From First Base: The Sequence of the Tip
of the X Chromosome of Drosophila melanogaster,
a Comparison of Two Sequencing Strategies
Panayiotis V. Benos,1,15 Melanie K. Gatt,2,11 Lee Murphy,3 David Harris,3
Bart Barrell,3 Concepcion Ferraz,4 Sophie Vidal,4 Christine Brun,4
Jacques Demaille,4 Edouard Cadieu,5 Stephane Dreano,5 Stéphanie Gloux,5
Valerie Lelaure,5 Stephanie Mottier,5 Francis Galibert,5 Dana Borkova,6
Belen Miñana,6 Fotis C. Kafatos,6 Slava Bolshakov,6,7 Inga Sidén-Kiamos,7
George Papagiannakis,7 Lefteris Spanos,7 Christos Louis,7,8 Encarnacio´n Madueño,9
Beatriz de Pablos,9 Juan Modolell,9 Annette Peter,10 Petra Schöttler,10
Meike Werner,10 Fotini Mourkioti,10 Nicole Beinert,10 Gordon Dowe,10
Ulrich Schäfer,10 Herbert Jäckle,10 Alain Bucheton,4 Debbie Callister,11
Lorna Campbell,11 Nadine S. Henderson,11 Paul J. McMillan,11 Cathy Salles,11
Evelyn Tait,11 Phillipe Valenti,11 Robert D.C. Saunders,11,12 Alain Billaud,13
Lior Pachter,14 David M. Glover,2,11 and Michael Ashburner1,2,16
1EMBL Outstation, The European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD,
UK; 2Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK; 3Sanger Centre, Wellcome Trust Genome
Campus, Hinxton, Cambridge, CB10 1SA, UK; 4Montpellier University Medical School, IGH-Institut de Ge´ne´tique
Humaine-CNRS, 34396 Montpellier Cedex 5, France; 5UPR 41, CNRS, Recombinaisons Ge´ne´tiques, Faculte de Medecine,
35043 Rennes Cedex, France; 6European Molecular Biology Laboratory (EMBL), D-69117 Heidelberg, Germany; 7Institute of
Molecular Biology and Biotechnology, FORTH, GR-71110 Heraklion, Greece; 8Department of Biology, University of Crete,
71409 Heraklion, Crete, Greece; 9Centro de Biologı´a Molecular Severo Ochoa, CSIC and Universidad Auto´noma de Madrid,
28049 Madrid, Spain; 10Max-Planck-Institut fu¨r biophysikalische Chemie, Department of Molecular Developmental Biology,
D-37070 Go¨ttingen, Germany; 11Department of Anatomy and Physiology, CRC Cell Cycle Genetics Group, University of
Dundee, Dundee, DD1 4HN, UK; 12Department of Biological Sciences, The Open University, Milton Keynes, MK7 6AA, UK;
13Fondation Jean Dausset-CEPH (Centre d’Etude du Polymorphisme Humain), 75010 Paris, France; 14Department of
Mathematics, University of California at Berkeley, California 94720-3840, USA
We present the sequence of a contiguous 2.63 Mb of DNA extending from the tip of the X chromosome of
Drosophila melanogaster. Within this sequence, we predict 277 protein coding genes, of which 94 had been
sequenced already in the course of studying the biology of their gene products, and examples of 12 different
transposable elements. We show that an interval between bands 3A2 and 3C2, believed in the 1970s to show a
correlation between the number of bands on the polytene chromosomes and the 20 genes identified by
conventional genetics, is predicted to contain 45 genes from its DNA sequence. We have determined the
insertion sites of P-elements from 111 mutant lines, about half of which are in a position likely to affect the
expression of novel predicted genes, thus representing a resource for subsequent functional genomic analysis.
We compare the European Drosophila Genome Project sequence with the corresponding part of the
independently assembled and annotated Joint Sequence determined through “shotgun” sequencing. Discounting
differences in the distribution of known transposable elements between the strains sequenced in the two
projects, we detected three major sequence differences, two of which are probably explained by errors in
assembly; the origin of the third major difference is unclear. In addition there are eight sequence gaps within
the Joint Sequence. At least six of these eight gaps are likely to be sites of transposable elements; the other two
15Present address: Department of Genetics, School of Medicine, Washington University, 4566 Scott Avenue,St. Louis, MO 63110 USA.
16Corresponding author.
E-MAIL m.ashburner@gen.cam.ac.uk; FAX 44-1223-333992.
Article and publication are at www.genome.org/cgi/doi/10.1101/gr.173801.
Letter
710 Genome Research 11:710–730 ©2001 by Cold Spring Harbor Laboratory Press ISSN 1088-9051/01 $5.00; www.genome.org
www.genome.org

are complex. Of the 275 genes in common to both projects, 60% are identical within 1% of their predicted
amino-acid sequence and 31% show minor differences such as in choice of translation initiation or termination
codons; the remaining 9% show major differences in interpretation.
[All of the sequences analyzed in this paper have been deposited in the EMBL-Bank database under the following
accession nos.: AL009146, AL009147, AL009171, AL009188–AL009196, AL021067, AL021086,
AL021106–AL021108, AL021726, AL021728, AL022017, AL022018, AL022139, AL023873, AL023874,
AL023893, AL024453, AL024455–AL024457, AL024485, AL030993, AL030994, AL031024–AL031028,
AL031128, AL031173, AL031366, AL031367, AL031581–AL031583, AL031640, AL031765, AL031883, AL031884,
AL034388, AL034544, AL035104, AL035105, AL035207, AL035245, AL035331, AL035632, AL049535,
AL050231, AL050232, AL109630, AL121804, AL121806, AL132651, AL132792, AL132797, AL133503–AL133506,
AL138678, AL138971, AL138972, and Z98269. A single file (FASTA format) of the 2.6-Mb contig is available
from ftp://ftp.ebi.ac.uk/pub/databases/edgp/contigs/contig_1.fa.]
Less than 90 years have elapsed since Alfred H. Sturte-
vant presented the world with the first-ever genetic
map of six visible markers on the X chromosome
of Drosophila melanogaster (Sturtevant 1913). The ex-
traordinary achievement of determining the en-
tire euchromatic DNA sequence of D. melanogaster
(Adams et al. 2000) now gives us the potential to iden-
tify every single coding region within this gene-rich
region.
The first tentative steps towards sequencing the
complete genome of Drosophila were taken 10 years
ago with the construction of a physical map of the X
chromosome (Side´n-Kiamos et al. 1990; Madueño et al.
1995) and the explicit declaration of the objective of
whole-genome sequencing. Since then, both the Euro-
pean and Berkeley Drosophila Genome Projects (EDGP
and BDGP) (Saunders et al. 1989; Kafatos et al. 1990;
Rubin 1996, 1998; Louis et al. 1997) and, more recently
Celera Genomics, have worked towards the common
goal of completing the sequence of the entire genome
of this fly. An essentially complete sequence of the
euchromatic genome of D. melanogaster has now been
published by the Celera Genomics/BDGP/Baylor Col-
lege of Medicine collaboration with some input from
EDGP; in this paper we call this the Joint Sequence (see
Methods) (Adams et al. 2000; Myers et al. 2000; Rubin
et al. 2000a).
We present an ∼2.7 Mb region accurately se-
quenced and analyzed independently of the Joint Se-
quence. This is only the second detailed molecular
analysis of a genomic sequence of several megabases
from Drosophila, and it offers some interesting con-
trasts with the 3 Mb region of an autosome, whose
analysis has been published recently (Ashburner et al.
1999). It also gives an opportunity to compare the re-
sults and analysis of a sequence obtained by the widely
adopted clone-by-clone approach to those obtained
from the whole-genome shotgun approach adopted by
Celera and their collaborators (Venter et al. 1998). We
also report the collection of ∼6 Mb discontinuous se-
quence from divisions 4 – 10, which was obtained by
sequencing at 1.5-fold coverage a collection of 29 BAC
clones representing a minimal tiling path.
The tip of the X chromosome of D. melanogaster is
a region of some sentimental, as well as much scien-
tific, interest to geneticists. It includes the locus of the
gene white, whose mutation was the first clear visible
mutation found in Drosophila (Morgan 1910) and
whose study led to the discovery of sex-linked inherit-
ance and, hence, to the proof of the chromosome
theory of heredity (Bridges 1916). It also includes a
region, between the genes zeste and white, which was
intensively studied by Burke Judd and colleagues (Judd
et al. 1972) in an attempt to analyze the relationship
between polytene chromosome bands and genes.
There are two classic genetic complexes at the tip of the
chromosome — the achaete-scute complex, whose phe-
notypic effects have long fascinated geneticists and
generated much theoretical speculation (Agol 1929;
Garcı´a-Bellido 1979), and the broad complex (Zhimu-
lev et al. 1995). The physical bases for the complexities
in genetic analysis are quite different in these two cases
(see below). Cytologically, the region includes, of
course, theXL telomere, perhaps the best-characterized
telomere in Drosophila (Biessmann and Mason 1997) as
well as a region of polytene banding complexity that
had indicated to Bridges (1935) the presence of a long
reverse-repeat (Benos et al. 2000).
The main part of the sequence is contiguous, con-
sisting of a single contig of 2,626,764 bp. The rest con-
sists of a cosmid clone (23E12) that contains a number
of Drosophila subtelomeric repeats (EMBL accession no.
L03284) and thus represents the most distal part of the
X chromosome. The two parts are separated by an un-
specified number of repeats, and together amount to
2,664,670 bp.
RESULTS AND DISCUSSION
Linking the Genetic Map of the X Chromosome
to a Molecular Framework
A decade ago, the founding members of the EDGP ar-
gued the case for constructing an accurate physical
map of the genome of D. melanogaster linked to the
genetic map (Side´n-Kiamos et al. 1990). To this end,
cosmid clones were selected by hybridization with
Sequencing the Drosophi la X Chromosome
Genome Research 711
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PCR-amplified DNA microdissected from each of the
100 individual divisions of the major polytene chro-
mosome arms. A physical map was generated by deter-
mining overlaps between the cosmids based on the
shared fragments generated by restriction endonucle-
ase digestion (Sulston et al. 1988). The localization of
cosmids was verified by in situ hybridization to the
polytene chromosomes and by determining STSs of
cosmid end sequences (Louis et al. 1997). This physical
map, and the cosmid library on which it was based, are
available as a public resource (http://www.hgmp.
mrc.ac.uk/Biology/descriptions/drosophila.html).
A physical map was also constructed by the BDGP
(Kimmerly et al. 1996) based on segments of DNA
cloned in a P1 phage vector that were aligned using
PCR based STS content mapping. However, it was clear
that both the cosmid and P1 maps would be an incom-
plete resource for sequencing the genome. Moreover,
although the YAC map of Ajioka et al. (1991) does give
good coverage, in our hands YAC clones were imprac-
tical for DNA sequencing purposes. We therefore un-
dertook to build another map based on BAC clones
because these vectors can, in principle, accommodate
larger inserts of DNA. The generation of these BAC
clones, that give an approximately 10-fold coverage of
the genome, will be described in detail elsewhere. The
library is available as a public resource (http://www.
hgmp.mrc.ac.uk/Biology/descriptions/dros_bac.html).
Clones from both this and a BAC library of partial
EcoRI digestion products of DNA constructed for the
BDGP (Hoskins et al. 2000) were physically ordered
and linked by hybridization with a total of 647 hybrid-
ization probes each of 40 nucleotides in length corre-
sponding to sequences distributed along the length of
the X chromosome. The resulting maps, whose full de-
scription will also be provided elsewhere, allowed us to
determine a minimal tiling path of clones for sequenc-
ing purposes. We selected such a minimal tiling path
extending through polytene divisions 4–10, and deter-
mined the sequence of these clones at ∼1.5-fold cover-
age (http://edgp.ebi.ac.uk/cgi-bin/progress.pl). This
provided a skeletal sequence scan of ∼6 Mb of the chro-
mosome that was made available to the Celera/BDGP/
Baylor shotgun sequencing project for use as an assem-
bly scaffold.
The accurate sequencing of polytene divisions 1–3
was initiated on a minimal tiling path of cosmid
clones, subsequently extended using the BAC clones to
fill gaps in the cosmid map. The clones selected for
sequencing are presented in Figure 1A, and the as-
sembled nonredundant sequence can be directly ac-
cessed at http://edgp.ebi.ac.uk/cgi-bin/progress.pl,
which links to the EMBL-Bank deposits.
General Features of Gene Content
As explained in Methods, we have used two general
classes of computational method to predict genes in
this chromosome region: similarity-based methods
and ab initio methods. Together these two approaches
have enabled us to predict 277 protein-coding genes
overall, of which 94 (33.9%) had been sequenced pre-
viously by the community (Table 1; Figure 1B). A total
of 25 genes (9%) were predicted solely by ab initio
methods, a lower fraction than in the Adh region
(19%). A possible reason for this difference is that we
used a stricter criterion for accepting a gene predicted
only by an ab initio method than did Ashburner et al.
(1999). Of the predicted genes, 205 have matches with
ESTs from the BDGP (Rubin et al. 2000b) and NIH (An-
drews et al. 2000) projects. The fraction of previously
known Drosophila genes that had EST matches (77.1%)
is the same as that of the genes predicted by sequence
similarity (77.2%), and is very similar to the proportion
of matches from the Adh region (71%). Assuming that
the criteria used to predict genes are adequate, these
figures provide a good indication for the proportion of
Drosophila genes currently represented in EST collec-
tions. Presumably the shortfall reflects mainly that the
cDNAs used to generate the ESTs have been derived
from a restricted number of developmental stages. The
value of ESTs in confirming gene identity and splicing
patterns provides a strong argument to extend the gen-
eration of EST data to other developmental stages and
tissues (Andrews et al. 2000; Rubin et al. 2000b). Based
on the analysis of EST hits, we identified nine genes
that are alternatively spliced in their coding regions,
and thus able to direct the synthesis of two or more
different proteins (Table 1, asterisks). It is striking that
of the 183 newly predicted genes, 55% have significant
similarities with sequences in other organisms thus in-
dicating the extent of conserved function.
The average size of the coding regions of the genes
predicted in the tip of the X chromosome is 1.8 Kb,
with 2.7 introns per gene. The gene with the highest
Figure 1 Physical maps of the interval 1A–3C. (A) Minimal tiling pattern of clones sequenced in divisions 1A–3C. BACR clones are
indicated in red; BACN and BACH clones are indicated in green; cosmid clones are indicated in blue; redundant clones sequenced are
indicated in pink; a few small regions were sequenced from other clones, these are indicated in yellow. The BACR, BACN, and BACH clones
are from the same strain as that sequenced by the BDGP and Celera; the cosmids are from a different strain (see Methods). Scale divisions
are 10 Kb. (B) Genes, transposable elements, and P-element insertions in divisions 1A–3C. Known genes are shown in red; genes with
significant protein similarities to nondrosophilid proteins are shown in blue; predicted genes with EST hits are shown in yellow; predicted
genes with no EST hits are shown in green; predicted genes with protein motif matches are shown in pink. Transposable elements are
shown in orange within the sequence coordinate line. The sites of P-element and EP-element insertions are indicated by gray triangles. The
large square brackets from 2100 to 2480 Kb embrace the zeste-white region (Figure 2). Scale divisions are 10 Kb (bold) and 1 Kb (regular).
Benos et al.
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Table 1. Genes Identified or Predicted in the 1A–3C Interval
Cytology Gene symbol Gene HMMER EST
Matching
gene(s)
EDGP
vs. joint
sequence
EG:23E12.1 PF01019: G_glu_transpept GH10105 CG17636 0
EG:23E12.5 GH15984 CG17617 0
EG:23E12.2 PF00169: PH LD22360 CG17960 B

