recombinant clones, a sample of which were analysed and shown to contain Drosophila DNA which hybridises in situ to the region of section 3 of the X chromosome. With this technique we can isolate clones from any desired region as small as 200 kb from the euchromatic arms of polytene chromosomes.

A cloned 340‐bp DNA fragment excised by EcoRI from the Chironomus pallividittatus genome has been localized to the telomeres by in situ hybridization as well as to connectives between telomeres. No hybridization was observed in other regions of the chromosomes. Another cloned EcoRI fragment, 525 bp long has also been studied. This represents a partial duplication of the 340‐bp sequence. Genomic blot hybridization experiments show that the 340‐bp sequence is a representative monomeric unit of tandemly repeated arrays which account for 1.2% of the Chironomus genome, on average 300 kb per telomere. The repeat unit contains two types of subrepeats each present twice per repeat unit. Northern blot hybridization experiments show that the telomere‐associated sequences are transcribed into a discrete RNA species approximately 20 kb in size. The evolution of this telomere‐associated DNA is discussed.

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We provide evidence that a chromosome end in the dipteran Chironomus pallidivittatus contains 340-bp tandem repeats reaching the extreme terminus of the chromosome. After adding synthetic oligonucleotide tails to DNA extracted from the microdissected right end of the fourth chromosome, we could demonstrate that the blocks of repeats were tailed at only one end, the chromosome terminus, the interior of the arrays being unavailable for tailing. Using PCR, we furthermore showed that the added tails were connected to 340-bp repeat DNA directly, i.e., without intervening DNA of any other kind. The tailed repeats belong to a subfamily previously known to be the most peripheral one of the different types of 340-bp units. Using plasmid controls, we could also make certain that we did not amplify rare or nonrepresentative DNA termini.Telomeres of most investigated eukaryotic species terminate with short repeats, usually with guanine excess in the 3Ј-terminating strand (4). Such repeats, whether regular, as in most cases (5,17,21,25), or irregular, as in Saccharomyces cerevisiae (29) among others, are likely to result from telomerase activities (6,11,16,31).Short repeats are also found in several insect orders (23), but for the dipteran Drosophila melanogaster a different mode of chromosome termination has been established. Here terminally deleted chromosomes can be capped by non-long-terminal-repeat retrotransposons (2,3,30,33), which can also be recovered at the ends of normal chromosomes (15).No other type of ending is known for nuclear chromosomes, but the linear mitochondrial DNA from Tetrahymena thermophila ends with Ͼ50-bp-long repeat units (19,20). We present evidence that this alternative, here in the form of 340-bp tandem repeats (27), is used by nuclear chromosomes in the dipteran Chironomus pallidivittatus.The 340-bp tandem repeat family is localized at seven of the eight (pairs of) chromosome ends in Ͻ200-kb blocks. Although the blocks are close to the chromosome ends as revealed by Bal 31 digestions (34), it has not been known whether they reach the extreme termini. The repeat family contains four different subfamilies, three of which, D1, D2, and D3, are derived from the fourth one, M1, each by a cluster of mutations in short regions designated ds1, ds2, and ds3, respectively (7). In the case of D3 there is a second difference, i.e., one large region has become substituted for another one. Telomeres differ in subfamily composition (8,34). When D3 is present together with other kinds of repeats, it is always the most peripheral unit (34).Briefly, our strategy was to gently release DNA from single microdissected right ends of the fourth chromosome and provide it with tails. Anchors added to the tails and primers for the 340-bp repeat were used for PCR amplifications. We could demonstrate that no internal breaks were tailed within the repeat blocks and that the tails were added directly to the 340-bp repeat DNA. We also showed that only DNA from a specific subfamily, previously shown to be distal (34), was amp...

Tandem repeats, 340 bp long, have been shown to terminate the chromosomes in Chironomus pallidivittatus and similar DNA may be used for this purpose by related insects. In view of the importance of Chironomus in telomere studies, representing in principle a third system after short repeats and Drosophila telomeric retrotransposons, we have investigated the related Chironomus dilutus, to learn what DNA structures are conserved at the chromosome ends. Interspersed subrepeats in the telomeric repeats, which contain a long palindrome, and a zone of about 100 bp of relatively constant subtelomeric DNA towards the junction to the telomeric DNA, are characteristic for C. dilutus as for previously investigated species. C. dilutus has similar subtelomeric DNA at all chromosome ends, but typical telomeric repeats in only seven of the pairs since the eighth telocentric pair contains centromere-specific repeats.