The bithorax complex (BX-C) of Drosophila, one of two complexes that act as master regulators of the body plan of the fly, is included within a sequence of 338,234 bp (SEQ89E). This paper presents the strategy used in sequencing SEQ89E and an analysis of its open reading frames. The BX-C sequence (BXCALL) contains 314,895 bp obtained by deletion of putative genes that are located at each end of SEQ89E and appear to be functionally unrelated to the BX-C. Only 1.4% of BXCALL codes for the three homeodomaincontaining proteins of the complex. Principal findings include a putative ABD-A protein (ABD-AII) larger than a previously known ABD-A protein and a putative glucose transporter-like gene (1521 bp) located at or near the bithoraxoid (bxd), infra-abdominal-2 (iab-2) boundary on the opposite strand relative to that of the homeobox-containing genes.

We describe here a transposon-based DNA sequencing strategy that allows the introduction of sequencing priming sites throughout a target sequence by bacterial mating. A miniplasmid was designed to select against transposon insertions into the vector. Sites of transposon insertion are mapped by the polymerase chain reaction with bacterial overnight cultures providing the templates. A small set of plasmids with transposons spaced several hundred base pairs apart can then be sequenced. Sequencing primers corresponding to the transposon ends allow sequencing in both directions. Thus, the entire sequence of both strands can be easily determined.One ofthe major problems in DNA sequence analysis oflarge or even moderately sized fragments is how to position unsequenced regions next to known priming sites. A variety of techniques have been developed for this purpose including random shotgun subcloning, unidirectional deletions and subcloning, and the continued synthesis of additional oligodeoxynucleotide primers (1-4). These methods are expensive or require many molecular manipulations.A number of strategies employ bacterial transposons to generate priming sites within a target DNA sequence (5-10). Several criteria exist for an efficient transposon-based sequencing strategy: (i) Mobilization of the transposon must be relatively simple. (ii) Selection for transposon insertions into the plasmid as opposed to the bacterial chromosome must be efficient. (iii) The transposon must insert into the target sequence and not into the plasmid vector. (iv) The transposition sites must be easily mapped to minimize the number of required sequencing reactions. In this paper we describe a transposon-based strategy that meets these criteria.We employ y6, which belongs to the Tn3 family of transposons (11) and which has been used previously in transposon-facilitated strategies (8,20). The members of this family contain 38-base-pair (bp) terminal inverted repeats and transpose by a replicative mechanism. Donor and target sequences are joined in an intermediate structure termed a cointegrate. The cointegrate, which contains two copies of the transposon, is rapidly resolved by a site-specific recombination system. The resolvase is encoded by the transposon and acts at the 120-bp res site located within the mobile element.

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We describe a method of screening for transposon insertions in or near Drosophila loci that correspond to cloned DNA sequences. We mobilize a modified P element transposon that carries a bacterial plasmid origin of replication and a drug-resistance marker. The genomic sequences flanking each transposon insertion site can then be rescued as a plasmid in Escherichia coli. Libraries of such plasmids, representing pools of transposon-mutagenized individuals, are used as hybridization probes against cloned sequences to determine whether a transposon has inserted next to a particular site in the genome. The number of loci that can be screened simultaneously by this procedure is quite large. We have screened an array of cDNA clones representing almost 700 distinct loci against libraries representing 760 mutagenized flies, and we obtained hybridization signals to 7 different cDNAs. Three of these events have been analyzed in detail and represent genuine insertions near genomic sequences that correspond to the cDNAs.

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