PF00620: RhoGAP
PF00621: RhoGEF
EG:23E12.3 CG17707 0
EG:BACR37P7.1 PF01762: Galactosyl_T CK01556 CG3038 0
EG:BACR37P7.2 PF00856: SET LD10743 CG2995 0
PF00023: ank
1A8 †EG:BACR37P7.3 †cin PF00994: MoCF_biosynth GH09380 CG2945 0
EG:BACR37P7.9 PF00106: adh_short CG13377 A

1A8 EG:BACR37P7.7 ewg AF171732 CG3114 B+
EG:BACR37P7.8 PF00071: ras CG13375 B

EG:BACR37P7.5 bs28b06 CG12470 0
EG:125H10.1 LP06894 CG3777 0
1B1 EG:125H10.2 y CG3757 0
1B1 EG:125H10.3 ac PF00010: HLH CG3796 0
1B2 EG:198A6.1 sc PF00010: HLH CG3827 0
1B3 EG:198A6.2 l(1)sc PF00010: HLH CG3839 0
1B4 EG:EG0001.1 pcl PF00026: asp CG13374 0
1B4 EG:165H7.2 ase PF00010: HLH CG3258 0
EG:165H7.1 Cyp4g1 PF00067: p450 GH20504 CG3972 0
1B4 EG:165H7.3 l(1)Bb LD14543 CG3923 D+*
EG:171D11.6 LD04586 CG13372 D*
CG18166
CG18273
EG:171D11.2 PF00664: ABC_membrane LD18126 CG3156 A

PF00005: ABC_tran
†EG:171D11.1 PF00171: aldedh GM07535 CG17896 0
EG:171D11.5 CG17778 0
1B7 †EG:171D11.3 †svr PF00246: Zn_carbOpept LD28490 CG4122 D*
CG18503
EG:171D11.4; EG:65F1.3 arginase PF00491: arginase GH02581 CG18104 C+
1B8 †EG:65F1.2 †elav PF00076: rrm HL03451 CG4262 0
EG:65F1.1 GH24496 CG4293 0
1B8 EG:65F1.5 Appl PF02177: A4_EXTRA HL03850 CG7727 A+
1B9 EG:118B3.1 vnd PF00046: homeobox CG6172 0
EG:118B3.2 PF00307: CH GH04661 CG13366 0
1B13 EG:115C2.5 mod(r) LP01383 CG17828 0
EG:115C2.1 PF00294: pfkB LP11157 CG13369 0
EG:115C2.12 LP11709 CG18451 0
EG:115C2.6 PF00096: zf-C2H2 LD23988 CG17829 0
1B13 EG:115C2.7 RpL36 PF01158: Ribosomal_L36e LD01128 CG7622 0
1B13 EG:115C2.2 l(1)lBi LD09823 CG6189 0
1B13 EG:115C2.9 Dredd PF00655: ICE_p10 LD14339 CG7486 B

PF00656: ICE_p20
1B13 EG:115C2.3 su(s) LD06838 CG6222 0
EG:115C2.8 GH16756 CG13367 A

EG:115C2.11 GH22310 CG16982 B+
EG:115C2.10 PF00856: SET LD03312 CG13363 C

1B13 EG:115C2.4 skpA PF01466: Skp1 LD03188 CG16983 0
1B14-C1 EG:BACR19J1.1 sdk PF00041: fn3 GM02010 CG5227 B+
PF00047: ig
EG:BACR19J1.2 PF00153: mito_carr LD09021 CG5254 0
EG:BACR19J1.3 GH28702 CG5273 0
EG:BACR19J1.4 RpL22 PF01776: Ribosomal_L22e LP05628 CG7434 0
EG:34F3.2 PF00784: MyTH4 LD11354 CG12467 D
PF00169: PH
EG:34F3.1 LD26268 CG12467 D
EG:34F3.8 PF00957: synaptobrevin LD05791 CG7359 0
EG:34F3.10 CG13358 A+
EG:34F3.9 CG13359 B

(Table continues on pp. 714–718.)
Sequencing the Drosophi la X Chromosome
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Table 1. (Continued )
Cytology Gene symbol Gene HMMER EST
Matching
gene(s)
EDGP
vs. joint
sequence
1B14-C1 EG:34F3.3 Rbf PF01858: RB_A LP07395 CG7413 0
PF01857: RB_B
EG:34F3.4 LD26306 CG16989 0
EG:34F3.5 LP04844 CG13360 0
EG:34F3.7 PF02366: PMT LP01681 CG12311 A
C

EG:34F3.6 fz3 PF01534: Frizzled CG16785 A

PF01392: Fz
EG:BACR7A4.2 bs33b10 CG3713 0
EG:BACR7A4.3 PF00089: trypsin CG11664 0
EG:BACR7A4.19 PF00651: BTB LP01394 CG3711 0
PF01344: Kelch
EG:BACR7A4.6 1.82 CG3034 0
EG:BACR7A4.18 PF00956: NAP_family GH17085 CG3708 A+
EG:BAC7A4.20 LP11534 CG3706 0
EG:BACR7A4.5 LP07093 CG11642 0
EG:BACR7A4.17 LD33276 CG3704 0
EG:BACR7A4.16 LD03548 CG3026 0
EG:BACR7A4.15 GH12139 CG3703 A

EG:BACR7A4.14 PF00106: adh_short LP06734 CG3699 D
*
PF00678: adh_short_C2
EG:BACR7A4.13 PF00083: sugar_tr GH13765 CG3690 0
EG:BACR7A4.7 PF02268: TFIIA_gamma GM03032 CG11639 0
EG:BACR7A4.12 PF00036: efhand bs03d05 CG11638 A++
1E1-4 EG:BACR7A4.8 anon-1Ed LD29918 CG3021 C

EG:BACR7A4.11 CDC45L LD08729 CG3658 0
1E1-4 EG:BACR7A4.9 anon-1Eb GH11273 CG14630 A

1E1-4 EG:BACR7A4.10 su(w[a]) PF01805: Surp SD01276 CG3019 B

EG:103E12.2 GH24974 CG14629 0
EG:103E12.3 LD08339 CG3655 0
EG:BACR42I17.12 PF00076: rrm CG14628 0
EG:BACR42I17.1 PF01652: IF4E bs10b09 CG11392 B+
EG:BACR42I17.2 LP03214 CG11378 0
EG:BACR42I17.3 CG11384 0
EG:BACR42I17.4 bs31h12 CG11379 0
EG:BACR42I17.5 CG14627 A++
EG:BACR42I17.6 CG14626 A

EG:BACR42I17.7 CG11380 A++
EG:BACR42I17.8 CG14625 A+
EG:BACR42I17.9 CG11381 D
CG14624
EG:BACR42I17.10 LP08751 CG11382 0
EG:BACR42I17.11 PF00096: zf-C2H2 CG11398 0
EG:33C11.3 LP06890 CG3638 A+ C


