Research Problem, Spring 2011

The scientific problem we will tackle during spring 2011 is a question of comparative genomics: can we distinguish heterochromatic and euchromatic domains based on sequence organization and/or characteristics of the genes in these different environments? What insight can we gain concerning the evolution of the fourth chromosome, and the genes it contains? In Drosophila (the fruit fly), the small fourth chromosome (sometimes called the "dot" chromosome) is unusual in that it appears to be essentially heterochromatic — packaged in a relatively condensed form, replicated late in S phase, exhibiting no meiotic recombination, etc. An examination of the DNA sequence indicates that the 1.2 Mb arm has a normal gene density (~80 genes), but a ten-fold higher frequency of repeated sequences (generally remnants of transposable elements) than the other chromosome arms, which are euchromatic. Many of the genes on the fourth chromosome are associated with silencing marks, but can be expressed in this heterochromatic environment. Recent studies have suggested that heterochromatin formation is targeted by the presence of repetitious sequence elements, although it appears that not all repetitious elements can trigger heterochromatin formation.

During the years 2004-2006, students enrolled in Bio 4342 sequenced and annotated the dot chromosome of Drosophila virilis. This analysis has recently been completed, and we have published a manuscript on the findings, with all contributing students as co-authors (Leung et al, 2010). This year we are going to be adventurous, and attempt to improve the sequence of the dot chromosome of D. ananassae, a strange species with a very large dot chromosome. During 2007-2010 we worked on the dot chromosomes of D. mojavensis and D. grimshawi, the Hawaiian fruit fly, and a control euchromatic domain in D. mojavensis. Sequencing of these is almost complete, but annotation remains to be done. Each student in Bio 4342 will take on the challenge of annotating a fosmid from this set (about 40 - 50 kb). We will be able to compare our results with data from the other fly species mentioned. The comparative analysis should tell us much more about this interesting chromosome.

In annotating your fosmid, you will want to address the following questions: What genes and/or pseudogenes are present? What models can you construct — can you find homologues for all to isoforms reported in D. melanogaster? What is the gene density? What types of repetitious elements are present, at what density? (We will use both RepeatMasker and an internal comparison to address this question.) What can be said from a comparison of this region (defined by the genes present) among D. melanogaster, D. virilis, D. mojavensis and D. grimshawi (your data), and any other Drosophila species for which sequence data are available? Is the region syntenic? Can any non-coding conserved sequences be detected? We will then want to pool our data, and consider the answers to these questions for the pooled data, in particular looking at the relative spacing of genes and repetitious elements. As time permits, we will want to look at codon bias and AT-composition, as these are reported to differ between regions with high rates of recombination (euchromatin) and regions with low rates of recombination (heterochromatin). If we can, we will also want to look for potential regulatory elements, in particular noting similarities among the 5' upstream regions of dot chromosome genes, based on the hypothesis that genes that function within a heterochromatic environment might exhibit special characteristics. The answers to these questions should tell us more about the relationship between DNA sequence organization, chromatin packaging, and gene regulation.