Analysis of DNA sequence variation among alleles of Cecropins, Attacins, and other pathogen-defense molecules produced by insects is revealing how insects are able to deal with diverse bacterial pathogens. We are quantifying patterns of DNA sequence variation in most of the known antibacterial genes and have found that, as a class, they exhibit high levels of variability and an unusual pattern of clustered singleton sites. We are developing the theory and analyses to determine the causes for these patterns of variation. Both a model of simple hypervariability and an "arms race" model can be firmly rejected, leaving models that rely on spatial structuring as the best explanation for observed patterns of variation. Many antibacterial genes occur as small gene families, and whereas the Cecropin cluster exhibits fairly rapid birth-and-death of genes, the Attacin cluster has definite intergenic conversion events. Functional aspects are being studied as well, including variation in transcriptional response to infection, and variation in the dynamics of intra-fly bacterial growth, and whole-fly survival. Variation in bacterial virulence in Drosophila is also being studied. The primary question is, “How can insects, whose immune system lacks memory, evade bacterial pathogens?”
Perhaps our most exciting recent success in genomics was to develop a method for identification of genes imbedded in heterochromatin. This method has already led to the discovery of eight previously unknown protein-coding genes on the Y chromosome of D. melanogaster (Carvalho et al., 2000, 2001). The basic idea of the method is to apply BLAST searches with any and all known proteins as the query, searching for alignments with the unmapped scaffolds left over from the whole genome shotgun assembly. Celera's original assembly had 631 unmapped scaffolds, and it was thought that these might be parts of genes imbedded in heterochromatin, because heterochromatin has such low cloning efficiency. Our method reveals BLAST hits with these unmapped scaffolds which we then piece together by spanning the gaps with reverse-transcription PCR. Having this wealth of Y-linked sequence is allowing us to now pursue the evolutionary questions regarding the dynamics of nucleotide sequence change on the Y chromosomes, and the deeper molecular evolutionary questions about the origins and divergence of these Y-linked genes.
When females are able to store sperm for extended periods, it is possible that sperm from more than one male may be “competing” for use in fertilization. In Drosophila, later-mating males have an advantage, but there is extensive genetic variation in this character. Sperm competition has profound evolutionary consequences, and we are studying the molecular basis for the phenomenon as well as the population genetic aspects of it. We have implicated the Accessory Gland Proteins (Acps) in the “defense” component of sperm displacement, and more recently, in a collaborative study with Willie Swanson, Chip Aquadro and Mariana Wolfner, we began an exhaustive identification of Acps by sequencing EST clones derived from the male accessory gland and tested for male-specific expression. This survey not only identified a large new set of accessory gland proteins, it also demonstrated that as a class they undergo accelerated amino acid replacement, and the hit rate of the clones suggests we are near exhaustive coverage of the genes. Functional tests of sperm competition provide our means to analyze the male–female chemical communication in gamete use. Quantification of sperm displacement in the field are surprisingly effective by scoring microsatellites (due to allele multiplicity), and we have completed three surveys of a brood-structured samples (with Larry Harshman and Jørgen Bundgaard). Models for Bayesian inference of sperm competition parameters by Monte Carlo Markov chain are being developed with Beatrix Jones.