Cytogenetic Analysis of the Third Chromosome Heterochromatin of Drosophila melanogaster

Corresponding author: Patrizio Dimitri, Università di Roma “La Sapienza,” P.le Aldo Moro 5, 00185 Rome, Italy., patrizio.dimitri{at}uniroma1.it (E-mail)

Communicating editor: K. GOLIC

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Previous cytological analysis of heterochromatic rearrangements has yielded significant insight into the location and genetic organization of genes mapping to the heterochromatin of chromosomes X, Y, and 2 of Drosophila melanogaster. These studies have greatly facilitated our understanding of the genetic organization of heterochromatic genes. In contrast, the 12 essential genes known to exist within the mitotic heterochromatin of chromosome 3 have remained only imprecisely mapped. As a further step toward establishing a complete map of the heterochomatic genetic functions in Drosophila, we have characterized several rearrangements of chromosome 3 by using banding techniques at the level of mitotic chromosome. Most of the rearrangement breakpoints were located in the dull fluorescent regions h49, h51, and h58, suggesting that these regions correspond to heterochromatic hotspots for rearrangements. We were able to construct a detailed cytogenetic map of chromosome 3 heterochromatin that includes all of the known vital genes. At least 7 genes of the left arm (from l(3)80Fd to l(3)80Fj) map to segment h49–h51, while the most distal genes (from l(3)80Fa to l(3)80Fc) lie within the h47–h49 portion. The two right arm essential genes, l(3)81Fa and l(3)81Fb, are both located within the distal h58 segment. Intriguingly, a major part of chromosome 3 heterochromatin was found to be "empty," in that it did not contain either known genes or known satellite DNAs.

CONSTITUTIVE heterochromatin represents a considerable portion of the genome in higher eukaryotes that can comprise specific chromosomal segments or even entire chromosomes. Heterochromatin exhibits similar cytogenetic and molecular properties in animals and plants. Its characteristics include late DNA replication, a relatively compact state throughout most of the cell cycle, low levels of genetic recombination, an enrichment of repeated DNA sequences (satellite DNAs and transposable elements), and a low density of genes (reviewed in GATTI and PIMPINELLI 1992 ; WEILER and WAKIMOTO 1995 ; ELGIN 1996 ; WALLRATH 1998 ; ZHIMULEV 1998 ).

The heterochromatin of Drosophila melanogaster accounts for 33% of the length of mitotic chromosomes (GATTI et al. 1976 ). In the last two decades, high resolution chromosome banding techniques have rendered the heterochromatin of Drosophila mitotic chromosomes amenable to cytogenetic studies. In particular, this material has been differentiated into 61 regions (h1–h61). Y chromosome heterochromatin is subdivided into 25 regions (h1–h25), while that of the X chromosome contains 9 regions (h26–h34). As for the autosomal heterochromatin, chromosomes 2 and 3 are both subdivided into 12 regions (h35–h46 and h47–h58), while chromosome 4 contains only 3 regions (h59–h61; GATTI et al. 1994 ). A detailed cytogenetic map of the heterochromatin of chromosomes X, Y, and 2 has been constructed, which relates these regions to the location of satellite DNAs and transposable elements (LOHE et al. 1993 ; PIMPINELLI et al. 1995 ; MAKUNIN et al. 1999 ; ABAD et al. 2000 ), the breakpoints of several chromosome rearrangements and the positions of genes and other functional elements. In particular, about 30 genetic loci have been localized to the mitotic heterochromatin of those chromosomes. The X chromosome contains the following loci: the bb gene (RITOSSA and SPIEGELMAN 1965 ); the meiotic pairing site col (MCKEE and LINDSLEY 1987 ); cr, which modulates the replication of ribosomal RNA genes (HILLIKER and SHARP 1988 ); Rex and Su(Rex), affecting the crossing over between two nucleolar organizers at the same chromosome (RASOOLY and ROBBINS 1991 ); Zhr, restoring viability of interspecific hybrids of Drosophila (SAWAMURA and YAMAMOTO 1993 ); and, finally, components of the ABO (PIMPINELLI et al. 1985 ) and crystal-Stellate (PALUMBO et al. 1994 ) systems. Within the entirely heterochromatic Y chromosome have been mapped six male fertility factors (GATTI and PIMPINELLI 1983 ), the bb gene (RITOSSA and SPIEGELMAN 1965 ), the meiotic pairing site col (PIMPINELLI et al. 1985 ), and genetic elements of the ABO (PIMPINELLI et al. 1985 ) and crystal-Stellate (LIVAK 1990 ) systems. Finally, chromosome 2 heterochromatin contains 16 vital genes (DIMITRI 1991 ), as well as the E(SD) and Rsp loci of the Segregation Distortion system (PIMPINELLI and DIMITRI 1989 ; DIMITRI 1991 ), and components of the ABO system (PALUMBO et al. 1994 ).

