Telomere elongation (Tel), a New Mutation in Drosophila melanogaster That Produces Long Telomeres

Corresponding author: James M. Mason, National Institute of Environmental Health Sciences, 111 Alexander Dr., PO Box 12233, Research Triangle Park, NC 27709-2233., masonj{at}niehs.nih.gov (E-mail)

Communicating editor: K. GOLIC

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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.

TO maintain stable telomere length, all eukaryotes must regulate the addition of new telomeric sequences to counterbalance the loss of terminal sequences occurring during DNA replication. In most organisms telomere length depends on proper activity of telomerase and on telomere structure. For example, telomere length in yeast is regulated by Rap1p, which recruits other proteins to telomeric DNA. When yeast telomeres are bound by a high number of Rap1 polypeptides, they assume a conformation that renders them inaccessible to telomerase. When the number of Rap1 proteins decreases below a certain threshold, yeast telomeres become accessible for elongation by telomerase (MARCAND et al. 1997 ). A similar mechanism for regulation of telomere length has been proposed for human cells, in which the switching mechanism is governed by the TRF1 and TRF2 proteins (VAN STEENSEL and DE LANGE 1997 ; SMOGORZEWSKA et al. 2000 ). It has been suggested that a high local concentration of these telomere-binding proteins promotes the looping of telomeric DNA, making it inaccessible to telomerase (GRIFFITH et al. 1999 ; SMOGORZEWSKA et al. 2000 ).

Telomere maintenance in Drosophila melanogaster, however, does not depend on the action of telomerase. Instead, two families of non-LTR retrotransposable elements, HeT-A and TART, maintain telomere length by transposing specifically to chromosome ends (BIESSMANN et al. 1990 ; LEVIS et al. 1993 ; MASON and BIESSMANN 1993 , MASON and BIESSMANN 1995 ). HeT-A elements have been shown to transpose to a single chromosome end with frequencies ranging from 10^-1 to <10^-4 (BIESSMANN et al. 1992 ; KAHN et al. 2000 ; GOLUBOVSKY et al. 2001 ), although nothing is known about the control of transposition. Telomeric arrays in Drosophila may also accumulate transposable elements via gene conversion under some circumstances (KAHN et al. 2000 ). The relative importance of transposition and conversion in telomere length maintenance is not known.

Here, we provide the first evidence for genetic regulation of telomere length in Drosophila. We have characterized a D. melanogaster strain, Gaiano, which has unusually long telomeres. This strain contains a dominant genetic factor, Telomere elongation (Tel), that maps to 69 on chromosome 3 and causes addition of both HeT-A and TART elements to all Drosophila telomeres. The chromosome ends with long telomeres associate with each other in both polytene and mitotic cells, but the end-to-end associations between metaphase chromosomes are resolved during anaphase.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks and genetic crosses:
All genetic markers and special chromosomes are described by LINDSLEY and ZIMM 1992 . The T(2;3)ap^Xa/CyO; TM6 stock was a generous gift of I. Boussy. Oregon-R is a standard laboratory wild-type strain. The multiply marked stocks ru h th st cu sr e^s ca/TM3 and ru h th st cu sr e^s Pr ca/TM6B were provided by the Bowling Green stock center and have been in the lab for many years. The stocks containing C(1)DX, y f, Sco/SM1, or Sb/TM6 were generated in crosses to Oregon-R to replace the unmarked chromosomes in these stocks with chromosomes from Oregon-R. The original Gaiano (Gaiano-O) was a generous gift of J. Lim. Stocks labeled Gaiano-I, Gaiano-II, or Gaiano-III were generated as shown in Fig 1. To examine females carrying two Gaiano-I X chromosomes, the X chromosome from Gaiano-I was reextracted into a homozygous stock.

View larger version (22K): In this window In a new window Download PPT slide   Figure 1. Genetic crosses made to generate Gaiano-I, Gaiano-II, and Gaiano-III stocks. Many of the symbols and special chromosomes are described by LINDSLEY and ZIMM 1992 , with the exceptions that G represents Gaiano; OR represents Oregon-R; and I, II, and III represent chromosomes X, 2, and 3, respectively.

All stocks were maintained and crosses were made on standard Drosophila medium at 25°. Stocks were kept on a 2-week cycle, 26 transfers per year.

The position of Tel along chromosome 3 was determined by allowing free recombination in Tel/ru h th st cu sr e^s ca females. Recombinant chromosomes were collected in males over ru h th st cu sr e^s Pr ca and placed into stock by crossing these males to Sb/TM6, Ubx females and interbreeding Ubx Pr⁺ sibling progeny. Presence of Tel on the recombinant chromosomes was determined after 50 generations by backcrossing to Sb/TM6 and examining the 2L tips in salivary gland cells for uneven telomere length between the homologs.

