Using a recently completed combined-evidence annotation of the Release 4 genome sequence 10, we revised estimates of the overall abundance of TE sequences in D. melanogaster (Table 1) from those based on the Release 3 sequence 2. Excluding foreign elements based on query sequences from other species (see Materials and methods), the estimated number of TEs in the D. melanogaster Release 4 genome sequence (n = 5,390) is over three-fold higher than in Release 3 (n = 1,572). In contrast, the amount of sequence annotated as TE increased by only approximately 44% in Release 4 (6.51 Mb, 5.50% of genome) relative to Release 3 (4.51 Mb, 3.86% of genome). (We note that the proportion of the Release 4 genome estimated here as TE is calculated as the sum of non-redundant annotation spans including unique sequences inserted into TEs; this procedure differs slightly from our previous estimates for Release 4, which only included sequences strictly homologous to TE query sequences 10.) The discrepant changes in these two metrics of TE abundance across releases results from the fact that almost all new TEs in Release 4 are either small fragments and/or annotations of the highly abundant but degenerated INE-1 element (also known as DINE-1 or DNAREP1_DM) 14, a family that was omitted from the Release 3 annotation. The inclusion of these new small fragments is also reflected in the fact that the proportion of TEs estimated to be full-length (defined as ± 3% of the canonical element including the length of inserted sequences) has declined from 30.5% in Release 3 to 9.83% in Release 4. The number of TEs involved in nests (n = 785) has more than doubled in Release 4 relative to Release 3 because of newly annotated sequences and improved joining of TE fragments belonging to the same insertion, although the estimated proportion of TEs involved in nests (14.6%) in Release 4 has decreased relative to Release 3 as a consequence of the increased total number of TEs annotated.

The major patterns of TE abundance identified in previous releases of the D. melanogaster genome sequence 2781516 are also observed in Release 4, suggesting that these trends are stable features of the D. melanogaster genomic landscape. As shown in Figure 1, both the pericentromeric regions of the major chromosome arms and the entirety of chromosome 4 have higher densities of TE insertions, relative to non-pericentromeric regions 2715. Densities over the non-pericentromeric regions are roughly equal, with no general increase in TE density in telomeric regions (Figure 1) 715, excluding TEs that are directly involved in telomere structure/function or in the subtelomeric arrays (see below). There is no general decrease in the abundance of TEs on the X chromosome 215, as expected if TE insertions generate deleterious recessive mutations 17. Long terminal repeat (LTR) retrotransposons occupy the greatest proportion of the genome sequence (3.29%), as has been observed previously 27, but the current annotation reveals that the INE-1 family is the most numerous category of TEs (n = 2,238) in the D. melanogaster genome 16. (We note that throughout this work, non-LTR retrotransposon is abbreviated as 'non-LTR', which is referred to as LINE-like in 27.) INE-1 has previously been suggested to be a retrotransposon on the basis of homology to the D. virilis Penelope element 16; however, we found that this reported homology between Penelope and INE-1 is spurious and restricted to flanking sequences in GenBank:U49102 (see also 18). From the percent genome sequence occupied, our analysis indicates that INE-1 distribution most closely fits the terminal inverted repeat (TIR) transposon class of TEs (Table 1), supporting the conclusion that INE-1 is a TIR element based on structural features of an improved consensus sequence 19.