The cytogenetic map of chromosome 3 mitotic heterochromatin has been less extensively determined and, to date, includes only a detailed localization of satellite DNAs and transposable elements (ABAD et al. 1992 , ABAD et al. 2000 ; LOHE et al. 1993 ; CARMENA and GONZALES 1995 ; PIMPINELLI et al. 1995 ; LOSADA et al. 1999 ). Although a large number of chromosome rearrangements with breakpoints located within the pericentric polytene chromosome regions 80 and 81 is available (FLYBASE 1999 ), their breakpoints are unknown at the level of mitotic chromosomes. As for genetic functions, a group of 12 vital genes, identified by EMS, P-element, and -ray mutagenesis, have been associated with the heterochromatin of chromosome 3 (MARCHANT and HOLM 1988B ; SCHULZE et al. 2001 ). Three of these genes from the left arm, l(3)80Fh, l(3)80Fi, and l(3)80Fj, have been cloned and found to correspond to single-copy sequences. Interestingly, l(3)80Fh and l(3)80Fj genes appear to be members of the trithorax group (trxG) genes, while the l(3)80Fi gene may have key functions in growth and development (SCHULZE et al. 2001 ). In addition, other single-copy genes such as -Cat, rp21, SCP, DSK, QIII, ziti, Dbp80, and PARP map to regions 80 and 81 (KELLY et al. 1977 ; SINCLAIR et al. 1981 ; KAY et al. 1988 ; NICHOLS et al. 1988 ; ODA et al. 1993 ; DEJ and SPRADLING 1997 ; EISEN et al. 1998 ; HANAI et al. 1998 ), but it is unclear whether or not they are allelic to the genes described by MARCHANT and HOLM 1988B . The mutual arrangement of these genes is inferred from indirect genetic evidence based on the frequency of breakpoints between loci (MARCHANT and HOLM 1988A ), yet their mapping along the mitotic heterochromatin of chromosome 3 remains uncertain.

To fill this gap in our knowledge of chromosome 3 heterochromatin, a series of chromosome rearrangements with presumed heterochromatic breakpoints were mapped to mitotic chromosomes by 4',6-diamidino-2-phenylindole (DAPI) and N-banding techniques. The genetic behavior of some of those rearrangements allowed us to determine the location of genes within the heterochromatin of chromosome 3. Our findings suggest that the functional organization of chromosome 3 heterochromatin is highly discontinuous, with specific segments that are gene rich, while large chromosomal regions contain no essential loci.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks:
Fly stocks used are listed in Table 1. All the stocks were from the Bloomington Stock Center. All the deficiencies used were tested by inter se complementation analysis, and their genetic behavior was found to be fully consistent with the data by MARCHANT and HOLM 1988A . Genetic markers, mutations, and balancer chromosomes are described in LINDSLEY and ZIMM 1992 . Cultures were maintained at 25° on standard cornmeal-sucrose-yeast-agar medium.

Cytology:
Techniques for mitotic chromosome preparations, DAPI, and N staining were described in GATTI et al. 1994 .

Microphotography:
Chromosome preparations were analyzed using a computer-controlled Zeiss Axioplan epifluorescence microscope equipped with a cooled CCD camera (Photometrics, Tucson, AZ). Fluorescence was visualized using the Pinkel No. 1 filter set combination (Chroma Technology, Brattleboro, VT). The fluorescent signals were recorded by IP Spectrum Lab Software and edited with Adobe PhotoShop 5.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cytological map of chromosome 3 heterochromatin:
In previous studies based on DAPI and N-banding techniques, the mitotic heterochromatin of chromosome 3 was subdivided into 12 regions, from h47 to h58 (GATTI et al. 1994 ). Our investigations substantially confirm these earlier observations. DAPI staining reveals a bright fluorescent band (h48), three moderately fluorescent regions (h52, h54, and h56) and six dull fluorescence regions (h47, h49, h50, h51, h55, and h58). Region h57 is DAPI negative, N-banding positive, while the centromere-containing region h53 is negative with both DAPI and N-banding staining (Fig 1, a and b). We differ with the previous report only in that we designate region h50 as a dull fluorescent region less bright than h52 (Fig 1A, and particularly in Fig 2A), while both these regions were previously regarded as moderately fluorescent. Most of the blocks can be clearly distinguished on prometaphase chromosomes. However, the small regions h47, h49, h51, and h55 are not easily detected, even in very elongated wild-type chromosomes. Moreover, in more condensed chromosomes adjacent bands such as h54–h56 cannot be resolved (see, for example, Fig 2G and Fig 3C).