EG:33C11.2 GM08856 CG11403 0
EG:33C11.1 A3-3 PF00170: bZIP GH24653 CG11405 0
EG:114D9.1 PF00036: efhand CG11408 0
EG:114D9.2 PF02181: FH2 LD26058 CG14622 A


EG:190E7.1 CG18091 0
EG:8D8.1 GM13066 CG11411 0
EG:8D8.2 LD34263 CG11409 0
EG:8D8.6 PF00583: Acetyltransf LD06467 CG11412 B+
EG:8D8.8 GM12784 CG11418 0
EG:8D8.7 PF00335: transmembrane4 LP04678 CT11415 0
EG:8D8.3 PF00324: aa_permeases LD15480 CG12773 0
EG:8D8.4 LD08351 CG11417 0
EG:8D8.5 png PF00069: pkinase CG11420 0
EG:132E8.1 PF00076: rrm LD09340 CG3056 0
1F EG:132E8.2 SNF1A PF00069: pkinase GH05909 CG3051 0
EG:132E8.3 PF00085: thiored LD03613 CG3719 0
EG:132E8.4 CG11448 0
EG:49E4.1 futsch GH21135 CG3064 D*
EG:BACN32G11.1 CG18531 0
EG:BACN32G11.2 GH10964 CG14785 0
Benos et al.
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Table 1. (Continued )
Cytology Gene symbol Gene HMMER EST
Matching
gene(s)
EDGP
vs. joint
sequence
EG:BACN32G11.3 PF01535: PPR LD01992 CG14786 0
EG:BACN32G11.4 PF00378: ECH LP07530 CG14787 A

EG:BACN32G11.5 PF01926: MMR_HSR1 HL05876 CG14788 0
EG:BACN32G11.6 GH07929 CG14789 A

EG:80H7.10 GH22272 CG14777 0
EG:80H7.1 PF00089: trypsin —
EG:80H7.2 LD18706 CG14779 0
EG:80H7.3 PF00089: trypsin CG14780 0
EG:80H7.4 PF00071: ras GM10914 CG14791 B

EG:80H7.11 LD02045 CG14781 B+
EG:80H7.5 PF01363: FYVE GM03532 CG14782 0
PF00169: PH
2B1-2 EG:80H7.6 sta PF00318: Ribosomal_S2 LD27557 CG14792 A
B C+
EG:80H7.7 PF00060: lig_chan CG14793 D*
EG:196F3.1 —
EG:196F3.3 CG14795 A+
EG:196F3.2 PF02214: K_tetra LD05656 CG14783 C+
EG:56G7.1 PF01607: Chitin_bind_2 CG14796 0
2B5 †EG:123F11.1;
EG:17A9.1; EG:25D2.1
†br PF00651: BTB
PF00096: zf-C2H2
LP05017 CG11491 0
2B6 EG:171E4.1 dor LD12589 CG3093 0
EG:171E4.4 CK00326 CG3740 D*
EG:171E4.2 PF00560: LRR CG3095 A+ C+
EG:171E4.3 CG3737 0
EG:73D1.1 LD24507 CG3791 0
2B6-7 EG:9D2.1 b6 HL05401 CG3100 0
EG:9D2.2 GH23439 CG3783 D*
2B6-8 EG:9D2.3 a6 LD13641 CG3771 C

EG:9D2.4 PF00089: trypsin CG3795 0
EG:4F1.1 GH21860 CG14808 0
EG:BACN35H14.1 Adar PF02137: A_deamin LD31451 CG12598 A+
PF00035: dsrm
EG:137E7.1 LD19625 CG17968 0
EG:131F2.2 PF00929: Exonuclease CG14801 A

EG:131F2.3 LP07325 CG14812 0
EG:63B12.10
COP LD30910 CG14813 0
EG:63B12.6 GM12676 CG14814 A

EG:63B12.13 GH20211 CG14802 0
EG:63B12.5 PF00515: TPR GH08708 CG14815 0
EG:63B12.9 LD13889 CG14803 B+
EG:63B12.4 PF00300: PGAM LD30851 CG14816 0
EG:63B12.8 LD10891 CG14804 0
EG:63B12.11 GH01621 CG14817 0
EG:63B12.7 PF00400: WD40 LD02447 CG14805 B+
EG:63B12.12 LP05103 CG14818 0
2B15 EG:63B12.3 trr PF00856: SET GM10003 CG3848 B++
2B15 EG:63B12.2 anon-2Bd PF00252: Ribosomal_L16 GH05976 CG3109 B+
2B15 EG:86E4.6 arm PF00514: Armadillo_seg LD10209 CG11579 A+
EG:86E4.2 PF01532: Glyco_hydro_47 LD21416 CG3810 C+
EG:86E4.3 PF00400: WD40 CG17766 A

EG:86E4.4 LD27573 CG3480 0
2B15 EG:86E4.1 eIF-2b PF02020: W2 LD26247 CG3806 0
PF00132: hexapep
EG:86E4.5 PF00783: IPPc GH18456 CG3573 0
EG:39E1.1 LD22420 CG11596 0
EG:39E1.3 LP09039 CG3857 0
EG:39E1.2 LD09945 CG3587 0
EG:BACH61I5.1 CG3600 0
EG:133E12.2 PF00104: hormone_rec CG16902 D*
PF00105: zf-C4
EG:133E12.3 PF01650: Peptidase_C13 CG4406 A+
EG:133E12.4 east LD33602 CG4399 0
Sequencing the Drosophi la X Chromosome
Genome Research 715
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Table 1. (Continued )
Cytology Gene symbol Gene HMMER EST
Matching
gene(s)
EDGP
vs. joint
sequence
2C3 †EG:133E12.1 †Actn PF00307: CH HL01581 CG4376 0
PF00036: efhand
PF00435: spectrin
2C3 EG:22E5.1 usp PF00104: hormone_rec LD09973 CG4380 0
PF00105: zf-C4
EG:22E5.12 PF00097: zf-C3HC4 CG4325 0
EG:22E5.11 PF00001: 7tm_1 CG4322 C+
EG:22E5.10 PF00001: 7tm_1 GM02327 CG4313 0
EG:22E5.8 PF00069: pkinase GH06888 CG4290 0
EG:22E5.7 LD08665 CG4281 D*
EG:22E5.5 PF00355: Rieske GH11732 CG4199 A+
PF00070: pyr_redox
EG:22E5.6 LD31238 CG4194 0
EG:22E5.3 PF01137: RCT GH07716 CG4061 0
EG:22E5.4 PF02390: Methyltransf_4 GM01339 CG4045 C+
EG:22E5.9 LP10820 CG4025 0
EG:67A9.2 LD01561 CG16903 C


EG:67A9.1 CK00561 CG3981 A

2D3 †EG:BACN25G24.2 †csw PF00017: SH2 HL03192 CG3954 0
PF00102: Y_phosphatase
2D3 EG:BACN25G24.3 ph-d PF00536: SAM GH08934 CG3895 A

B+ C+
2D3 EG:87B1.5 ph-p PF00536: SAM GH19743 D*
EG:87B1.3 PF01565: FAD_binding_4 GH17284 CG3835 0
2D6 EG:87B1.4 Pgd PF00393: 6PGD GH13486 CG3724 0
2D6 EG:87B1.6 bcn92 CG3717 0
2D6 EG:87B1.2 wapl LD29979 CG3707 A+
2D6 EG:87B1.1 Cyp4d1 PF00067: p450 GH01333 CG3656 0
EG:152A3.3 HL02445 CG3630 0
EG:152A3.7 anon-2Db CG3621 0
EG:152A3.2 Cyp4d14 PF00067: p450 HL05508 CG3540 0
2E1 EG:152A3.4 Cyp4d2 PF00067: p450 GH09810 CG3466 A

2E1 EG:152A3.6 Cyp4ae1 PF00067: p450 GH24265 CG10755 0
2E1 EG:152A3.5 pn GM10090 CG3461 0
2E3 EG:152A3.1 Nmd3 LD13746 CG3460 0
EG:17E2.1 LD17911 CG3457 B

2E3 EG:103B4.3 Mct1 PF01587: MCT LP01643 CG3456 A

EG:103B4.2 LP02712 CG18031 D
2E3 EG:103B4.4 msta GH20239 CG18033 0
2E3 EG:103B4.1 Vinc PF01044: Vinculin LD16157 CG3299 0
2E3 EG:30B8.4 pcx LD27929 CG3443 B


2F1 EG:30B8.2 kz GH21962 CG3228 0
2F1 EG:30B8.5 fs(1)K10 LD08992 CG3218 0
2F1 EG:30B8.7 Or2a CG3206 C
2F1 EG:30B8.1 crn PF02184: HAT LP05055 CG3193 0
EG:30B8.3 PF00650: CRAL_TRIO GM01086 CG3191 0
EG:30B8.6 GH06335 CG3078 D
EG:25E8.3 PF00400: WD40 LD29959 CG3071 B+
EG:25E8.2 PF00179: UQ_con LD09991 CG2924 A+ C

EG:25E8.1 PF00012: HSP70 GH11566 CG2918 0
EG:25E8.6 CG2879 D
EG:25E8.4 GH04956 CG2865 0
EG:BACH48C10.1 CG14050 0
EG:BACH48C10.2 GH19593 CG2854 C

2F6 EG:BACH48C10.3 phl PF00130: DAG_PE-bind GH03557 CG2845 B+
PF02196: RBD
PF00069: pkinase
EG:BACH48C10.6 CG14048 0
2F6 EG:BACH48C10.5 ptr GH02860 CG2841 A+
EG:BACH48C10.4 GH27724 CG14047 D
EG:BACH7M4.1 SD05785 CG14045 A


EG:BACH7M4.2 PF00168: C2 CK01827 CG14045 A
C

PF00505: PDZ
EG:BACH7M4.4 CG12496 C

Benos et al.
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Table 1. (Continued )
Cytology Gene symbol Gene HMMER EST
Matching
gene(s)
EDGP
vs. joint
sequence
3A2 EG:BACH7M4.5 gt CG7952 0
3A3 †EG:BACH59J11.1 †tko PF00164: Ribosomal_S12 GM03810 CG7925 0
EG:BACH59J11.2 PF00041: fn3 SD01373 CG13756 B+
3A3 EG:BACH59J11.3 z CG7803 0
EG:BACR25B3.11 pcan PF0008: EGF GM03359 CG7981 D*
PF00047: ig
PF00054: laminin_G
PF00057: ldl_recept_a
EG:BACR25B3.10 PF00047: ig GM02481 CG7981 D*
EG:BACR25B3.1 PF00047: ig GM06086 CG7981 A++ C

PF00052: laminin_B
PF00053: laminin_EGF
PF00057: ldl_recept_a
EG:BACR25B3.2 PF00057: ldl_recept_a CG12497 A+ B+
EG:BACR25B3.3 PF00002: 7tm_2 CG13758 D
EG:BACR25B3.4 PF01813: ATP-synt_D GH28048 CG8310 D
EG:BACR25B3.5 GH02552 CG13759 B+
EG:BACR25B3.6 LD41675 CG13760 A


