Map Position and Expression of the Genes in the 38 Region of Drosophila

Corresponding author: Beat Suter, Department of Biology, McGill University, 1205 Dr. Penfield Ave., Montreal, QC H3A 1B1 Canada., beat_suter{at}maclan.mcgill.ca (E-mail)

Communicating editor: T. SCHÜPBACH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION APPENDIX LITERATURE CITED

With the completion of the Drosophila genome sequence, an important next step is to extract its biological information by systematic functional analysis of genes. We have produced a high-resolution genetic map of cytological region 38 of Drosophila using 41 deficiency stocks that provide a total of 54 breakpoints within the region. Of a total of 45 independent P-element lines that mapped by in situ hybridization to the region, 14 targeted 7 complementation groups within the 38 region. Additional EMS, X-ray, and spontaneous mutations define a total of 17 complementation groups. Because these two pools partially overlap, the completed analysis revealed 21 distinct complementation groups defined by point mutations. Seven additional functions were defined by trans-heterozygous combinations of deficiencies, resulting in a total of 28 distinct functions. We further produced a developmental expression profile for the 760 kb from 38B to 38E. Of 135 transcription units predicted by GENSCAN, 22 have at least partial homology to mobile genetic elements such as transposons and retroviruses and 17 correspond to previously characterized genes. We analyzed the developmental expression pattern of the remaining genes using poly(A)⁺ RNA from ovaries, early and late embryos, larvae, males, and females. We discuss the correlation between GENSCAN predictions and experimentally confirmed transcription units, the high number of male-specific transcripts, and the alignment of the genetic and physical maps in cytological region 38.

DROSOPHILA is an outstanding model system for the study of gene activity in higher eukaryotes, and much of what we know about genetic pathways and how they function to build a complex organism rests upon work carried out in flies. Its utility is rooted in the experimental genetics that has attained an extraordinarily high level of sophistication over nearly a century of continuous development. Recently, a milestone was reached with the sequencing of the Drosophila genome (ADAMS et al. 2000 ), which removes the need for cloning and sequencing individual genes. Biological information must now be extracted from the genome sequence by systematic functional analysis of genes. Computer analysis alone can reveal only some of these functions. The large majority of genes have either no obvious function that can be predicted from their sequence or only a very general one, such as RNA binding, which gives no insight into a gene's specific developmental and biological role. Over the years a huge number of mutant fly strains have accumulated in numerous different laboratories. These mutants are the primary resource for functional analysis of these open reading frames (ORFs) and genes.

Outside of genome-based efforts, much of the data created in characterizing a specific gene is not relevant for this particular gene and is therefore often lost, even though it may become interesting for someone else later on. Nonsystematic analyses can often be redundant as well. For instance, the gene neb (= Klp38B = Mothra) at chromosomal location 38B4 was cloned and genetically characterized by several laboratories (ALPHEY et al. 1997 ; MOLINA et al. 1997 ; OHKURA et al. 1997 ; RUDEN et al. 1997 ). This sort of laborious effort can be rendered obsolete by genome-scale mapping projects. As many existing mutant strains were generated outside of coordinated genome efforts, and have been mapped to varying degrees of precision, we started to systematically collect and exhaustively map available mutants in the proximal 2L region. Here we report the genetic map of the cytological interval 38, the alignment of the genetic and physical maps, and the experimental identification of transcription units in the region.

Because much of the focus on Drosophila research is on identifying developmental processes, which are well conserved between Drosophila and mammals (MERRIAM et al. 1991 ; RUBIN et al. 2000 ), we also analyzed the developmental expression pattern of the various transcription units within region 38. These expression data provide experimental support for computer-predicted transcripts and give valuable information to researchers who expect a specific expression pattern based on a mutant phenotype.

The Drosophila melanogaster genome consists of four chromosome pairs that can be visualized under the light microscope by looking at the polytene chromosomes of larval salivary glands. As early as the 1930s these polytene chromosomes were mapped according to the banding pattern seen by histological staining (BRIDGES 1935 ). This cytological map made Drosophila the first organism to have a physical map, albeit of a low resolution, 100 kb (RUBIN 1996 ). The cytological map divides the chromosomes into numbered regions, which in turn are subdivided into lettered intervals.