View larger version (44K): In this window In a new window Download PPT slide   Figure 1. Heterochromatin banding pattern of a wild-type chromosome 3. (a) DAPI (top) and sequential N-banding (bottom) staining of chromosome 3 from the Oregon-R strain; (b) diagram showing the distribution of DAPI (top) and N-banded (bottom) differentially stained regions (from GATTI et al. 1994 ); black color and hues of gray color correspond to the intensity of staining; (c) deficiency mapping of genetic loci in chromosome 3 heterochromatin (from MARCHANT and HOLM 1988A ).

View larger version (99K): In this window In a new window Download PPT slide   Figure 2. Mapping of the deficiencies. (a) Df(3L)2-66; (b) Df(3L)9-56; (c) Df(3L)1-16; (d) Df(3L)3-52; (e) DAPI staining and (f) N staining of Df(3R)10-65; (g) Df(3L)2-30; (h) Df(3L)6-61; (i) DAPI staining and (j) N staining of Df(3R)4-75; (k and l) DAPI staining and (m) N staining of Df(3L)6B-29 + Df(3R) 6B-29; (n) Df(3L)10-26 + Df(3R)10-26.

View larger version (98K): In this window In a new window Download PPT slide   Figure 3. Mapping of the inversions. (a) In(3R)hbbs23; (b) In(3LR)A114; (c) In(3L)C90; (d) In(3LR)gvU; (e) In(3R)258; (f) In(3R)GC18C23R; (g) In(3R)ScrWrv5; (h) DAPI staining and (i) N staining of In(3LR)bxd194.

Cytogenetic analysis of chromosomal rearrangements:
For this study we selected both deficiencies genetically assigned to heterochromatin (MARCHANT and HOLM 1988A ; see the scheme in Fig 1C) and inversions with breakpoints mapping to regions 80 and 81 of polytene chromosomes (FLYBASE 1999 ). Although almost all of these rearrangements were previously thought to be simple, with only two breakpoints, our observations showed that several chromosomes carrying deficiencies also contain inversions and duplications within heterochromatin. Due to the method by which the deficiencies were generated they may also carry duplications of heterochromatic material of chromosome 3 (MARCHANT and HOLM 1988A ), providing a possible explanation for the presence of additional blocks of heterochromatin.

Deficiencies: Df(3L)2-66 (Fig 2A) and Df(3L)9-56 (Fig 2B) clearly affect the dull fluorescent region h50. The clearest evidence for this interpretation comes from comparison of the 3Lh portion of both deficiencies with the corresponding, wild-type-appearing region of the TM3 balancer in the same nucleus (see the comparison in Fig 2A). Although the 3Lh regions spanning h48 to h52 are present in TM3, h48 and h52 are physically contiguous in 2-66, indicating that at least a major portion of h50 is absent. The small h49 and h51 neighboring bands are not easily detectable; thus it is uncertain whether or not they are affected by these deletions.

Chromosomes carrying Df(3L)1-16 clearly lack region h48. In addition, distal to h52 a dull fluorescent material (indicated by arrow) is present that may derive from regions h47 and/or h49–h51 (Fig 2C). Df(3L)3-52 is a complex rearrangement in that only three equally fluorescent blocks are seen in 3Lh whose brightness is comparable to that of h52 (Fig 2D). We believe that one of these blocks corresponds to the original block h52, while the segment spanning from h48 to h50 is replaced by unknown material. However, taking into account the very low brightness of h50, we cannot rule out that a residual portion of this region is still retained. The actual identity of the duplicated material cannot be unambiguously determined, but based on its brightness it is conceivable that it derives from h52.

DAPI and N-banding staining of Df(3R)10-65 showed that this chromosome carries full-sized h54, h55, h56, and h57 regions (Fig 2E and Fig F), while h58 is considerably reduced in comparison with wild type, suggesting that 10-65 removes a major part of h58.