Cytological procedures:
Colchicine-treated metaphase chromosome preparations, stained with either aceto-orcein or Hoechst 33258, and aceto-orcein-stained anaphase figures were obtained from larval brains, as described previously (GATTI and GOLDBERG 1991 ; GATTI et al. 1994 ). In Gaiano-II telomere-telomere associations (TAs) were examined in Hoechst-stained metaphases, where the second chromosome can be easily identified. Other cytological analyses of mitotic TAs were performed on orcein-stained brain squashes. Polytene chromosomes were stained with aceto-orcein or prepared for fluorescent in situ hybridization (FISH) according to MASON et al. 1984 and ENGELS et al. 1989 , respectively. Mitotic chromosome preparations for FISH were obtained according to GATTI et al. 1994 .

Fluorescent in situ hybridization:
The FISH procedure used is described by GATTI et al. 1994 . The probe used for HeT-A hybridization was a mixture of the 2-kb ApaI fragment of the 3' untranslated region (UTR) and the 23Zn-1 fragment containing the open reading frame (ORF; BIESSMANN et al. 1994 ; DANILEVSKAYA et al. 1994 ), while the 2.25-kb SstI fragment of the TART-A1 element was used for TART hybridization.

Chromosome preparations were analyzed using a Zeiss (Oberkochen, Germany) Axioplan epifluorescence microscope equipped with a cooled CCD camera (Photometrics, Woburn, MA). The fluorescent signals, recorded separately as gray-scale digital images by IP Lab Spectrum software, were pseudocolored and merged using Adobe Photoshop 3.0.

To estimate telomere length in Oregon-R, Gaiano-O, and Gaiano-III chromosomes we examined polytene chromosome preparations after in situ hybridization with HeT-A. Telomere length was quantified by measuring the fluorescence intensity of the HeT-A telomeric signals, using Adobe Photoshop. Measurements were taken from Oregon-R/Gaiano-O and Oregon-R/Gaiano-III heterozygous female polytene preparations, where the signals on the homologous telomeres were separate. For both Oregon-R/Gaiano-O and Oregon-R/Gaiano-III the values obtained were normalized with respect to the Oregon-R 2L telomeric signal, which was chosen as the telomere length unit. At least 10 chromosome ends were examined for each telomere. The differences in telomere length obtained between Gaiano-O and Gaiano-III were confirmed by measuring telomeric signals in Gaiano-O/Gaiano-III heterozygous female polytene preparations.

Immunostaining with HP1:
Polytene chromosome preparation and the immunostaining technique for HP1 are described in FANTI et al. 1998 . The polytene chromosomes were incubated overnight with the anti-HP1 C1A9 monoclonal antibody (JAMES et al. 1989 ) diluted 1:50 in PBS. This primary antibody was detected by a 2-hr incubation with fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse Ig (Jackson Laboratories, Bar Harbor, ME), diluted 1:20 in PBS.

Genomic DNA isolation, Southern blotting, and hybridizations:
Genomic DNA was isolated from adults according to standard procedures (LIS et al. 1983 ), as were DNA transfers from agarose gels to nylon membranes and hybridization with nonradioactive probes (SAMBROOK et al. 1989 ). The nonradioactive DNA probes from HeT-A, TART, and jockey elements were prepared by random priming, using a digoxygenin (DIG) DNA labeling and detection kit (Boehringer Mannheim, Indianapolis). Because the pericentromeric heterochromatin contains tandem arrays of the HeT-A 3' UTR (YOUNG et al. 1983 ; TRAVERSE and PARDUE 1989 ; DANILEVSKAYA et al. 1993 ), only the HeT-A ORF was used as a probe to minimize detection of HeT-A heterochromatic sequences. The pol ORF was used as a TART probe and a complete element was used as jockey probe. Blots were stripped and reprobed with the EcoRI/SacI fragment of the mini-w gene. Southern blots were optically scanned and the density of hybridization signal in each lane or individual band was determined using NIH Image 1.61 software. Comparisons between strains were made after normalization to the white hybridization signal in the same lane.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Gaiano-O stock was caught in the wild, probably near Endine Gaiano, a small town in the Bergamo province in northern Italy. The flies must have been collected on or before 1946, as a brief report mentioning this strain was published in November 1946 by BARIGOZZI and TRAPANI 1946 . The stock was subsequently kept at the Istituto di Genetica, Universita' di Milano for many years. It was "exported" to the United States in the 1970s and was propagated among several laboratories (S. BECKENDORF, personal communication). The Gaiano flies are phenotypically indistinguishable from Oregon-R or Canton-S wild-type flies. They also exhibit normal viability and fertility and have the same life span as Oregon-R flies (our unpublished observations).

Our interest in Gaiano arose as a result of J. Lim's observation that Gaiano polytene chromosomes display long telomeric regions that are often associated with each other. We, therefore, analyzed Gaiano telomeres by addressing three basic questions: (1) Does the Gaiano strain contain one or more genetic factors responsible for the increase in telomere length?, (2) are the telomeres in this strain particularly enriched in HeT-A and TART sequences?, and (3) what is the basis for TAs that are seen in the Gaiano stock?