EG:BACR25B3.7 wds PF00400: WD40 LD30385 CG17437 0
3A8 EG:BACR25B3.8 egh CG9659 0
3A8 EG:BACR25B3.9 Klp3A PF00225: kinesin 14 LD21815 CG8590 0
3A9 EG:BACR7C10.3 mit(1)15 LD31038 CG9900 0
EG:BACR7C10.4 Bzd PF01753: zf-MYND CG13761 C+
EG:BACR7C10.6 PF00335: transmembrane4 GH15125 CG10742 0
EG:BACR7C10.1 LD08769 CG9904 0
EG:BACR7C10.7 CG13762 B

EG:BACR7C10.2 PF00613: PI3Ka GH26308 CG10260 D
PF00454: PI3_PI4_kinase
3B1 EG:155E2.3 sgg PF00069: pkinas3 GM02018 CG2621 A+
3B2 EG:155E2.2 HLH3B PF00010: HLH CG2655 0
EG:155E2.5 GH07966 CG2652 0
3B2 †EG:155E2.4 †per PF00989: PAS GH01975 CG2647 A
B+
3B2 EG:155E2.1 anon-3B1.2 CG2650 B

EG:100G10.7 anon-3Ba PF0004: AAA GH01006 CG2658 0
PF01434: Peptidase_M41
EG:100G10.6 PF00628: PHD HL01595 CG2662 0
EG:100G10.5 anon-3Bb LD37122 CG2675 A+
EG:100G10.3 PF01008: IF-2B CG2677 0
EG:100G10.4 GH11163 CG2680 B+
EG:100G10.2 GH02982 CG2681 B

EG:100G10.1 LD25954 CG2685 0
EG:100G10.8; EG:95B7.10 LD34251 CG2695 0
3B4 EG:95B7.9 anon-3Bd GH08386 CG2701 0
3C1 EG:95B7.8 fs(1)Yb CG2706 0
3C1 EG:95B7.4 fs(1)Ya LD47547 CG2707 A

EG:95B7.5 CG2709 0
3C1 EG:95B7.6 dwg PF00096: zf-C2H2 LD08032 CG2711 0
EG:95B7.3 LD05179 CG2713 0
EG:95B7.7 anon-3Be PF00096: zf-C2H2 LD39664 CG2712 0
3C2 EG:95B7.2 crm PF00249: myb_DNA- LD09365 CG2714 0
binding
EG:95B7.1 PF00804: Syntaxin CG2715 0
EG:BACN33B1.2 HL08104 CG2766 D*
CG2716
3C2 EG:BACN33B1.1 w PF00005: ABC_tran GH06126 CG2759 0
EG:BACR43E12.1 CG12498 0
EG:BACR43E12.7 GM07661 CG14416 0
EG:BACR43E12.6 CG14417 0
EG:BACR43E12.5 CG14417 0
EG:BACR43E12.4 PF00569: ZZ GH01442 CG3526 A++
EG:100G7.6 CG3588 A