Polytene region 38 is situated on the left arm of the second chromosome. It is divided into six lettered intervals that are subdivided into a total of 45 numbered intervals: A, 1–8; B, 1–6; C, 1–10; D, 1–5; E, 1–10; and F, 1–6. Region 38 contains 1 Mb of genomic sequence. It was chosen for analysis in part as a result of its location adjacent to previously characterized regions, in particular the Adh region from 34C to 36A (ASHBURNER et al. 1999 ) and the 37 region around Ddc (37B/C; STATHAKIS et al. 1995 ). Collections of lethal, maternal effect lethal, and female sterile mutations were available for the mapping project (SCHUPBACH and WIESCHAUS 1989 , SCHUPBACH and WIESCHAUS 1991 ; ERDELYI et al. 1997 ; KOZLOVA et al. 1998 ; SPRADLING et al. 1999 ). Because these mutants were isolated in different laboratories, most of them had not previously been systematically mapped with respect to one another.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION APPENDIX LITERATURE CITED

Genetic analysis:
Genetic mapping of point mutations was achieved through complementation analysis with deficiency stocks that break within the 38 interval. With the exception of purple and valois, the mutants were mapped by their recessive lethal or recessive sterile phenotypes. Point mutations mapping to the same deficiency interval were subsequently tested for complementation. The majority of the tested alleles were created by either EMS or P-element mutagenesis. Deficiency chromosomes and point mutations were isolated in a number of different laboratories and are listed in Table 1 and Table 2.

Sequence analysis:
Our molecular work was based on P1 and bacterial artificial chromosome (BAC) genomic sequences produced by the Berkeley Drosophila Genome Project (BDGP; Table 3). Data were obtained from http://www.fruitfly.org/sequence/drosophila-regions.html.

The program GENSCAN (BURGE and KARLIN 1997 ) was chosen to analyze the 38 sequence (http://genes.mit.edu/GENSCAN.html). In this study the vertebrate option with default parameters (ISOCHORE 1) was used, except where indicated. The GENSCAN predictions were the basis for all transcript analysis within region 38 and probes were designed for predicted genes on the basis of probability scores and prediction of exon structure.

Peptide homologies and EST searches:
As GENSCAN results are based on gene prediction algorithms only, additional sequence annotations were carried out to identify sequence similarities with those in the public domain. Each predicted peptide resulting from a GENSCAN prediction was run through the National Center for Biotechnology Information BLAST server (http://www.ncbi.nlm.gov/BLAST) using the BLASTP program with default parameters to search for homology with other gene products. Matches with a smallest sum probability of <1 x 10^-4 were taken as having significant homology and were noted. Expressed sequence tagged (EST) databases were searched with the entire genomic sequence of the region. In each case, the appropriate section of genomic DNA was used to search the dbEST from the BDGP web server (http://www.fruitfly.org/blast/). ESTs matching in >90% of their length were noted.

Generation of sequence tag sites:
The P elements with flanking genomic DNA were recovered from the stocks by inverse PCR (J. Rehm; http://www.fruitfly.org/about/methods/inverse.pcr.html). The amplified sequences were cloned into vectors before being isolated for sequencing. Sequencing was done with an Applied Biosystems (Foster City, CA) ABI 373 DNA Sequencer using 250–500 ng of the sample DNA and 3.2 pmol of T7 primer.

Northern and Southern blots:
Northern blots were prepared and hybridized as previously described (SUTER et al. 1989 ). Probes were made from PCR-amplified recombinant P1 bacteriophages containing the genomic region of interest. PCR primers were designed in accordance to recommendations by Applied Biosystems with an oligonucleotide length of 19–23 bases containing 12 G or C nucleotides and 7–11 A or T nucleotides, with an A or a T nucleotide at the 3' end.

To produce short probes for predicted ORFs, the primers were designed to amplify sequences within a predicted internal exon and/or to the exon with the highest probability score. They were, on average, 100–200 bp apart and used to amplify directly from the appropriate P1 clone. These probes were primarily used for the detection of transcripts.