The Gaiano stock contains a genetic factor(s) that increases telomere length:
To identify and map any genetic factors that might be responsible for generating long telomeres in Gaiano, the major Gaiano chromosomes were extracted into an Oregon-R genetic background (Fig 1), generating three stocks, each containing a different Gaiano chromosome. Because of its small size, chromosome 4 was ignored in these crosses and in the following discussion. The stocks were examined after 60 and 130 generations for telomere length by in situ hybridization, using a HeT-A probe. In stocks carrying either the X chromosome (Gaiano-I) or chromosome 2 (Gaiano-II) from Gaiano, the length of the telomeres reflected their origins; chromosomes that originated from Gaiano had long telomeres, while chromosomes from Oregon-R had short telomeres (Fig 3 and data not shown). In the stock carrying chromosome 3 from Gaiano (Gaiano-III), however, all the major chromosomes had long telomeres that hybridized strongly with HeT- A (Fig 2). These findings indicate that exposure to chromosome 3 from Gaiano for several generations results in a dramatic growth in Oregon-R-derived telomeres. This suggests that there is a genetic factor(s) on the Gaiano chromosome 3 that induces HeT-A addition to chromosome ends at high frequency.

View larger version (35K): In this window In a new window Download PPT slide   Figure 2. Quantification of telomere length to polytene chromosomes by FISH. (A–H) Polytene chromosomes of GO/OR, GIII/OR, or GO/GIII females hybridized with either HeT-A (A–G) or TART (H) probes. (G) The 2L and 2R telomeres are connected by a thread of HeT-A-containing material. (I) The relative lengths of the Oregon-R, Gaiano-O, and Gaiano-III telomeres, estimated by measuring the intensities of the HeT-A telomeric signals. Abbreviations are as in Fig 1.

View larger version (29K): In this window In a new window Download PPT slide   Figure 3. Fluorescent in situ hybridization with a HeT-A probe (red) to mitotic chromosomes. Chromosomes were obtained from colchicine-arrested brain cells of Oregon-R (A), Gaiano-O (B), Gaiano-I (C), Gaiano-II (D), and Gaiano-III (E and F) larvae. The inserts in A, B, and F show Y chromosomes from Oregon-R, Gaiano-O, and Gaiano-III stocks, respectively. Note that the labeling pattern of all these Y chromosomes is similar, except for the tip of YL, which is labeled in Gaiano-O and Gaiano-III but not in Oregon-R, where the fluorescent signal is subterminal. Also note the polymorphic Het-A clusters in XR (arrowhead) and in the third chromosome pericentric heterochromatin (arrows). See text for further explanations. Bar, 5 mm.

Cytological characterization of Gaiano telomeres:
To assess relative degrees of telomere extension after 130 generations, we crossed Oregon-R females to either Gaiano-O or Gaiano-III males. The polytene chromosomes in the female progeny of these crosses contain an Oregon-R chromosome tightly synapsed with its Gaiano homolog, allowing direct comparison between telomeric regions. We also compared Gaiano-O and Gaiano-III telomeres by examining the polytene chromosomes of Gaiano-O/Gaiano-III females. These polytene chromosomes were examined using FISH with a HeT-A probe and counterstained with 4',6-diamidino-2-phenylindole (DAPI) to visualize the chromosomes. This procedure allowed unambiguous recognition of the telomeric regions enriched in HeT-A and a reliable estimation of their length.

Analysis of the Oregon-R/Gaiano-O polytene chromosomes revealed that all Gaiano-O telomeres are longer than their Oregon-R counterparts (Fig 2B and Fig I). However, the Gaiano-O chromosomes differ from each other in telomere length. In Gaiano-O the telomeres of the left arm of the X chromosome (XL) and the right arm of chromosome 3 (3R) are substantially longer than those of the other chromosome arms (Fig 2I). The telomeres of the Gaiano-III chromosomes are longer than both their Oregon-R and Gaiano-O counterparts. Recall that the Gaiano-III strain contains the X and second chromosomes from Oregon-R and the third chromosome from Gaiano-O (Fig 1). Thus, in these experiments the Oregon-R XL, 2R, and 2L telomeres display a dramatic increase in their length after 130 generations' exposure to the Gaiano third chromosome. Interestingly, the Gaiano-III 3L and 3R telomeres are also longer than their Gaiano-O counterparts (Fig 2E and Fig F). The lengths of all the Gaiano-III telomeres are comparable, regardless of their lengths at the time Gaiano-III was constructed (Fig 2I). The finding that Gaiano-O telomeres differ in length suggests that these flies may carry additional elements that regulate telomere extension, thus limiting telomere growth. These elements may also be present in Gaiano-III, as the amount of HeT-A, measured by Southerns, is similar after 85 and 130 generations (see below).

Hybridization of polytene chromosomes from Oregon-R/Gaiano-III females with a probe for TART, the other telomere-specific transposon, revealed that all the telomeres of Gaiano-III chromosomes are also enriched in TART sequences (Fig 2H). The intensities of the fluorescent signals generated by TART hybridization, however, were much lower than those observed after hybridization with HeT-A. This suggests that TART addition contributed to telomere elongation in Gaiano-III to a lesser extent than HeT-A addition.