This set of 5,390 TEs defined 4,684 TE-free regions (TFRs) 20 in the Release 4 genome sequence; 94.5% (111.9 Mb of 118.4 Mb) of the Release 4 genome sequence can be found in TFRs, with 89.8% (106.2 Mb) and 56.1% (66.4 Mb) of the genome found in TFRs of greater than 10 Kb (n = 1,393) and 100 Kb (n = 357), respectively. The longest TFR in D. melanogaster is 855,890 base-pairs (bp) in length on chromosome 2R from 14,374,883-15,230,772, contains 106 genes, and is over 10 times longer than the longest TFR in the human genome 20. The mean TFR length of 23,878 bp is consistent with the genome-wide minimum estimate of the distance between middle-repetitive interspersed repeats (>13 Kb) based on reassociation kinetics 21; however, the median TFR length of 1,992 bp is much smaller. The distribution of TFR lengths departs significantly from an exponential distribution parameterized on this mean length using an adjusted Kolmogorov-Smirnov test (D = 0.4513, p < 0.001), which is based on the maximal difference between observed and expected cumulative distributions and accounts for the fact that the rate parameter for the exponential distribution has been estimated from the data 22. Similar results are obtained if the rate parameter for the exponential is calculated from the number of TE insertions divided by the total length of TFRs (as in 20), both including (adjusted Kolmogorov-Smirnov test, D = 0.4719, p < 0.001) or excluding (adjusted Kolmogorov-Smirnov test, D = 0.4456, p < 0.001) TEs nested in other TEs. These results are not simply a result of a high density in pericentromeric regions (see below) and demonstrate that the location of TEs is non-randomly distributed at the level of the complete D. melanogaster genome sequence, confirming previous results 7815. We note that TFRs in the D. melanogaster genome are likely to vary among individuals since most TE insertions are not fixed in the species 23; however, these results should be representative of other strains to the extent that the TE composition of the genome sequence reflects general properties of the species 2.

Since non-random distribution of TEs can lead to greater than one order of magnitude differences in TE abundance in pericentromeric and non-pericentromeric regions 2781524, overall genome-wide summary statistics do not accurately reflect TE abundance for any region of the genome sequence. To account for this heterogeneity, we attempted to partition the major chromosome arms into regions of high (pericentromeric) and low (non-pericentromeric) TE density using an independent criterion that is not based on TE content. Our primary goal here was to estimate the TE content in non-pericentromeric regions of the genome as accurately as possible, to understand baseline levels of TE abundance throughout the majority of the genome. Initially we investigated using a partition based on the cytologically defined boundaries between euchromatin and β-heterochromatin estimated in Hoskins et al. 25. As shown in Figure 1 (red triangles), the cytologically defined limits of the euchromatin/β-heterochromatin boundaries correspond almost exactly to the most distal pericentromeric region of high TE density on chromosome arms 3L and 3R. However, on chromosome arms 2L, 2R and X the most distal pericentromeric regions of extreme TE density are up to 2 Mb from the estimated euchromatin/β-heterochromatin boundary. Thus, using this cytological criterion to partition the genome into regions of high and low TE density still leads to an over-estimate of the true TE abundance for the majority of the genome.

We next evaluated whether genetically defined regions of different recombination rates estimated by Charlesworth 26 could partition the genome into high and low TE density regions. For all chromosome arms (excluding the fourth chromosome), we found that the estimated boundaries between 'reduced' and 'null' (that is, very low) recombination rates in pericentromeric regions (Figure 1, orange triangles) were located extremely close to the cytologically defined boundaries between euchromatin and β-heterochromatin. Thus, the same tendency to bias estimates of TE abundance exists if the boundary between reduced and null recombination rates is used to partition the genome as for the cytological criterion above. In contrast, the estimated transitions between 'high' and 'reduced' recombination rates in pericentromeric regions (Figure 1, green triangles) are approximately 1 to 2 Mb distal to estimated euchromatin/β-heterochromatin boundaries for all major chromosome arms. Virtually all regions with high TE density were included in the 11% of the genome sequence labeled under this definition as 'pericentromeric' (Figure 1), and, therefore, this partition was used to estimate TE abundance in different regions of D. melanogaster genome. Because our aim was to estimate the TE content in non-pericentromeric regions as a baseline to identify regions of extremely high TE content elsewhere in the genome, the inclusion of some low TE content regions in pericentromeric regions on chromosome arms 3L and 3R using this partition should not bias estimates of the background TE abundance throughout the euchromatin.