For the 10-kb genomic fragments, primers were designed so that each set of primers overlapped by 100 bp. The fragments were amplified using the Expand Long Template PCR System (Boehringer/Roche Diagnostics) and the appropriate P1 clone as a template. The PCR reaction was performed with 1 ng DNA template, 2.5 µl buffer 3, 3.5 µl dNTP (2.5 mM), 2.5 µl each primer (300 mM), 0.5 µl Expand Taq (3.5 units/µl), H₂O to a final reaction volume of 25 µl. PCR cycles were 10 sec at 92° and 8 min at 68° (x30) and the reaction was done using a hot start. These probes served primarily for the detection of aberration breakpoints. Labeling of amplified fragments was done by incorporation of [-³²P]dCTP through random priming.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION APPENDIX LITERATURE CITED

The genetic map of 38:
Procedure and definition of distal and proximal ends of the genetic map: Cytological division 38 is completely eliminated by the deficiency Df(2L)TW65. We selected mutants that failed to complement this deficiency for detailed mapping, even though Df(2L)TW65 extends into division 39. The distal end of Df(2L)TW65 defines the distal end of our genetic map of 38 (Fig 1). A total of 41 deficiency stocks, providing a total of 54 breakpoints within or at the border of the interval, were used to genetically map all available lethal and sterile mutations to the smallest possible deficiency intervals. All mutations mapping to the same deficiency interval were tested against each other for cross-complementation. The most distal complementation group included in this genetic map is scw (Fig 1), which cytologically maps to 38A1-2 (ARORA et al. 1994 ). On the proximal end of the genetic map we included complementation groups that mapped to Df(2L)DS9 but are excluded from Df(2L)bur-K1. On the basis of these criteria, l(2)38EFd is the most proximal complementation group. This gene was mapped to 38F5-6 by in situ hybridization (SPRADLING et al. 1999 ).

View larger version (200K): In this window In a new window Download PPT slide   Figure 1. Genetic map of cytological region 38. The borders of 38 are indicated with two big arrowheads on top of the map. The in the "mostly used allele" row indicates that the complementation group is defined by overlapping deficiencies. The cytology of P-element alleles is according to SPRADLING et al. 1999 and for the deficiencies according to FlyBase. Bold column borders separate complementation groups that map to different deficiency intervals. Faint column borders indicate that neighboring complementation groups could not be mapped relative to one another because they map to the same deficiency interval. Such complementation groups may therefore trade places in the future. The results of the complementation analyses are shown as follows: "mutant" indicates lethality, sterility, or a visible mutant phenotype (for pr only) and "wt" means wild type. In general, no or only very few escapers were found. However, for some alleles [e.g., l(2)03552] escapers were often seen. A question mark means that the result was ambiguous and an asterisk means that the complementation test was done with an alternative allele and not with the mostly used one. "2nd" site means that the observed lethality is caused by a second-site mutation. It is not clear whether l(2)38Aa is a lethal allele of pr or a different gene.

Not counting P-element insertion lines already known to be allelic to other lines, a total of 45 independent P-element lines (supplemental material 1 at http://www.genetics.org/supplemental) and 48 EMS, X-ray, and spontaneous alleles suspected to represent 27 potentially different complementation groups were considered (supplemental material 2 at http://www.genetics.org/supplemental). Fourteen of the P-element alleles targeted genes within the 38 interval that are essential for viability or fertility. These alleles define 7 complementation groups. The EMS, X-ray, and spontaneous mutations were found to define a total of 16 complementation groups that map to the 38 region. In addition, the frequently used marker gene pr also maps to 38. Because of overlap between these classes of complementation groups, the completed analysis revealed 21 distinct complementation groups (Fig 1).

In addition to these 21 complementation groups, trans-heterozygous combinations of deficiencies reveal seven more regions containing genetic functions essential for viability or fertility. The results of these crosses are shown in the Appendix. Failure of complementation between Df(2L)pr8311, Df(2L)pr49, Df(2L)pr1122, Df(2L)TW1, and Df(2L)TW161, on one side, and Df(2L)Sd37 and Df(2L)TW50, on the other side, appears to define a lethal and sterile region in 38A/B. This noncomplementation does not seem to be caused by a second-site mutation on the Df(2L)TW50 or Df(2L)Sd37 chromosome in the proximal 38 region, because even deficiencies that eliminate a large region from barren into cytological region 39/40 [Df(2L)pr-M1, Df(2L)pr11163] complement Df(2L)TW50 and Df(2L)Sd37, whereas a comparatively small deficiency from barren to 38D/E (Df(2L)pr8311) does not. The fact that five deficiencies from different sources fail to complement at least part of the lethal and sterile region in 38A/B also argues against a second-site mutation in the 37D–38A region that causes the noncomplementation. The different phenotypes of trans-heterozygotes can best be explained by postulating the presence of four different subregions in the lethal and sterile region in 38A/B. From distal to proximal these are a lethal, a female sterile, a male sterile, and another lethal region (Fig 1).