Although HeT-A and TART are localized primarily at the telomeres, weak hybridization signals were also observed in the chromocenter, while the euchromatic arms were completely devoid of signals (data not shown). Because the chromocenter of Drosophila polytene chromosomes contains heterochromatin, which is underreplicated during polytenization (RUDKIN 1969 ; ZHIMULEV 1998 ), it is possible that the hybridization signals observed in the chromocenter of Gaiano-O and Gaiano-III polytene chromosomes reflect significant accumulations of HeT-A and TART elements in heterochromatic regions. Previous work has shown that HeT-A sequences (YOUNG et al. 1983 ; TRAVERSE and PARDUE 1989 ; DANILEVSKAYA et al. 1993 ), like other transposable elements (PIMPINELLI et al. 1995 ), are located in discrete clusters in both centric heterochromatin and in the heterochromatic Y chromosome.

To ask whether the Gaiano factor(s) that increases HeT-A addition to telomeres also affects the amount of HeT-A in heterochromatin, we hybridized HeT-A to mitotic chromosomes of the Oregon-R, Gaiano-I, Gaiano-II, and Gaiano-III stocks (Fig 3). These studies first established that the Gaiano factor also increases telomere length in mitotic chromosomes. In Oregon-R, weak telomeric signals are observed only at the 2R and XL telomeres, and these signals are detectable only under particularly favorable conditions (Fig 3A). Gaiano-O chromosomes exhibit clear signals at all but the 3L telomere, which is usually unlabeled (Fig 3B). In Gaiano-I and Gaiano-II, clearly visible telomeric HeT-A signals are restricted to the X chromosome and second chromosome, respectively (Fig 3C and Fig D). This is consistent with the origin of the chromosomes, as these are the only Gaiano-derived chromosomes in each stock. In Gaiano-III, however, all the telomeres are always clearly labeled after 130 generations (Fig 3E and Fig F).

In contrast, the Gaiano factor(s) has little effect on heterochromatic HeT-A accumulations. In Oregon-R, the Y chromosome displays a signal on the tip of the short arm (YS), two weaker signals in the interior of the chromosome, and a weak subtelomeric signal on the tip of the long arm (YL; Fig 3A, inset). Y chromosomes in Gaiano-O, Gaiano-I, Gaiano-II, and Gaiano-III exhibit the same signals in location and intensity observed in their Oregon-R counterpart. The Gaiano-O and Gaiano-III Y chromosomes, however, show an additional signal at the tip of YL (Fig 3B and Fig F, insets). While the signals observed at the YS ends of all the Y chromosomes may correspond to either subtelomeric HeT-A clusters or real HeT-A-enriched telomeres, the signal at the tip of the YL in Gaiano-O and Gaiano-III is likely to represent a genuine telomeric accumulation of HeT-A sequences.

The Oregon-R and Gaiano-O stocks are both polymorphic for a HeT-A cluster located in the short arm (XR) of the acrocentric X chromosome (Fig 3B). This polymorphism is also present in Gaiano-I and Gaiano-II, but not in Gaiano-III, where all individuals examined (n = 20) showed a clear signal at the XR tip. Thus, it is quite likely that in Gaiano-III the XR telomeres have acquired newly transposed HeT-A sequences that mask the polymorphism.

Finally, Oregon-R, Gaiano-O, and the derivative strains are polymorphic for a HeT-A cluster located near the centromere of chromosome 3 (Fig 3). The intensities of the signals associated with these clusters are comparable in all stocks, suggesting that the Gaiano factor had no effect on the HeT-A copy number in these heterochromatic regions.

In summary, the results obtained by FISH on mitotic chromosomes are fully consistent with the observations of HeT-A labeling at polytene chromosomes and provide two additional pieces of information. First, the Gaiano factor(s) causes the extension not only of euchromatic telomeres, but also of heterochromatic telomeres such as those on XR and YL. Second, this genetic factor(s) does not cause HeT-A accumulation in centric heterochromatin. We, therefore, conclude that the Gaiano factor specifically increases the rate of HeT-A addition to both euchromatic and heterochromatic telomeres.

Gaiano exhibits an increase in HeT-A and TART copy number:
To quantify the relative amount of HeT-A and TART sequences in Oregon-R and Gaiano-III, DNA from these stocks was blotted and probed with either the HeT-A ORF or the pol ORF of the TART element. Southern blotting experiments performed after 85 and 130 generations, following the Gaiano-III stock construction, gave similar results. These Southern blots showed a fourfold increase in HeT-A copy number and a twofold increase in TART copy number in Gaiano-III relative to Oregon-R (Fig 4). The relative increase in HeT-A copy number revealed by Southern blotting does not fully agree with the in situ hybridization data, which suggest that the Gaiano-III telomeres are approximately sevenfold longer than Oregon-R telomeres (Fig 2I). However, we believe that this discrepancy does not depend on the method used for HeT-A quantification, but rather on the presence of heterochromatic HeT-A sequences. We have shown that Oregon-R and Gaiano-III are both polymorphic for HeT-A heterochromatic clusters. If some of the HeT-A copies in these clusters were detected by Southern blotting, they would mask the difference in abundance of telomeric HeT-A sequences between the two stocks.