C+
EG:100G7.5 CG14424 0
Sequencing the Drosophi la X Chromosome
Genome Research 717
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number of introns is EG:BACR25B3.1 (26 introns in
the coding region). The average size of the introns is
475 bp, with the shortest being 26 bp (EG:63B12.3) and
the longest being 34,401 bp (sidekick [sdk ] ,
EG:BACR19J1.1). The calculated average number of in-
trons per gene in this chromosomal region is consis-
tent with previous studies that have indicated the ma-
jority of Drosophila genes contain one or two small
introns located near their 5 ends (although exon and
intron numbers will have been underestimated as ab
initio gene prediction methods will not predict un-
translated exons). There are, however, some exception-
ally large genes. These include sdk, which encodes an
immunoglobulin-C2 domain protein, and is required
to prevent the“mystery cell” of the developing eye disc
differentiating as a photoreceptor (Nguyen et al. 1997).
This gene, sequenced previously as a cDNA, covers 60
Kb and includes at least 14 exons. Another very large
gene is futsch (EG:49E4.1), covering 18 Kb and encod-
ing a protein of 5327 amino acids predicted to encode
a microtubule-associated protein, on the basis of its
similarity with human MAP1B (SWISS-PROT:P46821),
which is only half the size. Recently Hummel et al.
(2000) have shown that futsch encodes the well-known
Drosophila neural antigen 22C10. Four other genes
have large transcription units: Appl, 35.1 Kb; br, 27.7
Kb: EG25B3.1, 20.0 Kb; and csw, 17.4 Kb. The overall
GC content of this collection of genes from the tip of
the X chromosome is significantly lower (45.5%) than
the overall GC content of the genes in the Joint Se-
quence (56.1%).
One of the surprising results of the analysis of the
Adh region sequence (Ashburner et al. 1999) was the
number of genes predicted to be included within the
introns of other genes (8%). These were most fre-
quently, but not exclusively, arranged as anti-parallel
transcription units. The present analysis of the tip of
the X permits a comparison with another segment of
genomic DNA. We predict four nested genes. This cor-
responds to 1.4 % of all of the genes we identify. This
is probably an underestimate, because ab initio gene
prediction programs do not predict genes within
genes.
One group of duplicated genes worthy of specific
mention in this region are the cytochrome P450s,
small monooxygenases often involved in the metabo-
lism of xenobiotic compounds. Eighty-seven genes en-
coding these microsomal or mitochondrial enzymes
had been identified in the essentially complete Joint
Sequence of D. melanogaster (Nelson 2000). Only two
(l(2)35Fb in the Adh region [Ashburner et al. 1999] and
disembodied [Chávez et al. 2000]) have been associated
with a mutant phenotype, although polymorphisms at
others have implicated them in differential resistance
to DDT and other compounds (Berge et al. 1998). One
characteristic of the genes encoding these proteins is
that they often occur in small clusters, indicating an
expansion of the gene family by duplication. In region
1–3 we have identified five cytochrome P450-encoding
genes (Cyp4g1, Cyp4d1, Cyp4d2, Cyp4ae1, and
Cyp4d14); of these, the latter three are in tandem
within about 7.5 Kb at 2E1 and Cyp4d1 is some 12 Kb
distal at 2D6. The Cyp4g1 (at 1B4) gene appears to be
more abundantly transcribed than any other P450
gene in D. melanogaster, at least judging from the large
number of its EST sequences (59; Nelson 2000).
We have analyzed all of the known or predicted
proteins by several methods, most extensively by
BLASTP against data sets derived from SWISS-PROT
and TrEMBL sorted by taxonomic origin (see Ash-
burner et al. 1999). We have also analyzed all of the
protein sequences by various methods to detect pro-
Table 1. (Continued )
Cytology Gene symbol Gene HMMER EST
Matching
gene(s)
EDGP
vs. joint
sequence
3C5 EG:100G7.1 anon-3Ca CG18089 0
3C5 EG:100G7.2 anon-3Cb CG3591 0
EG:100G7.3 CG3598 0
All known or predicted genes have a symbol in the form EG:#, where the # indicates the clones on which they were first discovered
followed by a dot and integer. Genes previously known are also shown with their FlyBase symbols and, if determined, cytological
locations. The EST column indicates a matching EST sequence from either the BDGP collection or B. Oliver’s testes-derived EST
collection (as submitted to GenBank; see Andrews et al. 2000). Only one cDNA clone name is listed for each gene. The column headed
“Matching Gene(s)” indicates the matching gene from the Joint Sequence. The column headed “EDGP vs. Joint Sequence” indicates
the result of comparing the EDGP and Joint Sequence at the predicted protein level. In this column, 0 indicates identity or <1%
difference in sequence; A, that the sequences differ in their predicted start sites; B, that they differ in their predicted termination sites;
and C, that they differ by a predicted exon or intron. A ‘D’ indicates that the gene models predicted by us and by the Joint Sequence
differ very markedly; an accompanying asterisk indicates that we have evidence that the EDGP model is the more correct (see text).
A plus sign indicates the EDGP sequence is longer than the CG sequence; a minus sign indicates that it is shorter. For more details see
the supplementary data. Only positive hits of known or predicted proteins to PFAM are shown (see text). A dagger before a gene
symbol indicates a gene with alternatively spliced messages.
Benos et al.
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tein motifs, and domains. Overall, 71% of the known
or predicted proteins have a BLASTP match with an
expectation of 10
7 or less when compared with non-
drosophilid protein sequences. Similarly, 137 contain
at least one known motif or domain (other than the
PROSITE Nuclear Localization Signal profile) as deter-
mined by matches against InterPro (http://
www.ebi.ac.uk/interpro/). These numbers are, of
course, both preliminary and transitory. All of these
data have been communicated to FlyBase and can be
found in the supplementary data (see Methods). We
have chosen only to present the PFAM hits in Table 1,
as an indication of the data obtained.
As we have discussed previously (Benos et al.
2000), examples of 12 different transposable elements
were identified within the region analyzed: 412, roo,
Doc, FB, jockey, mgd1, Tirant, S-element, 1360, Burdock,
blastopia, and yoyo. It is possible that more transposable
elements may be present in the region; however, we
have not identified them molecularly.
Chromosomal Regions of Particular Interest
The achaete-scute Complex
The achaete-scute complex (AS-C) comprises a region of
∼95 Kb (between y and Cyp4g1; chromosomal bands
1B1–4) defined by the physical mapping of >110 ach-
aete (ac) and scute (sc) mutations associated with chro-
mosomal breakpoints or insertions of transposable el-
ements (Campuzano et al. 1985; Ruiz-Go´mez and
Modolell 1987). ac and sc alleles either suppress forma-
tion of combinations of bristles (and other cuticular
sensory organs) or cause the generation of ectopic
bristles (Garcı´a-Bellido 1979). Most mutant alleles of
these genes are viable, although an adjacent vital ge-
netic function, lethal-of-scute (l(1)sc) (Muller 1935), is
uncovered by internal deficiencies of the complex such
as Df(1)sc4Lsc9R. Embryos homozygous for these defi-
ciencies have a defective CNS. Another genetic func-
tion, asense (ase), has also been mapped within the
AS-C (Dambly-Chaudière and Ghysen 1987; Jime´nez
and Campos-Ortega 1987) and found to be important
for the development of the larval external sensory or-
gans. Previous molecular characterization of the AS-C
(for review, see Campuzano and Modolell 1992) have
shown that the functions defined by genetic analysis
correspond to single genes, arranged over 85 Kb in dis-
tal-proximal order: ac, sc, l(1)sc and ase. All four genes
encode related transcription factors of the bHLH fam-
ily, which are partially redundant in their functions,
being required for epidermal cells to become neural
precursors. They have evidently evolved by tandem
duplication.
Our new analysis of the sequence in the region
between y and Cyp4g1 predicts the existence of only
the four AS-C genes and the previously known pepsino-
gen-like (pcl) gene, a nonvital gene located between
l(1)sc and ase, which is expressed in the larval gut
(Campuzano et al. 1985; González 1989; S. Romani,
unpubl.). We have not been able to detect the exist-
ence of two postulated genes, anon-1Ba (=T7) near sc
and anon-1Bc (=T9), located just distal to Cyp4g1 (Vil-
lares and Cabrera 1987; Alonso and Cabrera 1988).
These genes were also not annotated in the Joint Se-
quence. A further gene (anon-1Be), predicted previ-
ously to be located between y and ac giving rise to
several transcripts (5–0.9 Kb) (Chia et al. 1986) present
in the nuclei of the embryonic vitellum (L. Balcells and
J. Modolell, unpubl.) has also not been confirmed by
either genomic annotation study. This is most likely a
nonvital gene as a large part of it is deleted in the viable
Df(1)ac1. Curiously, it harbors within its transcription
unit the enhancer that drives ac and sc expression in
the proneural cluster that gives rise to the dorsocentral
bristles (Garcı´a-Garcı´a et al. 1999).
The broad Complex
In region 2B1–10 of the polytene X chromosome, an
ecdysterone-induced puff forms in the late third instar
larva (Becker 1962; Ashburner 1969). A large number of
lethal and visible mutations were recovered by Kiss,
Zhimulev, and colleagues that mapped to this region
(Zhimulev et al. 1995). The visibles included mutations
that affected wing morphology (broad alleles) and
those that reduced the number of chaetae on the pal-
pus (rdp alleles). Several different lethal complementa-
tion groups were characterized and it became clear that
the visible alleles were simply hypermorphic alleles of
lethal loci. The complementation patterns between all
of the available alleles in what became known as the
broad complex suggested four loci, br, rdp, l(1)2Bc, and
l(1)2Bd, with several mutations failing to complement
mutations at more than one of these. This is not, how-
ever, the result of a complex of genes, rather of a single
gene (broad) with a complex pattern of alternatively
spliced transcripts. This gene encodes a family of C2H2
Zinc-finger transcription factors (DiBello et al. 1991),
the different isoforms being the products of differen-
tially spliced primary transcripts that share common
carboxy-terminal exons. In our analysis, this gene cov-
ers nearly 30 Kb and, judging from the available cDNAs
and EST sequences, encodes four different isoforms. It
is known that these have temporally and spatially dif-
ferent expression patterns (Bayer et al. 1996, 1997; Tzo-
lovsky et al. 1999). The differential effects of individual
mutations on these isoforms explains both the differ-
ent phenotypes and the apparent genetic complexity
of the broad locus.
The zeste-white Region
The discovery of polytene chromosomes in the larvae
of Drosophila in the early 1930’s was a major event in
Sequencing the Drosophi la X Chromosome
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the history of genetics. These chromosomes are char-
acterized by a nonperiodic pattern of darkly staining
bands and lightly staining interbands, reflecting differ-
ences in the degree of DNA packing. These patterns are
both colinear with the genetic map, as proven by
Bridges (1937) and extraordinarily stable; they can be
recognized in species that have diverged many mil-
lions of years ago. The detailed maps of Bridges (see
Lefevre 1976; Sorsa 1988) enumerated 5072 polytene
chromosome bands (and, hence, interbands). Bridges
suggested, somewhat tentatively, that there may be a
one-to-one correspondence between these bands and
genes, a hypothesis that became known as the “one
band/one gene hypothesis”. A prediction of this hy-
pothesis was that Drosophila had ∼5000 genes. This
idea was apparently supported by estimates of the
number of vital loci on the X chromosome, ∼1000 or
∼5000 for the genome as a whole (Lea 1955; Lefevre
and Watkins 1986). Further apparent confirmation of
the one band/one gene hypothesis came from a num-
ber of attempts to “saturate” small regions of the ge-
nome with mutations, and hence estimate the number
of genes in that region (Alikhanian 1937). Most famous
of these experiments was that of Judd and students
(Judd et al. 1972; Young and Judd 1978) who studied a
small region of the distal X chromosome between
bands 3A2 and 3C2. By saturation mutagenesis in this
16-band region, Judd and colleagues, and subsequent
studies (e.g., Lim and Synder 1974) defined 20 genes,
of which 15 were vital. A number of other studies also
concluded that the ratio between gene and band num-
ber was about one (Zhimulev 1999). It is now clear
that, although the number of vital loci in Drosophila is
indeed ∼5000, the use of lethal mutations to define
genes results in a substantial underestimate; only
about one-third of genes are vital.
The complete sequence of the tip of the X chro-
mosome now gives us the chance to review the impor-
tant study of Judd and colleagues with a molecular
perspective (see also Judd’s own recent historical re-
view, Judd 1998). The region between the genes giant
and white studied by Judd et al. (the zeste-white region)
is 360 Kb in length and is predicted to contain 45 genes
(Fig. 2). It is indeed remarkable that conventional ge-
netic analyses had identified 20 of these. Of these 20,
12 can be placed directly on the genetic map, by virtue
of identity of sequence; the remaining eight genes,
known only from lethal mutations, have not been se-
quenced independently.
Unraveling the famous zeste-white region in the
ultimate detail of its complete DNA sequence leaves
major questions concerning the chromomeric struc-
ture of polytene chromosomes unanswered, of course.
The banding pattern is attributable to aperiodicities in
the packing ratio of the DNA, associated with proteins,
in chromatin. Does this pattern have any functional
significance whatsoever? No answer to this question
can yet be given. How is the banding pattern deter-
Figure 2 The zeste-white interval. The top is a reproduction of Figure 5 from Judd et al. (1972) showing the polytene chromosome
region 3A–3C and the complementation groups discovered by mutational analysis. Below this projections are made onto the interval
2100 Kb to 2480 Kb of the EDGP sequence showing the correspondence between the genetic analysis and the genes known or predicted
in this region from sequence analysis.
Benos et al.
720 Genome Research
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mined? At one level the answer to this is obvious, by
the DNA sequence. We have already described an in-
verted repeat sequence in the chromosomal DNA
flanking the broad complex that could account for the
unusual chromosomal banding pattern of this region
(Benos et al. 2000). However, more subtle aspects of
DNA sequence may define the domains of the majority
of polytene chromosome bands, and the full answer to
this problem will require considerable further analysis.
P-element Insertions
The majority of P-element screens to have been carried
out to date have been performed on the autosomes.
Spradling and colleagues (1999) have described their
attempts to consolidate a number of such P-element
collections, including a large collection of lethal
P-element insertions on the second chromosome (To¨ro¨k
et al. 1993). Similarly, the EDGP have described a col-
lection of lethal insertions on chromosome 3 (Deak et
al. 1997). We have begun to generate a comparable
collection of P-element insertion mutants on the X
chromosome in anticipation of their value for func-
tional genomics. The initial group of mutants corre-
sponds to ∼500 lethal insertions that have been
mapped by hybridization of P-element probes to poly-
tene chromosomes in situ. The characterization of this
collection will be presented elsewhere. We have local-
ized the insertion sites for 64 P-element-induced lethal
mutations that map to divisions 1–3, and determined
the gene(s) whose function is likely to be affected by
each insertion (Table 2). We have carried out a similar
computational analysis on a collection of random EP-
element insertions sequenced by the BDGP (Rørth et al.
1998). Forty-seven of these had been mapped to divi-
sions 1–3 by in situ hybridization; this is a density of
one element per 55 Kb, about twice that found for
EP-elements in the Adh region (1/108 Kb). This differ-
ence in density is not due to the existence of major
hotspots for insertion of EP-elements on the X chromo-
some tip, nor to a higher proportion of the insertions
on the X tip being outwith genes (in both regions
∼47% of EP-element insertions are within genes).
From a total of 111 P-element insertions that we
have located within the region analyzed, 41% fall in
regions in which they are expected to affect the expres-
sion of genes already known, whereas 50% are ex-
pected to affect the expression of predicted genes.
These expectations are based on the positions of the
P-element insertion either within transcribed regions or
within 5 Kb 5 to these. Some insertions might affect
two different genes, one on either side of the insertion
(Table 2). Only 13 elements or clusters of elements
map more distantly, 7–33 Kb 5 to the nearest known
or predicted gene (footnotes in Table 2; of these, five
elements or groups were selected as lethal, but may or
may not cause the lethality).
Comparison with the Joint Sequence
The determination of the sequence and gene annota-
tion of chromosomal divisions 1–3 was completed and
submitted to the EMBL-Bank by February 7, 2000, six
weeks before the publication and release of the anno-
tated Joint Sequence of the D. melanogaster genome in
March 2000 (Adams et al. 2000). Although preexisting
gene features were taken into account during the
analysis of the Joint Sequence, these are essentially in-
dependent annotation experiments that can be com-
pared. Moreover, direct comparison of the nucleotide
sequence determined by the EDGP with the Joint Se-
quence, allows one to assess some of the strengths and
weaknesses of the two different sequencing strategies.
We have compared both individual gene predictions
and the overall sequence between these two studies.
Comparison of Gene Predictions
We have identified 277 protein coding genes in the
region 1A–3C, including 94 genes that had been
known previously. There are 275 genes common to
both studies; two, namely EG:80H7.1 and EG:196F3.1,
have no corresponding prediction in the Joint Se-
quence. Neither of these two predictions are very
strong (in terms of their GeneFinder and/or Genscan
scores; see Methods), but both contain trypsin protein
motifs (EG:196F3.1 has only a PROSITE match whereas
EG:80H7.1 has both PROSITE and PFAM matches).
There are 33 genes predicted on the Joint Sequence
that are absent from the EDGP annotation. Some (13)
of these predictions were also seen in the EDGP analy-
sis but were excluded due to their low scores and lack
of other supporting evidence (see Methods). We have
examined the data for the remaining 20 and consider
these to be overpredictions in the Joint Sequence, for a
variety of reasons (see supplementary data).
We have carefully compared the known or pre-
dicted amino acid sequence of all genes between
the annotated Joint Sequence and our analysis (Table
1). At the level of their predicted proteins, 60% of the
275 genes in common are identical or differ by no
more than 1% of their amino-acid residues (class 0);
31.3% have one or more minor differences, for ex-
ample in the choice of ATG or stop codon or in an
internal exon (classes A–C); 8.7% (24 genes) have ma-
jor differences in their structure between the two stud-
ies (class D). We have analyzed these 24 in detail; for 10
of them we cannot make a decision, based on the avail-
able data, as to which interpretation is the better. How-
ever, for the remaining 14 (i.e., 5.1% of the total num-
ber of genes) the EDGP model is the more correct,
based on the EST data. (Note that the Joint Sequence
analysis did not use all available ESTs, as noted in
Methods.) Some of the class C differences (Table 1) in
gene models may reflect different splice variants of the
same gene.
Sequencing the Drosophi la X Chromosome
Genome Research 721
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Table 2. P-element Insertions in Divisions 1–3
Insertion line EMBL-Bank accession no. Cytology Cosmid or BAC Hits to gene
l(1)G0142 AJ299992 1B1-2 BACR37P7 cin
l(1)G0399 AJ299993 — cos171D11 EG:171D11.6
EP(1)1320 AQ073187 1B5-6 cos171D11 EG:171D11.1
EP(1)1398 AQ073214 1B5-6 cos171D11 EG:171D11.1
EP(1)0356 AQ025323 1B7-8 cos171D11 svr
l(1)G0319 AJ299994 1B7-10 cos65F1 elav and arginase
l(1)G0031 AJ299996 1B cos65F1 elav and arginase
EP(1)1117 AQ025390 1B7-8 cos65F1 elav and arginase
EP(1)0452 AQ025344 1B7-8 cos65F1 elav and arginase
l(1)G0471 AJ299997 1B11-14 cos115C2 Between RpL36 and l(1)Bi
EP(1)1412 AQ025449 1B12-14 cos115C2 Dredd
EP(1)1216 AQ254762 1B13-14 cos115C2 EG:115C2.10
l(1)G0037 AJ300000 1C cos115C2 skpA
l(1)G0109 AJ299999 1C cos115C2 skpA
l(1)G0058 AJ299998 1C cos115C2 skpA
l(1)G0389 AJ300001 1C cos115C2 skpA
EP(1)0369 AQ025326 1C1-3 BACR19J1 sdk
EP(1)1467 AQ025484 1C1-3 BACR19J1 EG:BACR19J1.3
l(1)G0115 AJ300002 1C1-3 BACR19J1 RpL22
l(1)G0422 AJ300003 1C BACR19J1 RpL22
l(1)G0451 AJ300004 1C BACR19J1 RpL22
EP(1)1600 AQ025529 1D1-2 BACR7A4 —1
EP(1)1498 AQ073221 1D1-2 BACR7A4 —2
l(1)G0132 AJ300005 1D BACR7A4 EG:BACR7A4.6
l(1)G0452 AJ300006 — BACR7A4 EG:BACR7A4.5
l(1)G0296 AJ300008 1E BACR7A4 EG:BACR7A4.15
EP(1)1392 AQ025435 1E1-2 BACR7A4 anon-1Ed
EP(1)1594 AQ025523 1E3-4 BACR42I17 —3
EP(1)0773 AQ025356 1E3-4 BACR42I17 —4