The analysis of Df(3L)8A-80 (data not shown) did not reveal any significant changes in the heterochromatin pattern, although this rearrangement uncovers two heterochromatic genes (see Fig 1C). The size of this deficiency is therefore below the level of resolution of our cytological analysis. We also failed to detect any obvious deletion in the heterochromatic pattern of the Df(3L)2-30, Df(3L)6-61, and Df(3R)4-75 chromosomes. We did, however, detect other cytological changes in these chromosomes. Deficiency 2-30 carries an additional heterochromatic block, located between h48 and h50, which exhibits a fluorescence intensity similar to that of region h52 (Fig 2G). Although the actual nature of this block cannot be unambiguously identified, it is possible that it corresponds to h52. Deficiency 6-61 carries an inversion of region h52–h53 (Fig 2H). Finally, the analysis of 4-75 suggested that this rearrangement actually corresponds to a long pericentric inversion with one breakpoint located distal to h50 and the other falling within h58 (Fig 2I and Fig J).

The analysis of the available deficiencies was completed by the characterization of Df(3L)6B-29 + Df(3R) 6B-29 and Df(3L)10-26 + Df(3R)10-26. Genetic analysis has shown that these chromosomes simultaneously lack genes from heterochromatin of 3L and 3R (MARCHANT and HOLM 1988A ; see Fig 1C). The cytological analysis of deficiency 6B-29 showed that it indeed lacks both 3Lh and 3Rh regions. In 3Lh the h47–h48 portion is apparent and is adjoined by residual dull fluorescent material. The fluorescence intensity of this material suggests that it may correspond to h49 together with a small portion of h50. Finally, region h52 is clearly absent, as well as the entire 3Rh, including most of h58. This deficiency retains at least a portion of h53 that contains the centromere (Fig 2K, Fig L, and Fig M). Df(3L)10-26 + Df(3R)10-2 has a complex pattern, the origin of which is difficult to trace back (Fig 2N). In 3Lh we were able to distinguish h48, which is partially deleted, and h53. In between these regions a small dull fluorescent block is present. In 3Rh, beyond h53, a dull fluorescent region of unknown origin is also evident, which is followed by a block possibly corresponding to h54–h56 (Fig 2N). The h57 and h58 regions are clearly absent (data not shown).

Inversions: The analysis of In(3L)hb^bs23 (Fig 3A) and In(3LR)A114 (Fig 3B) showed that these rearrangements share the same heterochromatic breakpoint located within region h49. The heterochromatic breakpoints of both In(3L)C90 (Fig 3C) and In(3LR)gv^U (Fig 3D) map to h51. In addition, the latter rearrangement appears to be a paracentric inversion, although it was originally described as pericentric. Among the right arm inversions, the heterochromatic breakpoints of In(3R)258 and In(3R)GC18^C23^R map to h55 (Fig 3E and Fig F). The breakpoint of the short inversion In(3R)Scr^Wrv5 is located in the proximal part of h58 (Fig 3G). In(3LR)bxd¹⁹⁴ turned out to be a complex rearrangement. Three heterochromatic segments designated as I, II, and III in Fig 3H separated by euchromatic portions are apparent in bxd¹⁹⁴ (Fig 3H and Fig I). Segment I consists of two brightly fluorescent regions, segment II corresponds to a dull fluorescent block, while segment III carries one moderately and one dull fluorescent region, as well as an N-banded block that corresponds to h57. The actual pattern of this chromosome cannot be unequivocally determined; however, it is conceivable that segment III corresponds to 3Rh, while segments I and II are derived from 3Lh.

Gene distribution within chromosome 3 heterochromatin:
The cytological characterization of chromosome rearrangements performed in this study allowed us to map the heterochromatic genes of chromosome 3 with respect to differentially stained heterochromatic bands (Fig 4; Table 1). Unfortunately, the heterochromatin of chromosome 3 has a single N-banded region and several blocks with similar size and fluorescent intensity. This can represent a limitation in the determination of precise cytological extension of some deletions and consequently in the mapping of heterochromatic genes.