Df(2L)Fs(2)Ket-RX32 has its distal breakpoint between spir and l(2)38Db, and Df(2L)40 has its proximal break between these two complementation groups. These two deficiencies are lethal when trans-heterozygous (see the Appendix). With the caveats discussed above, this result indicates the presence of at least one more essential gene in the 38C region.

The phenotype of trans-heterozygous combinations of deficiencies with breakpoints between l(2)38Db and l(2)38Ea define another two genetic functions in the 38D/E region (see the Appendix). Df(2L)DS5 and Df(2L)DS9 have their distal breakpoints in this region and they were crossed to each of the following deficiencies that have their proximal breakpoints in this interval: Df(2L) pr9201, Df(2L)pr-A16, Df(2L)pr37, Df(2L)pr2b, and Df(2L) pr8311. The phenotypes of these various trans-heterozygous deficiency combinations can be best explained by making the following assumption: between the l(2)38Db and the l(2)38Ea complementation groups, from distal to proximal, there are a male sterile region and a (semi)lethal region with escapers being female sterile. This raises the total of genetically identified functions and complementation groups to 28. A small number of the results shown in the Appendix are inconsistent with the proposed genetic map. These inconsistencies presumably result from additional hits on the chromosome. This is likely because many of these strains have been kept balanced in the laboratory for many years and, without selection, they accumulate additional lethal mutations and dominant modifiers.

The stock l(2)38Aa[1] and some of the deficiency stocks used for the analysis, Df(2L)be408 and in particular Df(2L)Fs(2)Ket-RX32, had additional lethal mutations elsewhere on the chromosome (see the Appendix and supplemental material 2 at http://www.genetics.org/supplemental/), thus complicating the complementation analysis. The second-site hit for l(2)38Aa[1] is in l(2)37Ea. Noncomplementation due to this second-site hit with deficiencies in the 37 region is indicated on Fig 1 as such. According to FlyBase (SPRADLING et al. 1999 ), l(2)k13715 has several P-element hits, one in 38F1-2 [allelic with l(2)01528], one in 87C6-7, and another lethal hit in 39A [allelic with l(2)05287]. We do not have any indication that our l(2)k13715 chromosome carries more than the lethal mutation in 38F [allelic with l(2)01528] because it complements l(2)05287 and Df(2L)burK1, a small deficiency that removes l(2)05287.

Phenotypes of complementation groups in 38: A total of 23 of the 28 genetically identified functions shown in Fig 1 have recessive lethal phenotypes. Two are male sterile [the 38A/B region between the proximal breakpoint of Df(2L)TW50 and the distal breakpoint of Df(2L)pr8311 and the 38D/E region between the proximal breakpoint of Df(2L)pr-A16 and the distal breakpoints of Df(2L)DS5 and Df(2L)DS9] and three are female sterile [spire, vls, and the female sterile defined by the distal breakpoints of Df(2L)pr8311 and Df(2L)TW1 in the lethal and sterile region in 38A/B]. Several of the lethal complementation groups have additional phenotypes: a recessive female sterile allele [fs(2)ltoPP43] is allelic to l(2)38Ac, Ketel has dominant and recessive female sterile alleles, and the essential gene diaphanous has a male sterile allele [ms(2)04318] and a maternal effect lethal allele (CASTRILLON et al. 1993 ; AFSHAR et al. 2000 ). Hypomorphic mutations of the recessive lethal pr have a recessive visible phenotype. Various alleles of Bristle are either recessive lethal or female sterile and show different dominant and recessive visible phenotypes. spire and Bristle were mapped by their female sterility and vls by its grandchildless phenotype.

The physical and transcript map of 38:
Most of the work was done with the sequence provided by the BDGP (http://www.fruitfly.org), which initially subdivided the region in different contigs that were available from the BDGP website (http://www.fruitfly.org/sequence/drosophila-regions.html; Table 3). Once the complete genome sequence became available (ADAMS et al. 2000 ), this map was updated, the sequences of nine P1s and one BAC were fused, and transcription units renamed. We chose to set the limits for the physical map on the basis of contigs sequenced by BDGP. The entire region is 760 kb and covers the cytological regions 38B–38E (Fig 2). We have subdivided the physical map of 38 into the alphabetical subregions. The border between the subregions is placed according to the in situ mapping data for the P1 clones and BACs used to establish the sequence.

