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Comparative genomic approaches to gene and cis-regulatory prediction are based on the principle that differential DNA sequence conservation reflects variation in functional constraint. Using this principle, we analyze noncoding sequence conservation in Drosophila for 40 loci with known or suspected cis-regulatory function encompassing >100 kb of DNA. We estimate the fraction of noncoding DNA conserved in both intergenic and intronic regions and describe the length distribution of ungapped conserved noncoding blocks. On average, 22%-26% of noncoding sequences surveyed are conserved in Drosophila, with median block length approximately 19 bp. We show that point substitution in conserved noncoding blocks exhibits transition bias as well as lineage effects in base composition, and occurs more than an order of magnitude more frequently than insertion/deletion (indel) substitution. Overall, patterns of noncoding DNA structure and evolution differ remarkably little between intergenic and intronic conserved blocks, suggesting that the effects of transcription per se contribute minimally to the constraints operating on these sequences. The results of this study have implications for the development of alignment and prediction algorithms specific to noncoding DNA, as well as for models of cis-regulatory DNA sequence evolution.

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies--although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.

We report findings on naturally occurring variation in the regulatory region of even-skipped in Drosophila. This pair-rule gene encodes a homeobox-containing transcription factor, is expressed as a series of seven transverse stripes in developing embryos, and defines parasegmental boundaries. The 5' flanking region of the gene contains a 671-bp enhancer governing stripe 2 expression. The stripe 2 enhancer contains multiple binding sites for four transcription factors that provide positional information in developing blastoderm, the positive regulators bicoid and hunchback and the repressors giant and Kruppel. The study compares polymorphism and divergence in Drosophila melanogaster and Drosophila simulans for the enhancer region, the spacer between the enhancer and the transcription start site, the untranslated leader, the first exon and the intron of eve. Contrary to expectations, we find a relatively high level of variation in the stripe 2 enhancer region, including point substitutions and insertion/deletions in binding sites, and a comparable level of variation in the other noncoding regions. The patterns of variation and divergence within the enhancer region and between regions of the locus fit a model of neutral molecular evolution. We suggest that the multiplicity of binding sites in the enhancer provides a redundancy in function that allows flexibility in the sequence requirements and structural design of the promoter.

A filter hybridization method is described for identifying restriction-site and insertion/deletion variation by using restriction enzymes that recognize four-nucleotide sequences and denaturing polyacrylamide gels for separating fragments. Eighty-seven lines of Drosophila melanogaster representing two natural populations were surveyed over a 2.7-kilobase region encompassing the alcohol dehydrogenase locus. Fifty distinct haplotypes were identified from 17 restriction-site and 11 insertion/deletion polymorphisms and from one allozyme polymorphism. There was no evidence for genetic differentiation between an East-Coast and a West-Coast (North American) sample. This technique has widespread applications in screening for DNA polymorphism.