EP(1)1543 AQ073253 1E3-4 BACR42I17 —4
EP(1)1615 AQ025541 1E3-4 BACR42I17 —4
EP(1)1443 AQ254774 1E3-4 BACR42I17 —4
EP(1)1312 AQ073181 1E3-4 BACR42I17 EG:BAC42I17.10
EP(1)1090 AQ025382 1E3-4 cos33C11 EG:33C11.3
EP(1)1325 AQ073191 1E3-4 cos33C11 EG:33C11.3
EP(1)0964 AQ025366 1E3-4 cos33C11 EG:33C11.3
EP(1)1542 AQ073252 1F1-2 cos114D9 —5
l(1)G0302 AJ300009 — cos190E7 —6
EP(1)1336 AQ073199 1F1-2 cos8D8 EG:8D8.1
l(1)G0105 AJ300010 1F1 cos8D8 EG:8D8.8
EP(1)1419 AQ025455 2A1-2 cos132E8 —7
l(1)G0431 AJ300011 2A BACN32G11 EG:BACN32G11.5
l(1)G0044 AJ300013 2B1-4 cos80H7 EG:80H7.2
l(1)G0012 AJ300012 2A1-2 cos80H7 EG:80H7.2
l(1)G0130 AJ300015 2B1-4 cos80H7 sta
l(1)G0129 AJ300014 2B1-4 cos80H7 sta
l(1)G0448 AJ300016 2B1-4 cos80H7 sta
EP(1)1515 AQ073234 2B3-4 cos17A9 br
l(1)G0318 AJ300017 2B1-8 cos17A9 br
l(1)G0401 AJ300018 2B1-8 cos17A9 br
l(1)G0018 AJ300019 2B1-4 cos17A9 br
l(1)G0042 AJ300020 2B1-8 cos17A9 br
l(1)G02848 AJ300021 2B1-8 cos9D2 a6
AJ300022
l(1)G0051 AJ300023 2B cos131F2 EG:63B12.10
l(1)G0450 AJ300024 2B cos131F2 EG:63B12.10
l(1)G0301 AJ300025 2B cos131F2 EG:63B12.10
EP(1)1444 AQ025468 2B13-14 cos63B12 EG:63B12.4
EP(1)1190 AQ025400 2B13-14 cos63B12 EG:63B12.12
l(1)G0355 AJ300026 2C1-2 cos63B12 trr
l(1)G0192 AJ300027 2B cos63B12 arm
l(1)G0234 AJ300264 2B7-10 cos63B12 arm
l(1)G0410 AJ300028 — cos86E4 arm
l(1)G0220 AJ300029 2B13-C2 cos86E4 Between EG:86E4.2 and EG:86E4.3
EP(1)1232 AQ254763 2B16-18 cos39E1 —9
EP(1)0427 AQ025337 2C1-2 cos133E12 EG:133E12.3
l(1)G0014 AJ300031 2C1-2 cos133E12 east
l(1)G0500 AJ300032 2C1-2 cos133E12 east
l(1)G0100 AJ300033 — cos133E12 Actn
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Table 2. (Continued )
Insertion line EMBL-Bank accession no. Cytology Cosmid or BAC Hits to gene
l(1)G0077 AJ300034 2C cos22E5 Actn
EP(1)1193 AQ025401 2C7-8 cos22E5 usp
EP(1)1529 AQ073244 2C7-8 cos22E5 Between EG:22E5.11 and
EG:22E5.10
EP(1)1631 AQ025553 2C7-8 cos22E5 Between EG:22E5.11 and
EG:22E5.10
l(1)G0360 AJ300037 2C7-D4 cos67A9 Between EG:67A9.2 and
EG:67A9.1
l(1)G0310 AJ300038 2D cos67A9 Between EG:67A9.2 and
EG:67A9.1
l(1)G0066 AJ300039 2C cos67A9 Between EG:67A9.2 and
EG:67A9.1
l(1)G0333 AJ30040 — cos67A9 Between EG:67A9.2 and
EG:67A9.1
l(1)G0158 AJ300035 2D1-2 cos67A9 EG:67A9.1
l(1)G0170 AJ300041 2D1-2 BACN25G24 csw
l(1)G0171 AJ300042 2C7-D2 BACN25G24 csw
l(1)G0458 AJ300043 2E cos87B1 ph-d
l(1)G0385 AJ300044 2E cos87B1 Pgd
EP(1)1460 AQ025479 2F1-2 cos103B4 Between Vinc and pcx
EP(1)0426 AQ025336 2F1-2 cos30B8 pcx
l(1)G0144 AJ300045 2F cos25E8 EG:25E8.3
EP(1)1596 AQ025525 2F1-2 cos25E8 EG:25E8.2
EP(1)1125 AQ254758 2F4-5 cos25E8 EG:25E8.4
EP(1)1606 AQ025534 2F4-5 cos25E8 EG:25E8.4
l(1)G0226 AJ300046 2F cos25E8 EG:25E8.4
l(1)G0475 AJ300047 3A1-2 BACH48C10 phl
EP(1)1605 AQ025533 3A1-2 BACH48C10 ptr
EP(1)1174 AQ254760 3A1-2 BACH7M4 EG:BACH7M4.2
EP(1)1385 AQ025430 3A3-4 BACR25B3 —10
EP(1)1447 AQ025470 3A3-4 BACR25B3 pcan
EP(1)1619 AQ025543 3A3-4 BACR25B3 pcan
l(1)G0023 AJ300049 3A1-4 BACR25B3 —11
l(1)G0374 AJ300050 3A1-4 BACR25B3 —11
EP(1)1160 AQ025397 3A3-4 BACR25B3 —11
l(1)G0377 AJ300053 3A1-4 BACR25B3 —11
l(1)G0211 AJ300052 3A1-4 BACR25B3 —11
l(1)G0412 AJ300056 3A3-4 BACR25B3 —11
l(1)G0271 AJ300055 3A3-4 BACR25B3 —11
l(1)G0362 AJ300057 3A1-4 BACR25B3 —12
l(1)G0251 AJ300060 3A3-4 BACR25B3 EG:BACR25B3.7
EP(1)0804 AQ025360 3A5-6 BACR25B3 egh13
EP(1)1379 AQ073212 3B1-2 BACR7C10 sgg14
EP(1)1576 AQ025509 3A8-9 BACR7C10 sgg14
l(1)G0335 AJ300062 3B1-2 BACR7C10 sgg14
l(1)G0263 AJ300061 3B1-2 BACR7C10 sgg14
l(1)G0183 AJ300063 3A1-4 BACR7C10 sgg14
l(1)G0055 AJ300064 3B1-2 BACR7C10 sgg15
EP(1)1362 AQ025419 3B1-2 cos155E2 Between EG:155E2.5 and per
A list of the P-element insertions from the EP collection (Rørth et al. 1998) and the Go¨ttingen screen (see Methods) in region 1A–3C
of the X chromosome. For each element we show the EMBL-Bank accession no. of its flanking sequence, its cytological location, the
corresponding cosmid or BAC (see Fig. 1A), and the gene predicted, on the basis of its position, to be mutant (see text).
1EP(1)1600 lies ∼19 Kb from the 5 end of EG:34F3.1.
2EP(1)1498 lies ∼30 Kb from the 5 end of EG:BACR7A4.6.
3EP(1)1594 lies ∼11 Kb from the 5 end of EG:BACR42I17.2.
4These four EP-elements lie between two genes: ∼5 Kb from the 5 end of EG:BACR42I17.1 and ∼7 Kb from the 5 end of
EG:BACR42I17.2.
5EP(1)1542 lies between the 3 ends of EG:114D9.1 and EG:114D9.2. It is ∼33 Kb from the 5 end of EG:8D8.1.
6l(1)G0302 lies at the 3 end of EG:190E7.1. It is ∼14 Kb from the 5 end of EG:114D9.2.
7EP(1)1419 lies ∼19 Kb from the 5 end of EG:132E8.3.
8l(1)G0284 contains two P-elements 40 Kb apart.
9EP(1)1232 lies ∼11.5 Kb from the 5 end of EG:39E1.3.
10EP(1)1385 lies ∼15 Kb from the 5 end of EG:BACH59J11.2.
11This group of six P-elements plus one EP-element lie ∼10 Kb from the 5 end of EG:BACR25B3.1.
12l(1)G0362 lies ∼19 Kb from the 5 end of EG:BACR25B3.2
13EP(1)0804 lies ∼7 Kb from the 5 end of egh.
14This group of two EP-elements plus three P-elements lie ∼16 Kb from the 5 end of sgg.
15l(1)G0055 lies ∼12.5 Kb from the 5 end of sgg.
Sequencing the Drosophi la X Chromosome
Genome Research 723
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Since the submission of version 1.0 of the Joint
Sequence, some 263 “new” genes from across the ge-
nome have been sequenced by the community as a
whole (and submitted to EMBL-Bank, GenBank, or to
DDBJ). Of these, some 53% are essentially
identical in their protein coding regions to
the Joint Sequence predictions (M. Ash-
burner, unpubl.). It is of some interest that
both these community data and the EDGP
data indicate that ∼55% of the proteins pre-
dicted by the Joint Sequence are essentially
correct. This is a minimum figure, because it
takes no account of alternative splice forms
or the fact that some of the new commu-
nity data represent only partial sequences.
Overall Sequence Comparison
The Joint Sequence for region 1A–3C is
found on nine GenBank entries (Fig. 3). We
have compared it to the contiguous EDGP
sequence using the MUMmer program of
Delcher et al. (1999) (Fig. 3). At the nucleo-
tide level, the differences between our se-
quence and that of the Joint Sequence in
this region are of two types: small indels
and large (1 Kb or more) blocks of differ-
ence. Thirty large blocks of sequence are
present in only one of the sequences. Ten
of these blocks occur at identical nucleo-
tide positions in both the Joint and EDGP
sequences (Fig. 3). The null hypothesis is
that these pairs of blocks are independent.
As will be shown below, this is probably
not true for all. Excluding these, the differ-
ence between the two studies at the nucleo-
tide level is 3.03% (n = 2,568,355 common
nucleotides). This figure may seem high,
but over half (56%) of the EDGP sequence
was from clones derived from a very differ-
ent strain from that used for the Joint Se-
quence. We have partitioned this differ-
ence into that seen in known or predicted
coding exons, known or predicted introns,
and other sequences; the figures are 0.90%,
2.29%, and 3.98%, respectively.
Most of the 30 blocks of sequence that
appear to be absent from one or other se-
quence are either regions that have not
been elucidated fully in the Joint Sequence,
or correspond to transposable elements of
variable location and/or length. In particu-
lar, 17 blocks in one or the other sequence
correspond to recognizable transposable el-
ements of variable length and/or location
( in Fig. 3). These include two roo elements
of different length found at the same posi-
tion (nucleotide 572,960) in both se-
quences; five roo elements of variable loca-
Figure 3 Sequence comparisons. A comparison of EDGP sequence of the tip of
the X chromosome with that of the Drosophila Joint Sequence in the same region.
The comparison was made using the MUMmer program (see Methods). The Gen-
Bank accession numbers corresponding to the Joint Sequence are shown on
the left (AE003417–AE003425); note that this is part of a unitig (Myers et al.
2000). The blocks indicate regions of
1 Kb present in one sequence but not
the other. The position and length of each block of sequence
1 Kb that is
unique to one sequence is shown; each GenBank accession is numbered to the
left of the unitig, the corresponding base position within the EDGP sequence
is shown in italics to the right of the unitig. The EDGP sequence is numbered
continuously. The length of each block of unique sequence is in parentheses.
The nature of these sequence segments is shown in the center (note that a
segment may include sequences in addition to those identified here). The
segments corresponding to transposable elements are indicated in orange;
those corresponding to known genes are red; a gray “neck” depicts a sequence
interrupted by a large block of n’s of length N (nN). The Greek superscripts
(,,,,
) refer to the class of sequence difference (see text). Note that there are
an additional nine transposable elements in the EDGP sequence that are not
seen to differ in the Joint Sequence.
Benos et al.
724 Genome Research
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tion; and 10 single occurrences of other transposable
element families at unique locations (BEL, 412, FB4, I,
412-like and mgd1 in the Joint Sequence, and Doc, Ti-
rant, Burdock, and FB in the EDGP Sequence). It should
be noted that two of the long runs of n in the Joint
Sequence correspond to transposable elements in the
EDGP Sequence (see below). The 17 differences in
transposable elements are not surprising, as the major-
ity of the two sequences were derived from two quite
different fruitfly strains. In the EDGP sequence we
have identified 18 transposable elements or fragments
of elements and at least 7 of these differ in position in
the Joint Sequence.
Ten of the 30 blocks are long gaps in the Joint
Sequence (, ,  in Fig. 3), represented in the GenBank
accessions by long runs of n, with a total estimated
length of 39,938 nucleotides. For four of the 10 gaps
(), the length of the gap in the Joint Sequence is
considerably larger than the corresponding region in
the EDGP sequence; for example the run of 4722 n’s at
position 1,245,921 corresponds to 102 bp in the EDGP
sequence. We presume the reason for this is that the
gap in the Joint Sequence represents a transpos-
able element. Indeed, two gaps () are caused by trans-
posable elements: The 6353-bp gap at 2,294,896 corre-
sponds to a 6062-bp Burdock element in the EDGP se-
quence, and the 8060-bp gap at 2,511,915 corresponds
to a roo element in the EDGP sequence. Of the four
remaining gaps (), two are complex (at 237,007 bp
and 556,147 bp) and cannot be explained simply; one
corresponds to the ph-d/ph-p gene duplication (see be-
low), and the final gap, at 2,011,597 bp will be dis-
cussed below.
The remaining three long blocks (
in Fig. 3) of the
30 that differ between the two sequences are informa-
tive, and will be discussed more fully. Two are only
found in the EDGP sequence and are clearly the result
of misassemblies in the Joint Sequence. The first of
these is just 3 to the Actn gene and is 4.7-Kb long; the
probable explanation for it is that the Joint Sequence
has failed to properly assemble a duplicated sequence
that includes a partial duplication of the predicted
gene EG:133E12.4. This duplication was first indicated
by the matches of EST sequences (e.g., EMBL accession
no. AA202518, EMBL accession no. AA696909) to both
an exon of EG:133E12.4 and to a region between this
gene and Actn. The duplication is 4777 bp in length
and the two copies are only mismatched over a 77-bp
internal gap (1.5% mismatch). The second is in the
region of the duplicate gene pair ph-d and ph-p; the
Joint Sequence has an incorrect model for ph-p. That
this region includes a long tandem repeat is known
from the work of Deatrick et al. (1991).
The third region, at 2,011,597, is more complex.
There is an 18.5 Kb region (of which 7.1 Kb are n’s) in
the Joint Sequence absent from the EDGP sequence;
this sequence is not in the shotgun sequence of either
relevant EDGP clone, cosmid 82C7, or BACH48C10. In
addition, there is a 10.3-Kb sequence at the junction of
these clones in the EDGP sequence that is absent from
the Joint Sequence. Finally, 11 Kb of cosmid 82C7 is in
the opposite orientation when compared to
BACH48C10; note that the cosmid and BAC DNAs are
from different strains (see Methods).
These three major sequence differences could be
caused by polymorphisms; all occur within regions of
EDGP cosmid sequence. However we consider that the
hypothesis of misassembly, at least for the Actn and
ph-d/ph-p region differences, is the more likely. The
current “finishing” of the Joint Sequence by the BDGP
should settle these problems.
Repeated regions are well known to present a prob-
lem to the software used to build long contiguous re-
gions of sequence, and there is evidence of this in at
least two regions of the Joint Sequence. It is interesting
that in both cases the assembler appears to have had
difficulties with tandem near repeats of quite long re-
gions. Using statistical criteria, the software that as-
sembled the Joint Sequence was able to identify and
filter out the highly repetitive sequences, based on
their higher than expected representation (Myers et al.
2000). However, the low copy repetitive sequences
(such as the tandemly duplicated regions in these two
cases) are difficult to identify by these methods. If this
comparison of the X tip is typical of the genome as a
whole, then it indicates some 90 misassemblies in the
euchromatic sequence of the Joint Sequence.
The differences revealed by this comparison of the
genomic sequence from the two projects includes both
differences in sequencing method (clone-based in the
case of the EDGP, and shotgun in the case of the Joint
Sequence) and differences in strain from which the
DNA was derived. Even when the sequenced DNA is
from the same strain, but isolated some years apart,
there are differences in sequence and transposable el-
ements. For example, Myers et al. (2000) compared the
Adh region sequenced by the BDGP using predomi-
nantly P1 clones (Ashburner et al. 1999) with that from
the Joint Sequence. Although the differences are
smaller than in the comparison made in this study,
they are qualitatively very similar.
There are clear differences in gene predictions be-
tween the EDGP and Joint Sequence projects, both in
the existence of genes and in the precise models of
genes predicted in common. Again this is not too sur-
prising, given that the Joint Sequence was annotated
very largely by automatic methods, whereas the EDGP
had the luxury of time to make a more careful study of
each gene model. These differences point out that we
have a long way to go before the annotation of eukary-
otic sequences can be left entirely in the hands of com-
puter programs (Ashburner 2000; Lewis et al. 2000).
Sequencing the Drosophi la X Chromosome
Genome Research 725
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This analysis has, for obvious reasons, concen-
trated on the differences between the two available se-
quences of this chromosome region. This must not ob-
scure the fact that in general the two analyses are in
remarkable agreement, and point to the overall utility
of the “complete” genomic sequence now available for
D. melanogaster.
METHODS
Clone Libraries and Map Construction
DNA from two strains has been sequenced. About 44% of the
sequence is from BAC clones derived from the same strain as
that sequenced by the BDGP and by Celera; in contrast, the
cosmid clones sequenced were from a different strain (Fig. 1).
The relationship between these strains cannot be determined.
Both strains were free of P-elements.
The cosmid library used for the construction of the X
chromosome physical map was derived from a wild-type
(Canton-S) strain and described in detail by Side´n-Kiamos et
al. (1990). It has an estimated average insert size of 35 Kb and
contains ∼18,000 clones providing a fourfold coverage of the
genome. The library is available on high density double spot-
ted filters from the MRC HGMP Resource Centre (http://
www.hgmp.mrc.ac.uk/Biology/Bio.html).
Three BAC clone libraries were used; each was con-
structed from DNA from the y2; cn bw sp isogenic strain. Two
BAC libraries were made at CEPH (Centre d’Etude du Poly-
morphisme Humaine). One (BACN clones) was prepared with
NdeII inserts and the other (BACH clones) withHindIII inserts,
both in the vector pBeloBACII. These two libraries were made
with pools of size-fractioned DNA that gave mean insert sizes
of up to 90 Kb. The 23,400 clones gave ∼10-fold coverage of
the genome. The third library was of EcoRI digested DNA
(BACR clones) and was constructed in the vector pBACe.3.6
by Aaron Mammoser and Kazutoyo Oseogawa at the Roswell
Park Cancer Institute (Buffalo, NY) in collaboration with the
BDGP (Hoskins et al. 2000). This library gave an ∼17-fold cov-
erage of the genome with an average insert size of 165 Kb.
Sequencing
Cosmids and BACs were sequenced by a two-stage approach
involving random sequencing of sub-clones followed by di-
rected sequencing to resolve problems. DNA from cosmids
and BACs was sonicated and fragments of 1.4–2 Kb were
cloned into either M13 or pUC18 vectors. Clones were se-
quenced using dye-terminator chemistry and loaded on
ABI373 or ABI377 automated sequencing machines. Sequence
base calling and contig assembly was accomplished using
Phred/Phrap software (Ewing and Green 1998; Ewing et al.
1998) and editing took place in either Consed (Gordon et al.
1998) or Gap4 (Bonfield et al. 1995). Gaps were filled using a
combination of custom primer walking and PCR.
Cosmid and BAC DNAs were nebulized and end repaired.
Following agarose gel purification, fragments of ∼1500
nucleotides were ligated to linearized vector (pTZ19R or pCR-
BluntII) and cloned in the KK2186 strain of Escherichia coli.
Bacterial clones were picked at random and cultured over-
night. Plasmid DNAs were prepared by an alkaline lysis
method and purified using the QIAprep 96 Turbo Miniprep
kit (QIAGEN). Insert DNA were sequenced from both ends
using universal primers. Cycle sequencing was performed
with labeled terminators using AmpliTaq and the Big Dye
Terminator Cycle Sequencing Ready Reaction kit (Applied
Biosystems).
The Heidelberg group employed the RANDI strategy that
combines the advantages of RANdom and DIrected ap-
proaches. It involves systematic simultaneous sequencing on
both strands from clones of combined libraries without clon-
ing gaps. The random library fragments were generated by
separate partial digestion with two four-cutter restriction en-
zymes (Tsp, Sau3A), gel-purified and ligated into plasmid vec-
tor. In parallel, BAC or cosmid DNA was completely digested
with EcoRI (or HindIII) and fragments were isolated from aga-
rose gel and inserted into the pUC vector. Their sequences
served as a “scaffold” in the assembly of the complete se-
quence of the BAC genomic insert and also as templates for
primer walking in the finishing stage. Cycle sequencing of
plasmid DNA was performed with the AmpliTaqFS core kit
(Applied Biosystems), using forward and reverse primers la-
beled with FITC or CY5. An MJ Research PT-200 cycler was
used for 25 cycles (97°C, 15 sec; 55°C, 30 sec; 68°C, 30 sec).
Reactions were loaded off-gel on the 72-clone porous-
membrane combs, applied to 60-cm long polyacrylamide gels
(4.5% Hydrolink Long Ranger gel solution, FMC) and ana-
lyzed on the ARAKIS sequencing system with array detectors,
developed at EMBL (Erfle et al. 1997). This system allows si-
multaneous on-line sequencing of both strands (doublex se-
quencing), with the two sequencing products obtained in a
single sequencing reaction, each labeled with a different fluo-
rescent dye (Wiemann et al. 1995). Up to 2000 bases are thus
obtained simultaneously in one sequencing reaction, which
represents an efficient system for identifying large numbers of
long sequences in one run. Raw sequencing data were evalu-
ated, analyzed, and the consensus sequence assembled, using
the software packages (LaneTracker and GeneSkipper) de-
veloped at EMBL. Remaining sequencing gaps were covered
by primer walking (Voss et al. 1993). Direct cosmid or BAC
DNA sequencing was carried out essentially as described else-
where (Benes et al. 1997).
P-element Stocks and Mapping
A large-scale screen for insertions of the enhancer trap vector
P{lacW} (Bier et al. 1989) in essentialX chromosome genes has
been performed in H. Jäckle’s laboratory (Peter et al., in prep.).
Females homozygous for a male sterile insertion of the
P{lacW} element in chromosome 2 were crossed en masse to
w/Y; wg Sp/CyO; P{ry+=delta2–3}(99B) males. In the next gen-
eration five homozygous FM6 females were mated to two w/Y;
P{lacW}/CyO; P{ry +=delta2–3}(99B)/+ males. F2 daughters in
which the CyO and P{lacW} chromosomes had cosegregated
were individually mated to Fm7c/Y males. Lines that pro-
duced only FM6 sons in the F3 generation were kept as can-
didates for a lethal insertion. If these re-tested, then the lethal
insertion was kept in stock balanced with FM7c.
P{lacW} insertion sites were mapped by either plasmid
rescue or inverse PCR. DNA from adult flies was isolated using
a QIAGEN column, digested overnight with an appropriate
restriction enzyme, and then ligated under conditions favor-
ing intramolecular joining. For plasmid rescue, E. coli cells
were electroporated with the DNA and plated for the selection
of ampicillin resistant colonies. These were used to inoculate
small scale overnight cultures from which plasmid DNA was
then isolated. Cycle sequencing was performed with a primer
complementary to the 31-bp inverted repeat of the P-element
on an ABI373 DNA sequencer using dye terminator technol-
ogy. In the case of inverse PCR, we followed essentially the
Benos et al.
726 Genome Research
www.genome.org

