Molecular Biology & Biochemistry
Mapping and functional analysis of genes in Drosophila heterochromatin.
Our work has two long term objectives:
- to contribute to the physical mapping/ sequencing/ functional annotation of Drosophila centric heterochromatin, as a model for metazoan genomes, and
- in parallel, to study the structure, function and evolution of genes in centric heterochromatin.
A huge proportion, roughly one third of the total, or an estimated 60Mb, was not represented in the first “complete” sequence of Drosophila melanogaster reported by Adams et al in 2000 (Science 287: 2185). Most of the missing sequence derived from centric heterochromatin, a key component of the genome (~1/4 of autosomes, ~½ of X, most of 4 and all of Y) containing sequences vital for chromosome replication and cell division, as well as regions which silence euchromatic genes artificially juxtaposed nearby (Position Effect Variegation, PEV).
Paradoxically, a few essential genes are present in, and even appear to require, this inactivating, PEV-inducing environment, suggesting unusual properties and regulation for DNA sequences in heterochromatin. Mapping and cloning of heterochromatic sequences have historically been extremely difficult, and a number of problems–the absence of polytene banding, lack of standard meiotic recombination, the presence of numerous repetitive DNA sequences, and other complexities–have therefore made it difficult to make progress with a genetic and molecular characterization of centric heterochromatin, using standard methods. More recently Gary Karpen, Roger Hoskins, Chris Smith and colleagues have made significant progress with characterizing large portions of this heterochromatic DNA. Much work remains however to map, sequence and functionally annotate these regions of the genome.
We have been mapping heterochromatic DNA sequences against genetic deficiencies within centric heterochromatin on chromosome 3, in order to link the genetic and physical maps in these regions. A next logical step is to use these to proceed with functional annotation—link specific genetic loci to individual gene models, by isolating DNA from individual mutants, and looking for corresponding defects in the sequence of a gene model. A key unresolved puzzle for these heterochromatic regions is the large discrepancy between the number of genetically defined loci (dozens) versus hundreds of in silico gene models. Our results to date have provided mutations in genes involved in a number of critically important cellular processes; this provides us with well-characterized heterochromatic genes, to use as substrates to study their regulation/ expression. In some cases, these mutations in essential genes provide us with a powerful tool for further studies of the structure and function of key regulatory proteins.
We have also been looking at orthologues of identified D. melanogaster heterochromatic genes in the genomes of other Drosophila species . This can be a valuable tool to look for functional conservation of coding and regulatory sequences; moreover, our recent results suggest that we can make significant contributions to the annotation of these Drosophila genomes, and gain other valuable insights into the structure, function and evolution of heterochromatic genes.