With this improved calibration of the background TE abundance that is typical of the major chromosome arms, we sought to identify specific regions of the genome with an extremely high local TE density (we abbreviate such high-density regions as HDRs). We omitted INE-1 from this analysis to prevent this very abundant family from dominating the overall genomic trends. Additionally, since it has been postulated that INE-1 underwent a burst of transposition prior to speciation and has subsequently become immobilized 1632, INE-1 elements are predicted to be fixed (barring subsequent deletion). As such, their distribution in the sequenced strain should represent a more stable baseline of ancestral TE content to compare with other more recently active TE families. We identified 24 HDRs containing 10 or more (non-INE-1) TEs in a 50 Kb window, a cut-off of roughly 20-fold higher density of TEs than the majority of the genome (Figure 1, Table 2). Two HDRs have been previously reported: HDR8 at cytological division 38 33 and HDR3 at cytological division 20A, which is likely to be fixed in D. melanogaster 34.

As expected, nearly all HDRs are located in pericentromeric regions or on chromosome 4, consistent with the general observation that heterochromatic and/or low-recombination rate regions of the genome sequence have high TE densities (see above) 2715. Three HDRs (1, 16, 17) on the major chromosome arms are located in regions not defined as pericentromeric; however, HDR1 on the X-chromosome is found very close to the boundary demarcating these regions and could probably be classified as pericentromeric. HDRs total 4.27 Mb of sequence and, therefore, comprise only 3.6% of the genome, but contain one-third (1,822/5,390; 33.8%) of annotated TEs. Interestingly, one of the most extreme regions of localized TE density in the D. melanogaster genome sequence (HDR4) contains the insertion site for a P-element induced allele (flam^py+(P)) of the as-yet-uncharacterized gene flamenco 35, one of the few genetic loci shown to regulate the activity of transposable elements in Drosophila 36. HDR4 (which includes the physical gap in cytological division 20) occupies over 230 Kb of DNA and contains at least 104 TEs and 6 genes, including DIP1, which has been excluded as being the gene that is causal for the flamenco mutation 35. We note that the COM locus also in 20A2-3, which is known to regulate the ZAM and Idefix families of LTR elements, is genetically separable from flamenco 37 and, therefore, unlikely to correspond to the same region.

Two exceptional HDRs are found on chromosome arm 3R. HDR16 contains a set of duplicated, nested TEs in the intergenic region between Hsp70Ba and Hsp70Bb in division 87C (Figure 2a). This region contains the αβ repeat 38, which our results indicate corresponds to a duplicated nest of Dm88 and invader1 sequences (see also 3439. The fact that the αβ repeat is composed of TE sequences, as predicted by Hackett and Lis 40, explains the observation that components of the αβ repeat are dispersed in multiple heterochromatic locations 40 and share homology with 'clustered, scrambled' arrangements of middle repetitive DNA located elsewhere in the genome 41. This region also contains the non-coding RNA gene known as the αγ-element, which is transcribed in response to heat shock 3842 and is a chimeric transcript composed of Dm88 and invader1 sequences emanating from a fragment of the Hsp70 promoter 43. It is likely that the unusually high abundance of TE insertions in this region has arisen in part because of the unusual chromatin architecture of heat-shock promoters 4445. The peculiarity of this region is underscored by the fact that αβ repeat has evolved since the divergence of D. melanogaster from its sister species D. simulans 4246, but yet appears to be fixed in D. melanogaster 47.

The second exceptional HDR (17) on chromosome arm 3R corresponds to a tandemly duplicated array of invader4 elements embedded within the sub-telomeric mini-satellites called telomere-associated sequences ('TAS'). We also found that TAS repeats from chromosome arm 2R 48 and the original TAS repeat derived from the Dp1187 X-minichromosome 49 also contain invader4 sequences (results not shown), although no homology to invader4 (or any other TE) is observed in the TAS repeat derived from chromosome arms 2L or 3L 4850, suggesting that TE sequences are not functionally constitutive components of TAS repeats. The presence of mobile TE sequences in TAS repeats may explain non-telomeric hybridization signal to TAS probes in the chromocenter and basal euchromatic locations 49. No HDRs are observed at the ends of other chromosome arms, despite the fact that, in Drosophila, the retrotransposons Het-A, TART and TAHRE function as telomeric repeats to ensure proper integrity of the chromosome ends 515253. In the Release 4 sequence, only the X chromosome and fourth chromosome 9 terminate with small clusters of telomeric TE sequences.