View larger version (84K): In this window In a new window Download PPT slide   Figure 4. Genomic abundance of HeT-A, TART, and jockey in Oregon-R and Gaiano-III. Southern blots of genomic DNAs extracted from D. melanogaster stocks Oregon-R (OR) and Gaiano-III (GIII) and from D. virilis (DV), a species without HeT-A (BIESSMANN et al. 2000 ), were probed with the HeT-A ORF (A) or the pol ORF of TART (B) or jockey (C). DNA bands of different sizes are detected in A and B, which reflect a highly polymorphic HeT-A/TART terminal array. In A, a strong band at 3.0 kb is identified in both Oregon-R and Gaiano-III. This suggests that there is another BamHI site in the extreme end of the 3' UTR, which was not identified in the sequenced HeT-A clone (BIESSMANN et al. 1992 ). In B, two major bands at 3.4 and 4.2 kb are detected in both Oregon-R and Gaiano-III, with an additional intense band at 4.4 kb identified in Gaiano-III. The 3.4- and 4.2-kb bands were expected from the TART sequence (no. U14101), which predicts a restriction map with three BamHI sites. (C) DNAs from both Oregon-R and Gaiano-III exhibit a similar pattern of jockey element distribution. M shows the HindIII marker bands. The marker band profile in B and C is the same. To quantitate the relative amounts of HeT-A and TART DNA, the blots were scanned and the relative intensities of different lanes were determined and normalized to a white hybridization in the same lane. Abbreviations are as in Fig 1.

We also performed Southern blot analysis and in situ hybridization experiments using jockey, a D. melanogaster retrotransposon very similar in structure to HeT-A and TART (BIESSMANN et al. 1992 ; LEVIS et al. 1993 ). These experiments did not detect significant differences between Oregon-R and Gaiano-III in total jockey DNA (Fig 4C) or in the number of jockey insertions on polytene chromosomes (data not shown), suggesting that the Gaiano factor(s) is specifically involved in the control of HeT-A and TART copy number.

Telomere-telomere associations in Gaiano:
Examination of polytene chromosomes from Gaiano-III larvae revealed very frequent TAs. Analysis of 50 polytene nuclei hybridized with HeT-A showed that all of them exhibited at least one TA. In the majority of these nuclei most if not all of the telomeres were associated with other telomeres, thus giving rise to a chromocenter-like structure (Fig 5). In addition, in several cases telomeres were connected by thread-like structures that hybridized with the HeT-A probe (Fig 2G), as seen occasionally in wild-type larvae (RUBIN 1978 ). We believe that these HeT-A-positive threads result from the stretching of telomeric material caused by the squashing procedure used to obtain polytene chromosome preparations.

View larger version (80K): In this window In a new window Download PPT slide   Figure 5. Polytene chromosomes from the Gaiano-III stock stained with DAPI. Note that all the telomeres are fused (arrow), forming a structure reminiscent of a chromocenter (arrowhead).

The frequent TAs seen in Gaiano-III might be a direct result of the activity of the Gaiano factor(s), or they could be a consequence of the extraordinary length of Gaiano-III telomeres. To discriminate between these two possibilities, we examined the polytene chromosomes of the Gaiano-II stock, which contains second chromosomes with long telomeres but which lacks the genetic factor(s) on the Gaiano third chromosome. Of 50 Gaiano-II polytene nuclei hybridized with HeT-A, 26 displayed TAs, all involving second chromosome ends. This strongly suggests that long HeT-A-containing telomeric regions tend to fuse to each other, even in the absence of a putative genetic factor(s) that enhances HeT-A addition.

We next asked whether the long telomeres associate with each other in mitotic cells. Analysis of colchicine-arrested metaphases from larval brain cells revealed that the Gaiano-O and Gaiano-III stocks both display a significant frequency of TAs, reminiscent of those described in UbcD1 mutants (CENCI et al. 1997 ; Fig 6). Chromosome end associations are normally classified as either double telomere associations (DTAs), in which one pair of sister chromatids interacts with another pair, or single telomere associations, where the two sister chromatids behave independently (CENCI et al. 1997 ). Here we consider only DTAs, because they are cytologically unambiguous and almost completely absent in wild type. As shown in Table 1, metaphases of cells with Gaiano chromosomes exhibit 4.5–28 times the frequency of DTAs observed in the Oregon-R control. In addition, 8 of the 9 DTAs observed in Gaiano-II involved at least one of the two second chromosomes, while 7 of the 11 DTAs found in Gaiano-I females involved at least one of the X chromosomes. These data clearly indicate that long HeT-A-containing telomeres also tend to associate with each other in mitotic cells. Again, telomere length seems to be an important factor for the formation of TAs.