View larger version (49K): In this window In a new window Download PPT slide   Figure 2. Molecular map of the cytological region 38B–38E. The blue line represents the genomic DNA with a black mark every 10 kb. Triangles above the blue line indicate insertion sites for P elements for which the lethality or sterility was genetically mapped (see also Fig 1). The extension of the sequenced clones is shown with green arrows. The cytology of the P1s was used to define the cytology of the region. Hence, 38B is defined as starting with DS00863 and ending immediately before the start of DS01096 (position 1 78,614); 38C is defined by the beginning of DS01096 and it ends where DS08416 starts (position 78,615 406,914); 38D is defined by the beginning of DS08416 and ends immediately before DS04217 ends (position 406,915 616,781); and 38E is defined by DS04217 (position 616,782 759,580). A total of 759,580 bp (black line) were analyzed with the GENSCAN gene prediction program, and a total of 135 transcription units were predicted. According to the position of their 5' ends, they were named 38B.1–38B.23, 38C.1–38C.56, 38D.1–38D.32, and 38E.1–38E.24 (in this figure "38" is omitted from their name). These cytological designations may not always precisely reflect the cytological position of the transcription unit. *: 38B.23, 38D.32, and 38E.24 are nested genes identified by using an appropriate genomic fragment. Transcripts for these three genes identified by Northern blots may therefore originate from a neighboring gene. 38B.23 and 38E.24 were identified with the ISOCHORE2 of GENSCAN. A total of 63 transcription units are encoded on the positive strand (arrows above the black line) and 72 on the negative strand (arrows under the black line). The transcription units that correspond to known genes bear their names written in black, and the ones that correspond to known genes for which mutant fly stocks are available also bear their names in red below. The genomic region to which deficiency breakpoints were mapped through restriction fragment length polymorphism is indicated with dashed bidirectional brown arrows. The extent of the deleted genomic region of the corresponding deficiency is shown with a brown line. An arrowhead at the end of a brown line means that the deficiency continues beyond the analyzed region. A number of complementation groups shown in Fig 1 could not be mapped to a single transcription unit. The physical interval to which they were mapped by various methods is shown with red dashed bidirectional arrows.

Identification of transcription units: All mapping of transcription units was based on predictions by the gene-finding program GENSCAN (BURGE and KARLIN 1997 ). Additional information, such as homology to other polypeptides and EST hits, was determined separately by sequence database searches. Polypeptide homologies were established using the BLASTP program of BLAST (ALTSCHUL et al. 1990 ) and EST hits were identified using the BDGP BLAST server (http://www.fruitfly.org/blast/) against the Drosophila EST data set. The probability scores provided in the following text for each prediction refer to the exon to which the probe was designed; most of the time this is also the exon with the highest probability score for that particular prediction. Fig 2 shows the position and direction of the predicted transcription units. Table 4 and supplemental material 3 (at http://www.genetics.org/supplemental/) list their names, positions, homologies, probability scores, and other characteristics.

GENSCAN predictions and expression of predicted genes: GENSCAN predicted a total of 135 genes to lie within the 760 kb of sequence analyzed. Of these, 17 correspond to genes that have previously been characterized and another 22 are at least partially homologous to mobile genetic elements such as transposons and retroviruses (Table 5). To test these gene predictions and to determine the expression patterns of predicted genes, probes were designed for 121 known and predicted genes, and developmental Northern blots containing mRNA from six different stages and tissues were probed. The chosen stages reflect most of the fly life cycle plus isolated ovaries. In total, these experiments allowed us to determine the expression pattern for an additional 64 of the 96 potential new transcription units (in addition to the previously published ones and the mobile elements). GENSCAN predictions, the autoradiographs of Northern blots, and a summary table of their developmental expression profile can be seen on http://www.mcgill.ca/Biology/labs/MDGP/transcripts.html.

About half of the predicted genes, 68, had similarity to known proteins. Similarity was determined using BLASTP and matches with a smallest sum probability of <1 x 10^-4 were noted. Of the 46 predicted polypeptides with a homology other than to mobile elements, 45 or 98% were confirmed experimentally by detection of a signal on a Northern blot.

Thirty-six, or about one-half of the 67 predicted peptides with no significant homology, revealed detectable transcripts on Northern blots. EST searches carried out for the remaining 31 predictions validated an additional 3. In cases where probes were designed to an exon with a probability score of <0.5, 57% failed to give a signal on a blot. Most primers were designed to amplify one exon of each predicted ORF in order to generate a probe for use on developmental Northern blots. Lack of detection of transcripts by specific probes accordingly may be caused either by lack of mRNA expression or accumulation or by alternative processing that eliminates this particular exon in a specific stage and tissue.