Surprisingly, the improved resolution provided by our new annotation showed that TE density is not uniformly high in pericentromeric regions, nor is TE density simply an increasing function of proximity to centromeric regions (Figure 1, inset panels). This is especially true for chromosome arms X, 2L and 2R, where pericentromeric HDRs are interspersed with regions of normal TE density, creating a ragged, punctate increase in TE abundance in the direction of the centromere. Chromosome 4 also exhibits discrete regions of different TE density (Table 2), despite a higher overall level of TE abundance. Some HDRs (for example, 1, 8, 13, 16) clearly occur in regions of low INE-1 density, which suggests a recent origin for the high TE density in these regions, assuming that INE-1 represents the ancestral TE distribution at the time of its major burst activity prior to the split of D. melanogaster from its sister species D. simulans 1632. Other HDRs (9, 10, 15 and those on the fourth chromosome) co-occur with regions of high INE-1 density, suggesting these regions of the genome have permitted a high density of TEs, at least as far back as the ancestor of the D. melanogaster species subgroup 1632. This also is likely to hold true for HDRs 11, 12 and 14 at the bases of chromosome arms 2L, 2R and 3L, where non-INE-1 TEs occupy virtually all of the sequence, creating an apparent negative association with INE-1 density.

What evolutionary mechanisms cause such a localized pattern of extreme TE density? Clearly, transposition is the ultimate source of all TE insertions in the genome, and accordingly HDRs typically contain a mix of different TE families and nested elements (Table 2), both hallmarks of recurrent transposition. However, it is possible that other mechanisms of genome evolution - such as inversion or duplication - might have contributed to the origin of HDRs. To investigate whether this punctate pattern of HDRs arose from chromosomal inversions that bring TE-rich, heterochromatic DNA into euchromatic regions, we extracted orthologous regions from the D. yakuba genome sequence and assayed whether the unique sequences flanking HDRs are collinear in the two species. We found that unique sequences flanking HDRs were collinear for 15 of the 16 HDRs (93.8%) that are internal to the ends of the chromosome arms, for which both flanking sequences can unambiguously be identified (Table 2, Figure 3a,b). Intriguingly, HDR 13 does occur in the same region as an inversion breakpoint between D. melanogaster and D. yakuba, but outgroup analyses place this inversion event on the D. yakuba lineage, not the D. melanogaster lineage (JM Ranz, D Maurin, YS Chan, LW Hillier, J Roote, M Ashburner and CM Bergman, personal communication). Thus, we found no evidence indicating that inversions carrying TE-rich DNA from heterochromatic regions generate HDRs, but remarkably we did find evidence that a region of the D. melanogaster genome that permits a high TE density can tolerate inversion breakpoints in other Drosophila lineages. It is important to note, however, that the majority of HDRs do not correspond to inversion breakpoint regions and vice versa.

We did, however, find a relatively high incidence of duplicated sequences in HDRs, suggesting that tandem or segmental duplication plays an important role in the genesis of TE-rich regions of the genome: 13 of 23 HDRs show evidence of duplication (Table 2, Figures 2 and 3c,d). Duplications in HDRs can contain multiple TEs from different families, often nested, sometimes with different copies of the duplicated region containing additional TE insertions (Figure 2). Duplications in HDRs also amplified cellular genes as well as TE sequences: for example, eight partial and complete duplicates of the gene CG32381 are present in HDR1 (Figure 2b). HDRs may also include retrotransposed gene duplicates, such as the Mgst1-like CG12628 54, which is found in a nest of TEs in HDR11. The series of events leading to tandem duplication of TEs in HDRs is often highly complex, with repeat structures present at different scales (Figure 3c,d). Duplication of TE sequences could also be observed in other regions of the genome with lower TE density, such as duplication of Rt1c elements interspersed between the SDIC gene duplicates 5556. A more thorough analysis of the interplay between TEs and segmental duplications will be the subject of a separate study (A-S Fiston, D Anxolabehere and H Quesneville, personal communication).