protocol from the BDGP. We used their primers Plac1 and
Plac4 for the amplification of 5 sequences and primers Pry4
and Plw3–1 for the amplification of 3 sequences, respec-
tively. Sequencing was done as before with primer SP1 for 5
and primer SP6 for 3 analysis.
Sequence Analysis
Sequences were analyzed by the EDGP on a clone-by-clone
basis; i.e., only fully sequenced clones (cosmids or BACs) were
included. The overall analysis scheme is similar to that
adopted by other genome projects (e.g., C. elegans Sequencing
Consortium 1998).
tRNA genes were identified by tRNAscan-SE program, v.
1.0 (Lowe and Eddy 1997). Candidate protein coding genes
were predicted independently by GENEFINDER version 0.84
(P. Green, unpubl.) and the publicly available Genscan ver-
sion 1.0 (Burge and Karlin 1997). These two programs employ
fundamentally different algorithms and complemented each
other on gene discovery. GENSCAN and GENEFINDER had been
trained on a vertebrate gene set and a Drosophila-specific set
(compiled by G. Helt, pers. comm.), respectively. We mea-
sured the accuracy of prediction of the two programs with
already known Drosophila genes and we found them to be
comparable. However, each of them performed better on
a different set of genes. As expected, Drosophila-trained
GENEFINDER showed a preference for genes with fewer exons
and smaller introns when compared to the vertebrate-trained
GENSCAN.
Additional supporting evidence for the predicted genes,
as well as indications of their function, was obtained by simi-
larity searches against SWISS-PROT and TrEMBL protein da-
tabases (Bairoch and Apweiler 2000), Drosophila nucleic acid
sequences (derived from EMBL-Bank), andDrosophila EST sets,
generated by the BDGP (Rubin et al. 2000b) and by Andrews
et al. (2000). (Note that the annotation of version 1 of the
Joint Sequence did not use the entire BDGP EST data set; in
particular 4,654 3 ESTs, out of a total of 86,121, were not used
[S. Lewis, pers. comm.]). EST alignments were also used to
fine-tune the intron/exon boundaries of the predicted genes.
Simple repetitive sequences were filtered out by TANDEM, IN-
VERTED, and QUICKTANDEM programs (R. Durbin, pers.
comm..) whereas repeats of higher complexity were screened
out using similarity searches against Drosophila repetitive and
transposable element databases (see below). For protein and
nucleotide database searches we used BLASTX and BLASTN, v.
1.4.9. (Altschul et al. 1990), respectively.
Finally, protein domains/motifs of the predicted genes
were identified by PPSEARCH and HMMER (v. 2.1.1) programs,
scanning the PROSITE and PFAM databases, respectively.
PROSITE output was further filtered using the EMOTIF pro-
gram (Nevill-Manning et al. 1998).
All data generated by the automatic computational
analysis described above were parsed into an ACeDB-based
database (http://www.acedb.org/), XDrosDB, tailored to the
needs of the EDGP. The combined data were manually exam-
ined/analyzed using ACeDB software. During this analysis we
disregarded genes with a GENEFINDER score <50, if there was
no other supporting evidence for them (i.e., protein similarity
and/or EST matches). This cutoff is stricter than the one used
by the BDGP (cutoff = 20) for the analysis of the Adh region
(Ashburner et al. 1999); and, presumably, increases the num-
ber of rejected genes (false negatives). However, we chose to
set it this high to avoid overpredicting genes (false positives).
During the initial phase of our work, we, in collaboration
with the BDGP, created and subsequently curated three
datasets. One consisted of 1332 D. melanogaster coding se-
quences from genes that have been previously studied geneti-
cally and/or biochemically. This is a nonredundant set, i.e.,
only one copy of each gene is included in it. In case a gene
appears in multiple entries in the public databases (e.g., alter-
natively transcribed, submitted from more than one labora-
tory, etc.), we manually selected one copy (usually the best
documented or longest open reading frame). We used this
dataset to test the accuracy of the two chosen gene prediction
programs (GENEFINDER, GENSCAN), as well as a source for
hexanucleotides score calculation (GENEFINDER). This dataset
has been subsequently expanded/updated to include genes
identified by Drosophila genome projects (EDGP, BDGP, and
Celera), with the help of Leyla Bayraktaroglou (FlyBase at Har-
vard). Both the original and expanded versions, together with
information about their history, can be found at: ftp://
ftp.ebi.ac.uk/pub/databases/edgp/sequence_sets/or from
http://fruitfly.berkeley.edu/.
Similarly, a nonredundant collection of 47 D. melanogas-
ter transposable elements and another consisting of 96 mis-
cellaneous repetitive sequences were also assembled during
the initial phase of our project. These datasets were used to
identify complex repetitive regions, as described previously.
They are also available from the same ftp site or from the
BDGP site.
For clarity, we use the term “Joint Sequence” to refer to
v1.0 of the complete sequence of the genome of D. melano-
gaster (Adams et al. 2000) released on March 24, 2000 by Cel-
era. Comparisons of predicted, or known, protein sequences
from the EDGP project with those from the Joint Sequence
were done by CLUSTALW using the protein sequences of re-
lease 1.0 of the Joint Sequence (http://www.fruitfly.org/
sequence/sequence_db/aa_gadfly.dros of March 21, 2000).
These comparisons were then analyzed by hand. The com-
parison of the entire sequence of the X chromosome tip with
the sequence of the same region from the Joint Sequence was
done using the MUMmer program (Delcher et al. 1999), which
aligns long genomic regions by finding corresponding maxi-
mal unique matches. Nine separate alignments were done us-
ing the following GenBank accession nos.: AE003417,
AE003418, AE003419, AE003420, AE003421, AE003422,
AE003423, AE003424, and AE003425, each being matched
against the entire EDGP sequence. The resulting alignments
were analyzed by hand to find regions where the discrepan-
cies between the sequences were large. Figure 3 was drawn by
hand and is a graphic depiction of the alignment produced by
MUMmer. Large segments absent from one of the sequences
have been highlighted.
The results presented in this study were obtained by or
before February 7, 2000. However, if we had repeated the
same analysis today we would have assigned function (by
protein similarity) to 23 more of the predicted genes (raising
the percentage of the genes with significant protein similari-
ties to 66% of the 206 newly identified genes).
Supplementary data are available from ftp://ebi.ac.uk/pub/
databases/edgp/EDGP-GenomeResearch_suppdata_2001.
ACKNOWLEDGMENTS
This work was supported by a Contract from the European
Commission under Framework Programme 4 (coordinator
D.M. Glover), by a grant from the Medical Research Council,
London to M.A. and D.M.G., by a grant from the Direccio´n
General de Investigacion Cientı´fica y Te´cnica to J.M., by a
Sequencing the Drosophi la X Chromosome
Genome Research 727
www.genome.org