View larger version (21K): In this window In a new window Download PPT slide   Figure 4. Scheme summarizing the cytogenetic data on chromosome 3 heterochromatin obtained in this study and elsewere (LOHE et al. 1993 ; PIMPINELLI et al. 1995 ; LOSADA et al. 1999 ; ABAD et al. 2000 ). Gray and black lines indicate, respectively, the largest and the smallest limits of the deficiencies. (a) Flags indicate breakpoints of eu-heterochromatin inversions. Direction of the flags indicates the location of the second breakpoint. Brackets show heterochromatic inversions. Lines below designate the extent of the deficiencies. (b) Gene distribution within chromosome 3 heterochromatin. (c) Mapping of highly (top) and moderately (bottom) repeated DNAs in chromosome 3 heterochromatin.

Genes in 3Lh: The comparison of rearrangements 1-16, 2-66, 9-56, and 6B-29 allowed us to map the 10 known genes in 3Lh. Given the cytological complexity of 3-52, this rearrangement was not used for the purpose of this study. As shown in Fig 4, the 3 most distal genes, l(3)80Fa, l(3)80Fb, and l(3)80Fc, map to the segment h47–h49, between the distal borders of 1-16 and 6B-29, while the cluster of 3 most proximal genes, l(3)80Fh, l(3)80Fi, and l(3)80Fj, is included between the proximal borders of 1-16 and 2-66 and thus maps to the h50p–h51 portions.

The mapping of the remaining four genes, l(3)80Fd, l(3)80Fe, l(3)80Ff, and l(3)80Fg, rests upon the comparison between 1-16 and 6B-29. These chromosomes genetically lack all four genes and cytologically overlap at the level of the distal part of h50 (h50d). This suggests that l(3)80Fd, l(3)80Fe, l(3)80Ff, and l(3)80Fg map to h50d. However, this mapping apparently conflicts with the behavior of 2-66 and 9-56. These deletions genetically lack only l(3)80Fh, l(3)80Fi, and l(3)80Fj, while at the cytological level they overlap distally with both 1-16 and 6B-29. We were not able to evaluate exactly the right extent of these deletions or their positioning within the h49–h51 segment. One possible explanation for this apparent inconsistency is that deficiencies 2-66 and 9-56 are indeed located more proximally within h50 and do not affect h49 at all. That would place l(3)80Fd, l(3)80Fe, l(3)80Ff, and l(3)80Fg within the h49-h50d region in agreement with the comparison between 1-16 and 6B-29.

It is important to point out that some of the genes shown in clusters can be genetically resolved by specific deficiency breakpoints. For example, l(3)80Fh is separated from l(3)80Fi and l(3)80Fj by the breakpoint of 9-56. However, since the breakpoints of 9-56 and 2-66 are cytologically indistinguishable, it has not been possible thus far to establish whether l(3)80Fh cytologically maps to the left or to the right of the cluster. Further work will be needed to improve the resolution of the cytological analysis and to provide a more detailed mapping of the genes in 3Lh, for example, by characterizing the breakpoints at the level of extended chromatin fibers or after treatment with Hoechst 33258, which is known to induce decondensation of 3L heterochromatin (PIMPINELLI et al. 1975 ).

Genes in 3Rh: We were able to determine the location of l(3)81Fa and l(3)81Fb, the two genes genetically mapped in 3Rh, by analyzing 4-75, 10-65, 6B-29, and 10-26. We found that 10-65, 6B-29, and 10-26, three deletions that genetically lack the l(3)81Fa gene, all affect the h58 segment. In particular, 10-65, the shortest deficiency among those of 3Rh, removes the largest part of h58. This suggests that l(3)81Fa lies somewhere within h58; subsequently, l(3)81Fb must be located within h58 as well or, alternatively, in 3R proximal euchromatin. The analysis of 4-75 allowed us to better define the location of l(3)81Fb. This chromosome, which genetically uncovers lethal alleles of both l(3)81Fa and l(3)81Fb (Fig 1C), carried a pericentric inversion with a 3Rh breakpoint within h58 (Fig 2I and Fig J). The simultaneous inactivation of both l(3)81Fa and l(3)81Fb in 4-75 may result from a cytologically undetectable rearrangement associated with the breakpoint or from position-effect variegation due to changes in the amount of the heterochromatin in cis, as shown for 2Lh vital genes (HOWE et al. 1995 ). The data suggest that l(3)81Fb is located within h58 (Fig 4B).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The main result of this study is the mapping of 12 vital genes within the heterochromatin of chromosome 3 (Fig 4). Three distal genes from 3Lh map within segment h47–h49, while the remaining 7 proximal genes map to regions h49–h51. Both genes from 3Rh map to region h58. The distribution of these genes within heterochromatin correlates only partially with that based on the frequency of rearrangement breakpoints (MARCHANT and HOLM 1988A ). In particular, we have mapped 3 genes, l(3)80Fh, l(3)80Fi, and l(3)80Fj, in the middle of 3Lh, while according to MARCHANT and HOLM 1988A these genes are located within the most proximal 15% of 3L heterochromatin. Our present observations on the distribution of breaks within heterochromatin may contribute to reconciling this apparent discrepancy. We have found that heterochromatic breakpoints of randomly chosen inversions preferentially occur within regions h49, h51, h55, and h58. The occurrence of hotspots for breakpoints within heterochromatin (LIFSCHYTZ 1978 ) and/or the preferential recovery of rearrangements may underlie such a nonrandom distribution. For example, heterochromatic regions may be differentially sensitive to breakage events, or, alternatively, rearrangements with breakpoints that may inactivate aplo-insufficient loci would fail to be recovered. Whichever the responsible mechanism, it is clear that the frequency of breaks between heterochromatic loci cannot be taken as a measure of the physical distances separating them.