Regions of extremely high TE density typically contain a high proportion of TEs inserted into other TEs, and our new annotation allowed us to examine patterns of TE nesting in greater detail than has previously been possible. Few methods exist to analyze TE nesting, partly because of limitations in accurately joining fragments of a TE insertion that become separated in the genome by a subsequent nested TE insertion, and partly because analysis of TE nesting is complicated by the redundancies inherent in complex nesting relationships. For example, if one TE (A) is nested within a second (B) that is in turn nested within a third (C), simply analyzing overlapping ranges of TEs in the genome will erroneously yield three nesting events (A→B, A→C, and B→C), when only two occurred historically (A→B and B→C). We found that complex nesting relationships could best be analyzed by identifying 'primary' nesting relationships (A→B and B→C in the example above) and assembly of these simple binary events into more complex nesting relationships by applying concepts from network analysis to describe and quantify patterns of TE nesting. In this formulation of the problem, TE nesting relationships are represented as a graph having TEs as nodes and transposition events as directed edges. The directed nature of this graph implies both the spatial relationships of nested TEs in the genome as well as temporal relationships implied in TE nesting resulting from the fact that the outer TE in a nest must have existed in the genome prior to the insertion of the inner TE 57. This 'nesting graph' is amenable to standard computation and can be recast in several forms, since each annotated TE node can be analyzed at the individual, family or class level. (We chose not to analyze the degree of distribution of nesting graphs for 'small-world' properties because of biases resulting from duplicated nests, and because the subgraphs in the sequenced portion of the genome may not reflect properties of the entire nesting graph 58.)

At the individual TE level, nesting relationships form a sparse, acyclic graph of 785 nodes and 491 edges that provides a detailed overview of the global pattern of TE nesting in the D. melanogaster genome (Figure 4). These 785 TEs (14.6% of all 5,390 TEs annotated) are found in 294 distinct nests, which can be calculated from the number of sink TEs (nodes in the graph that have an out-degree of zero). These 294 nests are formed by 448 source TEs (nodes in the graph with an in-degree of zero), and 43 TEs that act as internal nodes in the graph (with both non-zero in-degree and out-degree). The vast majority of TE nests in D. melanogaster (263/294, 89.4%) are composed of simple 'primary' nests with a maximal path length of one, consisting mainly of one (203/263, 77.2%) or sometimes greater than one (60/263, 22.8%) inner TE nesting into a single outer TE. Of the 31 nests with more complex nesting relationships, 25 have a maximal path length of two ('secondary' nests), and only 6 have a maximal path length of three ('tertiary' nests) (Figure 4). Relative to the proportion of the genome in each compartment, nests are highly enriched in pericentromeric regions (215/294, 73.1%), as well as on the fourth chromosome (27/294, 9.2%), but rare in non-pericentromeric regions (52/294, 17.7%).

The nesting graph at the individual level provides details about the structures of all TE nests in the genome, but since individual TEs are members of distinct families nesting relationships can also be analyzed at the family level by relabeling nodes with family identifiers and collapsing redundant edges. Recasting the same set of TEs as a nesting graph at the family level provides novel means to study the physical proximity and historical co-existence of all TE families at a global level. Nesting relationships at the family level form a highly connected cyclic graph of 110 nodes and 334 edges (Figure 5), involving the vast majority (90.1%) of the 121 TE families represented in the Release 4 genome annotation. This result implies that nested TEs provide paths of sequences that connect virtually all families, and that a large diversity of novel chimeric sequences between different families exists in the junction regions between TEs in nests. Most TE families (80/110, 72%) are internal nodes in the graph acting as both inner and outer components of nests, with only 22 source families and 8 sink families. The majority of families (97/110, 88%) also form nested relationships with more than one other family. Fifteen families have members that transpose into another member of same family, forming self-loops (or cycles) in the graph. Self-nests require a genomic copy to be present into which another family member can insert, and are consistent with multiple bursts of transposition for a given family or a burst that extends over multiple host generations.