Large number of male-specific transcripts: The poly(A)⁺ mRNA used on each blot was isolated from 0- to 4-hr embryos, 8- to 20-hr embryos, the three larval instars, mature males, mature females, and ovaries. In general, the majority of transcripts detected were not ubiquitous to all stages and differential expression was detected in >80% of cases. No obvious clustering of transcripts with similar expression profiles was noted.

Of the 64 new predictions that we confirmed by Northern blots, 24 had at least one transcript that is expressed only in mature males. Nine of the probes did not detect any other transcript. The absence of transcripts in the other stages we analyzed may suggest that the gene has a male-specific role. However, the large number of such transcripts argues against this interpretation because only two genes with male sterile phenotypes are known to map to 38. One of these is an allele of diaphanous and the other is ms(2)38C (BRITTNACHER and GANETZKY 1983 ), which cytologically maps to 38C5-C10. While it is possible that phenotypes for the other genes may be found with appropriate mutant screens, it seems just as likely that adult males express many genes that are not essential for viability and normal development and are shut down in females and most other developmental stages.

Only one of the 10 predictions encoding male-specific transcripts, 38C.2, has homology to a known gene; 38C.2 is homologous to antigen 5-related, a gene encoded on the X chromosome of D. melanogaster, which in turn has partial homology to a sperm-coating protein from rat epididymis. However, a direct search with 38C.2 did not pick up the sperm-coating protein. There is one more prediction with homology to a male-specific protein, 38D.26, which is homologous to a human sperm acrosomal protein. Surprisingly, however, the 38D.26 transcript is expressed in all stages of development and much more strongly in females, ovaries, and 0- to 4-hr embryos than in males.

Genome organization in the 38 region: A heterochromatic region of close to 200 kb extending from proximal 38B to the 38C region [between l(2)38Ab and spir] contains a large number of mobile genetic elements. This region is highly repetitive in nature and contains no complementation group. On the basis of 81 confirmed transcription units that do not correspond to mobile elements (Table 4 and supplemental material 3 at http://www.genetics.org/supplemental), the overall gene density in the 38 region is approximately one gene in 9.2 kb. However, in some parts of the region the gene density can be much higher than that, reaching one gene per 3.2 kb in the central part of 38B.

Alignment of genetic and physical maps:
The alignment of the genetic and the physical map was achieved primarily by using two sources of sequence tag sites (STS). The cloning of a number of genes from the 38 region was reported in the past few years. These include barr (BHAT et al. 1996 ), pr (KIM et al. 1996 ), neb (ALPHEY et al. 1997 ; MOLINA et al. 1997 ; OHKURA et al. 1997 ; RUDEN et al. 1997 ), spir (WELLINGTON et al. 1999 ), Hr38 (KOZLOVA et al. 1998 ), Fs(2)Ket (ERDELYI et al. 1997 ), dia (CASTRILLON and WASSERMAN 1994 ), cad (MLODZIK et al. 1985), and others. Additional STS were obtained by sequencing the genomic regions flanking the P-element insertion sites. After eliminating known allelic P-element insertions, 14 P elements that cytologically mapped to the 38 interval were found to have their lethality in the 38 region. Of these 10 are alleles of either barren, nebbish, Hr38, or diaphanous, all of which are cloned and thus their positions already known on the physical map. Supplemental material 1 (http://www.genetics.org/supplemental) summarizes the information about these P elements. The BDGP has generated STS from the 5' ends of the P elements disrupting the complementation groups l(2)38Ac and l(2)38EFd. The remaining P-element lines are stocks containing two insertions and no STS were made from these lines. The first line, l(2)k09314, genetically maps within the 38B interval but proved difficult to map more finely as many crosses result in semilethal phenotypes. The second line, l(2)k13715, is allelic to l(2)01528 and together they define the new complementation group l(2)38EFg. We generated 3'-end STS for the P-element insertions in l(2)k09314 and l(2)k13715 (Fig 3). Each STS was tested for alignment to the 38 sequence. One of the STS generated from l(2)k09314 aligned between purple and nebbish (GenBank accession no. AZ575480). It localizes between the predicted nebbish promoter and the transcription start site (Fig 2). One of the STS from l(2)k13715 matched to the 38F region and is at the proximal end of the map in Fig 2 (GenBank accession no. AZ575479).