Our investigations reveal that the cytogenetic organization of chromosome 3 heterochromatin is similar to that of chromosome 2 (DIMITRI 1991 ), but it differs from that of sex chromosome heterochromatin. As in the case of heterochromatic vital genes of chromosome 2, we found that the vital genes mapping to the heterochromatin of chromosome 3 are clustered within discrete blocks, but do not correspond to the entire blocks themselves. Thus, the genes of chromosome 3 heterochromatin appear to be not as large as the Y chromosome fertility factors or the repetitive loci from the X chromosome heterochromatin (GATTI and PIMPINELLI 1983 ). Our findings concur with HILLIKER's (1976) suggestion and with subsequent molecular data showing that several vital genes in the autosomal heterochromatin, such as l(3)80Fh, l(3)80Fi, and l(3)80Fj from chromosome 3 and lt, cta, rl, and Nipped-B from chromosome 2, correspond to single-copy sequences (DEVLIN et al. 1990 ; PARKS and WIESCHAUS 1991 ; BIGGS et al. 1994 ; ROLLINS et al. 1999 ; SCHULZE et al. 2001 ). Although Y chromosome contains at least one single-copy gene mapped to the kl-5 fertility factor and encoding axonemal dynein (GEPNER and HAYS 1993 ), the structure of the gene is complex, with blocks of satellite DNA within huge introns (KUREK et al. 2000 ). In contrast, the X chromosome heterochromatin apparently does not contain any single-copy genes, while it harbors several repetitive loci such as bb, ABO, Ste, and SCLR (RITOSSA and SPIEGELMAN 1965 ; NURMINSKY et al. 1994 ; PALUMBO et al. 1994 ; TULIN et al. 1997 ).

Our cytogenetic analysis also implies that essential genes are not uniformly distributed throughout the heterochromatin of chromosome 3. In fact, most of the genes appear to be excluded from brightly and moderately fluorescent blocks as well as from N-banded regions. Interestingly, most of the autosomal heterochromatic genes thus far detected are located in dull fluorescent regions. Possible exceptions may be represented by l(2)41Ab and l(2)41Ad of chromosome 2 and perhaps by l(3)80Fa, l(3)80Fb, and l(3)80Fc of chromosome 3. The l(2)41Ab gene lies between the h39 DAPI-bright region and the N-band h40, l(2)41Ad maps to the moderately fluorescent h44 block (DIMITRI 1991 ), while l(3)80Fa, l(3)80Fb, and l(3)80Fc may be located in the bright fluorescent region h48. In addition to single-copy vital genes, dull fluorescent regions harbor several clusters of transposable elements, but they are devoid of satellite DNAs (PIMPINELLI et al. 1995 ; CARMENA and GONZALES 1995 ; DIMITRI 1997 ; Fig 4C). For example, segment h49–h51, which contains at least seven 3Lh vital genes, harbors clusters of known retroelements such as Doc, I, F, blood, gypsy, and copia. Autosomal heterochromatin is thus genetically and molecularly heterogeneous, with complex DNA regions composed of unique coding genes and transposable elements that alternate with blocks enriched in highly repeated, noncoding satellite DNAs.