Directed cycles other than those from self-nests were also observed in the family-level nesting graph, clearly indicating either continuous or discrete periods of overlapping transpositional activity for different TE families in the lineage leading to D. melanogaster. Exhaustive enumeration detected 12 distinct cycles of length two (A→B→A), and 43 distinct cycles of length three (A→B→C→A), in the family-level nesting graph, with tens of thousands of distinct cycles of length less than ten. The complexity of the family-level nesting graph is such that it is not feasible to enumerate all cycles in reasonable time; however, a set of independent cycles that do not use the same edge can be extracted efficiently. Figure 6 shows the set of edge-disjoint cycles of length greater than three in the family-level nesting graph, and provides examples of the complex periods of contemporaneous TE activity that must be invoked to explain the global pattern of nesting at the family level. These procedures detect many novel examples of nesting among families, in addition to classical examples such as NOF→FB nesting 5960.

The complexity of nesting among TEs observed at the individual and family levels simplifies at the class level (Table 3). The nesting graph at the class level is complete save for events involving the rare Foldback (FB) class of TEs, with instances of all possible types of nesting between LTR, non-LTR, TIR and INE-1 elements observed in the genome. The most frequent type of nesting event at the class level is LTR→LTR (151/491, 30.7%) and LTR elements form both the most frequent inner (233/491, 47.4%) and outer (207/491, 42.1%) components of nests, extending the finding based on Release 3 that LTR elements are most often involved in nests or clusters 2. The rank order of abundance for both inner and outer members of nests is LTR > non-LTR > TIR > INE-1 > FB, which follows the trend for amount of TE sequence in the genome by class rather than number of TEs, indicating that target size influences class level nesting patterns (Table 1). The observed number of nests for pairwise combinations of classes departs significantly from the random expectation based on the marginal counts of inner and outer nests for each class (χ² = 144.9, 16 degrees of freedom (df), p < 10^-16). Non-random patterns of nesting are observed just for the three major classes of TEs (χ² = 81.8, 4 df, p < 10^-16), suggesting that neither the low FB counts nor undue influence by INE-1 causes this pattern. The non-random pattern of nesting appears in large part to be the result of preferences for TEs to nest in other TEs of the same class, which may represent some sort of a 'homing effect' mediated through protein complexes shared by the TEs belonging to the same class. Alternatively, the non-random pattern of nesting among TE classes may also result from complex historical factors, including the total amount of pre-existing TE sequence present in the genome as targets for insertion, and/or a non-random order of transpositional events among families and classes of TEs.

The nature of the transition zone between euchromatin and heterochromatin in D. melanogaster has been the subject of much controversy, in part because heterochromatic regions (as defined in mitotic chromosomes) can be further subdivided into α-heterochromatin and β-heterochromatin 61. β-heterochromatic regions are cytologically visible in polytene chromosomes, although their banding pattern is 'diffuse' or 'mesh-like,' suggesting under-replication relative to the finely banded euchromatic regions (reviewed in 28). Under-replicated regions are observed elsewhere in polytene chromosomes and co-localize with regions referred to as 'weak points' or 'intercalary heterochromatin' that form ectopic contacts and are subject to chromosome breakage 6263. The amount and degree of polytenization in β-heterochromatic regions is subject to both environmental and genetic factors 64, as most conclusively shown by the appearance of several large banded regions in the chromocenter of salivary gland chromosomes of the Su(UR) mutant 65. Charlesworth et al. estimate that 10% of the D. melanogaster genome is composed of β-heterochromatin 24 and large amounts of β-heterochromatic DNA are found in pericentromeric regions of most (but not all) chromosome arms 2728, a fraction of which is captured in the Release 4 genome sequence (Figure 1).