View larger version (117K): In this window In a new window Download PPT slide   Figure 6. Telomere-telomere associations in larval brain cells from Gaiano stocks. (A–D) Colchicine-arrested metaphases. (E and F) Anaphases. (A) Normal male metaphase. (B) Male metaphase with a DTA involving two metacentric autosomes (arrow). (C) Male metaphase with a DTA involving a metacentric autosome and the long arm of the acrocentric X chromosome (arrow). (D) Female metaphase with a DTA between the XR telomeres (arrow). (E) A normal anaphase. (F) An anaphase showing a resolved chromosome bridge. Bar, 5 µm.

We also investigated the fate of TAs observed in Gaiano-III. Examination of non-colchicine-treated Gaiano-III brain cells revealed that the TAs seen in metaphase generate chromatin bridges in anaphase. However, most if not all of these bridges are resolved, as no lagging acentric fragments in anaphase figures were observed. In UbcD1 mutants TAs are similarly resolved in mitotic anaphases but are not resolved during male meiosis, giving rise to extensive chromosome breakage (CENCI et al. 1997 ). In contrast, we found no evidence for chromosome breakage during meiotic anaphase I or II in Gaiano-III males (data not shown).

HP1 localization in Gaiano telomeres:
Mutations in the Su(var)205 gene, which encodes heterochromatic protein 1 (HP1), cause extensive TAs that fail to be resolved during anaphase (FANTI et al. 1998 ). HP1 binds the chromocenter, the telomeres, and multiple euchromatic sites on all Drosophila chromosomes (JAMES et al. 1989 ; KELLUM et al. 1995 ; FANTI et al. 1998 ). In addition, HP1 binds the ends of terminally deleted chromosomes that lack HeT-A and TART sequences, indicating that HP1 telomere binding is independent of HeT-A and TART (FANTI et al. 1998 ).

To examine HP1 binding to the long Gaiano telomeres we immunostained polytene chromosomes of Gaiano-O/Gaiano-III heterozygous females. This analysis showed that HP1 binds both the Oregon-R and the Gaiano-III telomeres. However, while the Oregon-R telomeres usually exhibit a compact HP1 staining, the Gaiano-III telomeres often display a discontinuous HP1 labeling (Fig 7). Nonetheless, the amount of HP1 associated with the Gaiano-III telomeres is similar to, or only slightly lower than, that seen on Oregon-R telomeres. The reason for the discontinuous HP1 labeling at the Gaiano-III telomeres is unclear. This staining pattern may reflect either a squashing artifact or a peculiar structure of the Gaiano-III telomeres. For example, an uneven termination of the DNA strands at the Gaiano-III telomeres may result in a punctate and discontinuous HP1 localization.

View larger version (66K): In this window In a new window Download PPT slide   Figure 7. HP1 immunostaining of polytene chromosomes from Oregon-R/Gaiano-III hybrids. (A and B) DAPI staining. (C and D) HP1 immunostaining. (E and F) Merged images; the HP1 signal is yellow. Note the discontinuous HP1 staining on the long Gaiano-III telomeres.

Genetic mapping of the Gaiano factor:
To ask whether the Gaiano factor maps as a single genetic unit, we crossed the Gaiano-III stock to a strain carrying the markers ru h th st cu sr e^s ca, which span chromosome 3. After allowing free recombination in the heterozygous progeny females, 20 recombinant third chromosomes were recovered and placed in stock with non-Gaiano genetic backgrounds. As controls, two nonrecombinant chromosomes were also recovered, one with all of the markers from the multiply marked chromosome, the other with none of these markers. The former is identified as recombinant 16; the latter was lost. After 50 generations these stocks were outcrossed to the Sb/TM6, Ubx stock used in the last step in their construction, and the 2L telomeres of the heterozygous offspring were examined in orcein-stained polytene chromosomes (as shown in Fig 2, differences in telomere length can be clearly seen even in the absence of in situ hybridization with HeT-A). Length differences between homologous telomeres were evident in some recombinant lines, but not in others (Table 2). For most recombinants, the telomere phenotype is consistent with a single telomere-elongating genetic factor mapping between sr and e, at 69. One recombinant line, however, does not fit the pattern. According to its genetic markers, recombinant 7 should carry the telomere-elongating factor from Gaiano-III. Nonetheless, it does not exhibit long terminal arrays on chromosome 2. In addition, although the right arm of this recombinant chromosome should derive from the Gaiano-III parental chromosome, it does not display a long 3R telomere. It is possible that recombinant 7 was generated by multiple recombination events, including double crossing over between the sr and e^s markers. Alternatively, the telomeres of this recombinant line may be short for reasons independent of the presence of the telomere-elongating factor.

The ru h th st cu sr e^s ca chromosome has been kept in heterozygous condition for many years and has accumulated lethals and other deleterious mutations. In particular, we mapped a lethal mutation segregating on this chromosome very close to cu. Thus, several recombinant chromosomes, including 4, 10, 18, and 20, were homozygous lethal and were maintained over the TM6 balancer. Most of the other recombinant chromosomes, while not strictly lethal, required the TM6 balancer to maintain a healthy stock. As telomeres grew in these heterozygous lines, we conclude that the genetic factor responsible for telomere elongation is dominant. Taken together, our mapping data suggest that the process of telomere elongation observed in Gaiano-III is due to a dominant mutation in a single genetic unit, and we propose the name Telomere elongation (Tel) for this new gene.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Regulation of HeT-A and TART addition:
This article includes the first description of the genetic control of telomere length in Drosophila. We have found a mutation, Tel, at 69 on chromosome 3 that is correlated with increased addition of HeT-A and TART elements to chromosome ends and telomere elongation.