It has been estimated that the density of single-copy genes in heterochromatin is 1% of the density of genes within euchromatin (HILLIKER et al. 1980 ). This estimate is consistent with recent molecular data. The D. melanogaster genome contains 14,000 euchromatic genes and consists of 216.6 Mb of DNA (ADAMS et al. 2000 ). Euchromatin consists of 119.8 Mb, while the remaining 96.8 Mb correspond to heterochromatin, with the heterochromatic portions of chromosomes 2 and 3 accounting for 32.8 Mb. It follows that 1 Mb of euchromatin contains on average 117 genes (14,000/119.8), while only 0.8 genes (28/32.8) are found per 1 Mb of autosomal heterochromatin. Thus, the density of genes in heterochromatin is 0.7% of that in euchromatin. Because the distribution of genes within heterochromatin is not uniform, gene density in those regions of heterochromatin containing genes is somewhat higher. On the basis of the DNA content of autosomal heterochromatin (ADAMS et al. 2000 ) we estimated that the h49–h51 segment from chromosome 3 and the h35 region of chromosome 2 contain roughly 3 and 2 Mb of DNA, respectively. Together these regions, where most of the vital genes are clustered, contain at least 15 vital genes, corresponding to a gene density of 3% of that calculated for euchromatin.

Several factors might account for the low number of genes found in heterochromatin. First, heterochromatic genes can be larger than euchromatic ones. This has been previously shown for the Y chromosome, which contains a few extremely large genes (GATTI and PIMPINELLI 1992 ; KUREK et al. 2000 ), and is also true for the rolled gene, which was shown to span 80 kb (W. BIGGS and K. ZAVITZ, unpublished results). Second, classical mutagenesis methods might not allow the detection of all genes that exist in heterochromatin and hence alternative approaches may be needed to reveal the full complement of heterochromatic genes. This is certainly true for D. melanogaster euchromatin, where, for example, the X chromosome band 10A1-2 and the Adh gene region were found to contain more genes than those determined by genetic analysis (KOZLOVA et al. 1997 ; ASHBURNER et al. 1999 ), and can be also true for heterochromatin. Indeed, many predicted genes were identified in the pericentric regions of autosomes as well as in the Y chromosome (ADAMS et al. 2000 ; CARVALHO et al. 2000 ). Additional "cryptic" heterochromatic genes that are refractory to conventional analysis, like ABO and Rsp, might also exist (reviewed by GATTI and PIMPINELLI 1992 ).

In our map of chromosome 3 heterochromatin (Fig 4), the portion from h52 to h57 appears to be devoid of genetic loci. In addition, while transposable elements are widely distributed throughout chromosome 3 heterochromatin (PIMPINELLI et al. 1995 ; CARMENA and GONZALES 1995 ), satellite DNAs have been found only in regions h48–h49, h53, and h57 (LOHE et al. 1993 ; LOSADA et al. 1999 ; ABAD et al. 2000 ). Thus, both the genetic and molecular composition of the vast majority of chromosome 3 heterochromatin is unknown. Those regions may contain still undetected classes of highly repetitive DNAs, as well as novel genetic elements. Further cytogenetic and molecular studies, including analysis of the eu-heterochromatin junctions of chromosome rearrangements (HOWE et al. 1995 ; MAKUNIN et al. 1999 ), as well as the characterization of microdissected heterochromatin (Yu. M. MOSHKIN, S. N. BELYAKIN, N. B. RUBTSOV, E. B. KOKOZA, A. A. ALEKSEYENKO, E. I. VOLKOVA, E. S. BELYAEVA, I. V. MAKUNIN, P. SPIERER and I. F. ZHIMULEV, personal communication), should provide further insight into the nature of the apparently empty heterochromatin of chromosome 3.

	FOOTNOTES

This paper is dedicated to the memory of Franco Tatò.

	ACKNOWLEDGMENTS

We thank Elena S. Belyaeva, Silvia Bonaccorsi, Patrizia Lavia, and Mike Goldberg for critical reading of the manuscript and helpful comments. This work was partially supported by grants from the International Association for the promotion of cooperation with scientists from the New Independent States of the former Soviet Union (YSF-98-82, INTAS-99-1088), Russian State Programs Frontiers in Genetics (99-2-020), Russian Foundation for Basic Research (99-04-49270, 00-15-97984), and Ministero dell'Università e della Ricerca Scientifica e Tecnologica.

Manuscript received July 31, 2001; Accepted for publication November 5, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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