Analysis of the first draft of the D. melanogaster genome sequence offered the first glimpse of the contiguous molecular organization of β-heterochromatin, and suggested that "there is no clear boundary between heterochromatin and euchromatin" but rather that the transition is characterized by "a gradual increase in the density of transposable elements and other repeats" 66. The view that the β-heterochromatic regions exhibit a gradual increase in TE density has been subsequently reiterated 2567, although our results call this view into question for three of the five major chromosome arms. Far from a gradual transition, our high-resolution TE annotation provides evidence for discretely localized regions of extremely high TE density at the base of chromosome arms X, 2L and 2R overlain on a background of increased TE abundance, such that the increase in TE content is not monotonic in the direction of the centromere. This result represents the inverse of, and provides an explanation for, previous observations that the distribution of genes on these chromosome arms alternates between low and high density in the centromere proximal direction 6667. We note that the alternating pattern of high and low TE (versus unique) sequences reported here in β-heterochromatic regions differs from the 'islands' of complex (TE) sequences surrounded by 'seas' of satellite DNA observed deeper in α-heterochromatic regions 68.

How does the ragged, punctate pattern of TE density affect the interpretation of the transition zone between euchromatin and heterochromatin? If TE density is directly responsible for heterochromatin formation, then such discrete regions of extreme TE density may argue against a gradual transition between euchromatin and heterochromatin, and would support the model of Lifschytz 69, who suggested several distinct transitions between euchromatin and heterochromatin in cytological divisions 19-20 of the X chromosome. However, as noted by Yamamoto et al. 70, the interpretation of multiple, discrete transitions between euchromatin and heterochromatin by Lifschytz 69 was based indirectly on the distribution of X-ray induced deletions, rather than direct cytological evidence for heterochromatic properties. We integrated our annotation of HDRs in the Release 4 genome sequence with the genetic map of Lifschytz 69 and found a striking correspondence between our HDRs and 'hotspots' for X-ray induced deletions in his analysis. Based on the few complementation groups that can be mapped to the genome (A112 = Hlc, M67 = fog, and X-3 = stn), we hypothesize that our HDRs 2, 3, 4, 5, 6, and 7 correspond to hotspot intervals 18, 12, 11, 9, 7, and 6, respectively, in 69 (Figure 7). The major hot-spot for X-ray induced breakage in this region (interval 11 in 20A) most likely corresponds to HDR4, which we find to be the region of highest TE density in the genome sequence. Together, these results suggest that the Lifschytz 69 data may simply reflect preferential breakpoint use in TE-rich regions devoid of genic function, rather than multiple distinct transitions between euchromatin and heterochromatin. These conclusions support those of Ashburner et al. 71, who showed that the distribution of rearrangement breakpoints in the Adh region correlates with the amount of DNA in an interval rather than with any property of the sequence itself.

Further evidence that discrete regions of extreme TE density outside of β-heterochromatic regions may have unusual cytological properties can be found on chromosome 2. Discrete HDRs can be observed in the vicinity of the Histone cluster in 39E (HDRs 9+10) and just distal to the major tRNA cluster at 42A (HDR13). Both of these regions are known to be 'weak points' in polytene chromosomes, which form breaks and ectopic contacts with other weak points in the genome that are alleviated by the Su(UR) mutation, suggesting that these regions are under-replicated in polytene chromosomes 65. These observations, together with the generally poor banding patterns in high TE density pericentromeric regions and on the fourth chromosome, suggest that high TE density may directly interfere with the process of polytenization, either through stalling replication forks 72 or through DNA elimination 73. Thus, high TE densities may not be directly responsible for heterochromatin formation per se, but may simply inhibit the ability to detect bona fide euchromatic regions that are TE dense, at least in salivary gland polytene chromosomes. The formation of large blocks of TE-rich, banded material deep in heterochromatic regions in under-replication suppressing strains like Su(UR) supports this view 6574. Moreover, if regions of high TE density affect polytenization, ectopic contact among 'weak points' may occur via homology between sequences of the same TE family. Additionally, the inherent mobility of TEs provides a mechanism to explain differences in the presence or absence of β-heterochromatin on homologous chromosome arms among Drosophila species 28.

Although the predominant organization of middle repetitive DNA such as TE sequences in D. melanogaster is characterized by individu