Genomic organization of the Drosophila telomere retrotransposable elements

Abstract

The emerging sequence of the heterochromatic portion of the Drosophila melanogaster genome, with the most recent update of euchromatic sequence, gives the first genome-wide view of the chromosomal distribution of the telomeric retrotransposons, HeT-A, TART, and Tahre. As expected, these elements are entirely excluded from euchromatin, although sequence fragments of HeT-A and TART 3 untranslated regions are found in nontelomeric heterochromatin on the Y chromosome. The proximal ends of HeT-A/TART arrays appear to be a transition zone because only here do other transposable elements mix in the array. The sharp distinction between the distribution of telomeric elements and that of other transposable elements suggests that chromatin structure is important in telomere element localization. Measurements reported here show (1) D. melanogaster telomeres are very long, in the size range reported for inbred mouse strains (averaging 46 kb per chromosome end in Drosophila stock 2057). As in organisms with telomerase, their length varies depending on genotype. There is also slight under-replication in polytene nuclei. (2) Surprisingly, the relationship between the number of HeT-A and TART elements is not stochastic but is strongly correlated across stocks, supporting the idea that the two elements are interdependent. Although currently assembled portions of the HeT-A/TART arrays are from the most-proximal part of long arrays, approximately 61% of the total HeT-A sequence in these regions consists of intact, potentially active elements with little evidence of sequence decay, making it likely that the content of the telomere arrays turns over more extensively than has been thought.

Figure 1.

Elements in the HeT-A/TART array in the telomere of 4R. (A) Overview of elements in assembled sequence. Not drawn to scale. End of chromosome is on left. The right end begins with the most distal gene, CAPS, 425 bp from most proximal TART. All HeT-A and TART elements are in the same orientation, and except for those in the transition zone, all truncated elements have lost sequence from the 5 end. (For details on size and sequence content of particular elements, see B.) (B) Elements from the HeT-A/TART array in A. Each bar represents an element in the array drawn to scale with the total number of nucleotides indicated at the 5 end. The most distal element in the assembled array is on top, and the most proximal telomeric element is at the bottom. Because elements are variously truncated from the 5 end, they are aligned from the 3 end (at the right axis). In the array the 3 -most sequence of each element is actually connected to the 5 end of the element beneath it, but elements are shown separately for clarity. Open coding regions are light blue; complete coding regions are marked ORF. 3 ends marked A have an intact 3 end and oligo(A). HeT-A oligo(A) tails in this study range from 3 to 26 bp, averaging 8 bp; TART tails are longer, from 14 to 23 bp with an average of 18. Red “A” indicates elements thought to be “tags” (see text). Nontelomeric elements in the array (purple) are indicated by name on the right axis. Element marked “T” has an intact 3 end but has oligo(T) rather than oligo(A). Asterisk (*) indicates an element missing final GTT and oligo(A). Elements not marked “A” are 3 -truncated and shown displaced from the right axis to show their alignment with complete elements; in some cases there are enough missing 3 nucleotides to be seen as white space between the element and the right axis of the graph.

Figure 2.

An example of Southern blots used to measure genome content of telomere elements. Samples of DNA from the Oregon R and 2057 stocks probed with HeT-A ORF (lanes on left) and probed with TART ORF (lanes on right) were cut with BamHI (lane B), EcoRI (lane E), HindIII (lane H), or XhoI (lane X). Filters were first probed with sequence for rp49 as a loading control, and the relevant region of each filter is shown below the HeT-A/TART blots. Comparison of the restriction fragments shows that the two stocks differ significantly in the number and linear arrangement of different sequence variants of the telomere elements.

Figure 3.

HeT-A and TART ORF equivalents in D. melanogaster. Numbers of full-length ORF equivalents were calculated from measurements of Southern blots (see Fig. 2). Data were corrected for DNA loading by first hybridizing with a probe for a single copy gene (rp49). The relative activity of the two probes (rp49 and ORF) was determined by measurement of a dose curve for each probe on the same filter. Fly DNA was prepared from adult female heads. For each DNA sample, the first four bars show the results of separate experiments, each of which averages several measurements (light gray indicates HeT-A; dark gray, TART). The final two bars (rising stripes, HeT-A; falling stripes, TART) show the results of averaging all data for that DNA. Error bars indicate the standard deviation of these averages (defined as the standard error of the underlying population).

Figure 4.

Estimated number of complete HeT-A and TART elements in D. melanogaster. Two features illustrated by this graph are the under-replication in salivary glands over a data range so wide that the vertical axis has been chosen to be logarithmic, and also the near proportionality over such a wide range in the relationship between the number of HeT-A and TART elements across different stocks and tissues (correlation coefficient, r = 0.88–0.96, P = 0.07–0.0001, depending on tissue type) (see Supplemental material, Section 3.) Less obvious, but statistically significant, is the tracking of elements in heads vs. that same element in salivary glands (r = 0.95–0.99, P = 0.05 to <0.0001). Light gray indicates HeT-A; dark gray, TART.

Figure 5.

Salivary under-replication in female larvae. The ratio of the number of elements in salivary glands to the number in heads is shown for each stock and for an average over all stocks. Error bars indicate the standard deviations in these averages (see Fig. 3 legend). All are under- replicated except, perhaps, TART in 2057. Light bars are HeT-A; dark bars, TART.