View larger version (16K): In this window In a new window Download PPT slide   Figure 3. The stocks l(2)k09314 and l(2)k13715 have a P-element insertion in 38. DNA adjacent to the 3' end of these P elements was sequenced and the generated STS are shown. The sequences were submitted to GenBank [accession nos.: AZ575479 for l(2)k13715 and AZ575480 for l(2)k09314].

Physical mapping of selected deficiency breakpoints: We mapped selected deficiency breakpoints to further align the genetic and physical maps. To reduce the number of polymorphisms in the analyzed heterozygous stocks, all but two deficiency chromosomes were first crossed to an isogenic balancer chromosome (CyO bw). The exceptions are Df(2L)pr1 and Df(2L)pr11, which were induced on a CyO balancer chromosome. These deficiencies were kept over a Sco chromosome and were always blotted side by side where they could serve as controls for each other.

Genomic fragments of 10 kb were amplified and used to probe filters containing restriction-digested genomic DNA from balanced deficiency flies. A total of 317.5 kb of sequence was covered in these experiments. Five deficiency breakpoints were identified in this way, and their locations are shown in Fig 2. One of the breakpoints, distal Df(2L)pr1, breaks genetically between vls and pr in 38B. Two deficiency breakpoints, proximal Df(2L)TW9 and proximal Df(2L)pr1, fall between l(2)38Ab and spire. Identification of the physical location of these last two breakpoints places l(2)38Ab distal to position 70 kb and spire proximal to position 185 kb. The recent cloning of spire confirmed that it maps to the region between 260 kb and 290 kb (Fig 2 and WELLINGTON et al. 1999 ). Although the breakpoints of these two deficiencies are genetically in the same region, physically they are separated by 130 kb. Two other breakpoints were uncovered proximal to spire: proximal Df(2L)pr40 and proximal Df(2L)pr11. Identification of Df(2L)pr40 places the lethal region in 38C distal to position 475 kb, and l(2)38Db, the lethal and sterile region in 38D/E, and l(2)38Ea proximal to position 450 kb (Fig 2). The molecular mapping of proximal Df(2L)pr11 contradicts the genetic mapping. Genetically, the deficiency removes Hr38, but its molecular mapping puts the breakpoint distal to Hr38. It is therefore possible that this deficiency chromosome has an additional mutation in the Hr38 gene.

Conclusion:
The expression profiling of the predicted transcripts of the 38 region provides experimental evidence for 81 of 113 predicted single copy genes. The developmental profile gives further useful information for researchers with interest in developmental biology. The high resolution genetic map of the 38 region presented here identifies the genetic breakpoints of 41 deficiency chromosomes. The analysis of the various types of genetic aberrations in the region revealed a total of 28 functions on this map. By creating new links between the genetic and the physical map we were able to further improve the genetic map's resolution. The detailed map now provides the D. melanogaster research community with the necessary information to more efficiently use the genetic resources available in region 38.

	FOOTNOTES

¹ Present address: FlyBase, Department of Genetics, University of Cambridge, Downing St., Cambridge, CB2 3EH, United Kingdom.
² Present address: DNA LandMarks Inc., St-Jean-sur-Richelieu, QC J3B 6X3, Canada.

	ACKNOWLEDGMENTS

We thank all our colleagues who supplied us with the mutant fly strains. We are grateful to the BDGP for making 38 a priority region for sequencing and for supplying us with P1s and numerous P elements. Special thanks to Amy Tan and Jonathan Spicer for their mapping efforts, and to all the present and past members of the group for their support. We also thank Michelle Peters-Akit for her help in putting together the manuscript. Our flies wish to acknowledge the gourmet fly food made by Nguyen Lee. This work was supported by a CGAT and Canadian Institute of Health Research genomics grant and by a grant from the Fonds pour la formation de Chercheurs et l'aide à la Recherche to P.L. and B.S. P.L. and B.S. were Research Scientists of the National Cancer Institute of Canada supported by funds from the Canadian Cancer Society and are now Canadian Institute of Health Research Investigators. R.D. was supported in part by a postdoctoral fellowship from the Swiss National Science Foundation.

Manuscript received December 14, 2000; Accepted for publication May 10, 2001.

	APPENDIX

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION APPENDIX LITERATURE CITED

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION APPENDIX LITERATURE CITED

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