The mechanism by which Tel mediates HeT-A and TART addition to chromosome ends is not known. One possibility is that Tel increases the rate of HeT-A and TART transposition to the telomeres. Previous studies have clearly shown that these elements can specifically transpose to chromosome ends. Starting with a terminal deficiency chromosome broken in the yellow (y) gene, BIESSMANN et al. 1990 found that HeT-A elements transposed to the tip of the X chromosome and attached to the y sequence by their oligo(A) tails. New HeT-A elements then attached to the first in a head-to-toe fashion (BIESSMANN et al. 1992 ).

Various HeT-A elements have either a single ORF (BIESSMANN et al. 1994 ) or two overlapping ORFs (DANILEVSKAYA et al. 1994 ) that may encode RNA-binding proteins. These retroelements, however, do not encode a reverse transcriptase (RT), and it has been proposed that an RT is provided in trans, possibly by the host, as a means of controlling telomere length (BIESSMANN et al. 1994 ). Tel may increase, directly or indirectly, the activity of an RT that could promote both HeT-A and TART transposition, leading to telomere elongation. RT-like proteins also regulate telomere length in other eukaryotes. In these organisms telomere extension is modulated by levels of telomerase catalytic subunits (LINGNER et al. 1997 ; BODNAR et al. 1998 ), which contain conserved domains common to all known reverse transcriptases, including those encoded by non-LTR retrotransposons, such as HeT-A and TART (EICKBUSH 1997 ). Alternatively, Tel may encode a product that increases the accessibility of an RT to both HeT-A and TART RNAs or a transposition intermediate to chromosome ends. At present, however, we cannot distinguish among these alternatives, nor can we establish whether a putative Tel product is encoded by the host genome per se or by another transposable element.

Alternatively, Tel might increase HeT-A and TART addition to telomeres by promoting recombination/gene conversion events at chromosome ends. Telomere recombination may be the primary mechanism for maintaining chromosome length in some organisms that lack telomerase, for example, in other dipteran insects, such as the mosquito Anopheles (ROTH et al. 1997 ) and the midge Chironomus (LOPEZ et al. 1996 ). Even in organisms that normally rely on telomerase to extend chromosome ends, telomeres may be elongated by recombination when telomerase is defective or missing. In the yeasts Saccharomyces (LUNDBLAD and BLACKBURN 1993 ), Schizosaccharomyces (NAKAMURA et al. 1997 ), and Kluyveromyces (MCEACHERN and HICKS 1993 ) most cells lacking a component of telomerase senesce and die. Survivors, however, arise relatively frequently in all three organisms, and in both Saccharomyces and Kluyveromyces survival requires a RAD52-dependent recombination mechanism. In Saccharomyces, two classes of survivors are found. Type I survivors carry tandem duplications of the subtelomeric Y' elements, possibly generated by gene conversion (LUNDBLAD and BLACKBURN 1993 ). Kluyveromyces (MCEACHERN and BLACKBURN 1996 ) and Saccharomyces (TENG and ZAKIAN 1999 ) type II survivors carry very long terminal repeat arrays that resemble the long terminal arrays found in human tumors (BRYAN et al. 1997 ) and immortalized cultured cells (BRYAN et al. 1995 ) that lack telomerase. These immortalized telomerase-negative human cells use an alternative mechanism to lengthen telomeres (ALT) and carry distinctive nuclear bodies that contain telomeric DNA, telomere-binding proteins TRF1 and TRF2, as well as recombination proteins RAD51 and RAD52 (YEAGER et al. 1999 ), consistent with the idea that human telomeres are elongated by recombination in the absence of telomerase. Direct evidence has been found for the transfer of a plasmid DNA tag from one terminal repeat array to another in ALT cells, as expected for a recombination or gene conversion mechanism (DUNHAM et al. 2000 ).

Recent work has suggested that telomere elongation in Drosophila can also occur via a recombination/gene conversion pathway. It has been shown that broken X chromosomes that terminate in the y gene (MIKHAILOVSKY et al. 1999 ), or in a HeT-A element directly attached to the y gene (KAHN et al. 2000 ), can also be extended by a gene conversion mechanism that adds y sequences or HeT-A sequences to the terminus, respectively. Moreover, P. GEORGIEV (personal communication) found that chromosome 3 from Gaiano carries a dominant genetic factor(s) that increases the rate of gene conversion. However, it remains to be seen whether this factor(s) is Tel.

The basis for telomere-telomere associations:
Both the polytene and mitotic nuclei of the Gaiano stocks display frequent TAs. These associations preferentially involve long telomeres and occur in the absence of Tel. Thus, the formation of TAs in Gaiano stocks depends primarily on telomere length rather than the genetic activity of Tel.