grant from the Hellenic Secretariat General for Science and
Technology to K.L., and by a grant from the Deutsche
Humangenomprojekt to H.J. R.D.C.S. was supported by a
Wellcome Trust Senior Fellowship. We thank many col-
leagues for their help. We are grateful to Gerry Rubin and his
colleagues at the BDGP, particularly Suzanna Lewis, Sima
Misra, and Susan Celniker (and, of course, Gerry himself) for
the exchange of materials, information, and ideas over the
years. Greg Helt of the BDGP was very helpful in providing us
with the initial Drosophila gene training set. We also thank
Rolf Apweiler and his SWISS-PROT/TrEMBL team at the EBI,
particularly Alexander Kanapin and Wolfgang Fleischmann
for their help with the protein motif analysis. We also thank
Rolf Apweiler, head of that team, for his blessings. Richard
Durbin’s group at the Sanger Center have been extraordinar-
ily helpful; in particular, Daniel Lawson gave tremendous
help with ACeDB despite having to bend double at times. Kim
Rutherford of the Pathogen Sequencing Unit at the Sanger
Center provided the software to draw Figure 1; without this
we may have been lost. We thank Brian Oliver of the NIH,
Bethesda for a pre-print copy of his paper on testis ESTs, Leyla
Bayraktaroglou (FlyBase group, Harvard) for her help in the
curation of reference sequence data sets, and David Judge of
the Cambridge School of Biological Sciences Biocomputing
Unit for help.
The publication costs of this article were defrayed in part
by payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 USC
section 1734 solely to indicate this fact.
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