Telomeric associations have been observed both in mammals and Drosophila. In mammals frequent TAs are present in cells with short telomeres, such as senescent human fibroblasts (BENN 1976 ), tumor cells (DE LANGE 1994 ), and fibroblasts from mice lacking functional telomerase (HANDE et al. 1999A ). TAs are also found in human cells overexpressing a dominant negative allele of the telomere-binding protein TRF2 (VAN STEENSEL et al. 1998 ). It has, thus, been suggested that short telomeres become fusigenic because they cannot recruit a sufficient amount of TRF2 (VAN STEENSEL et al. 1998 ). The TAs induced by inhibition of TRF2 are due to covalent fusions of telomeric DNA and are not resolved during anaphase, thus leading to chromosome breakage (VAN STEENSEL et al. 1998 ).

Frequent TAs have also been found in mouse cells carrying mutations in components of the DNA-protein kinase (PK) complex (SLIJEPCEVIC et al. 1997 ; BAILEY et al. 1999 ; HANDE et al. 1999B ; DIFILIPPANTONIO et al. 2000 ; SAMPER et al. 2000 ). This complex consists of a catalytic subunit (PKcs) and a heterodimeric regulatory factor, Ku, containing the Ku70 and Ku80 proteins (JEGGO 1997 ). Interestingly, the TAs observed in mice deficient for either PKcs or Ku80 do not result from telomere shortening, as these mice have longer telomeres than their wild-type counterparts (SLIJEPCEVIC et al. 1997 ; HANDE et al. 1999B ; SAMPER et al. 2000 ). It is unclear whether these TAs are resolved during anaphase.

In Drosophila frequent TAs have been observed in UbcD1 and Su(var)205 mutants (CENCI et al. 1997 ; FANTI et al. 1998 ). The UbcD1 gene encodes a ubiquitin-conjugating (E2) enzyme (CENCI et al. 1997 ), while Su(var)205 encodes HP1, a protein enriched in both centric heterochromatin and telomeric regions (EISSENBERG 1989 ; FANTI et al. 1998 ). In larval brain cells of Su(var)205 mutants, TAs result in anaphase bridges leading to extensive chromosome rearrangements and may, therefore, involve covalent fusions of telomeric DNA (FANTI et al. 1998 ). In contrast, TAs between mitotic chromosomes of UbcD1 mutants are resolved during anaphase without causing chromosome breakage or nondisjunction (CENCI et al. 1997 ), suggesting that these TAs are due to proteinaceous linkages between telomeres that result when one or more telomere-associated proteins fail to be degraded via ubiquitin-mediated proteolysis (CENCI et al. 1997 ).

Because they can be resolved, the TAs observed in Gaiano brain cells are similar in strength to those elicited by the UbcD1 mutations. The basic mechanisms that give rise to telomeric associations may, nonetheless, differ. In Gaiano stocks those chromosomes that exhibit an increase in HeT-A and TART at chromosome ends preferentially form TAs. This suggests that an increase in length, and more specifically acquisition of HeT-A and TART, make a Gaiano telomere sticky. In contrast, telomeres in UbcD1 mutants, even those devoid of HeT-A and TART sequences, participate in telomeric associations with high frequency, suggesting that the presumptive UbcD1 substrates bind chromosome ends independently of DNA sequence (G. CENCI, G. M. SIRIACO and M. GATTI, unpublished data). It is, thus, unlikely that Gaiano telomeres are sticky because these UbcD1 substrates accumulate on them. It is also unlikely that HP1 plays a role in mediating telomere associations in Gaiano, as HP1 binds chromosome ends independently of the presence of HeT-A and TART sequences (FANTI et al. 1998 ). Accordingly, the amount of HP1 localized to the long Gaiano telomeres is comparable to that associated with the Oregon-R telomeres. Finally, TAs in HP1 mutants are not resolved in anaphase, indicating that the mechanism of fusion is not the same as in Gaiano. Thus, it appears that the mechanism of TA formation in Gaiano is distinct from those involving either UbcD1 or HP1. Given that the Gaiano TAs are weak associations, it is likely that they are mediated by a HeT-A and/or TART binding protein(s) or by DNA hydrogen bonding rather than covalent DNA-DNA bonds.

	FOOTNOTES

¹ Present address: Dipartimento di Biologia, Ecotekne, Universita' di Lecce, Lecce, Italy 73100.
² Present address: Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA 23501.

	ACKNOWLEDGMENTS

We are grateful to Johng Lim for the Gaiano-O stock; Robert Levis for the gift of the TART plasmid; Harald Biessmann for the jockey plasmid; Sally Elgin for the C1A9 antibody; and Kevin Lewis, Elena Kurenova, and Mike Goldberg for critically reading the manuscript. G.S. received support from a Training and Mobility of Researchers (TMR) grant of the European Union. This work has been supported in part by a grant from the Associazione Italiana per la Ricerca sul Cancro to M.G.

Manuscript received May 20, 2001; Accepted for publication October 23, 2001.

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