Genetic and Molecular Analysis of Essential Genes in Centromeric Heterochromatin of the Left Arm of Chromosome 3 inDrosophila melanogaster

Abstract

A large portion of the Drosophila melanogaster genome is contained within heterochromatic regions of chromosomes, predominantly at centromeres and telomeres. The remaining euchromatic portions of the genome have been extensively characterized with respect to gene organization, function and regulation. However, it has been difficult to derive similar data for sequences within centromeric (centric) heterochromatin because these regions have not been as amenable to analysis by standard genetic and molecular tools. Here we present an updated genetic and molecular analysis of chromosome 3L centric heterochromatin (3L Het). We have generated and characterized a number of new, overlapping deficiencies (Dfs) which remove regions of 3L Het. These Dfs were critically important reagents in our subsequent genetic analysis for the isolation and characterization of lethal point mutations in the region. The assignment of these mutations to genetically-defined essential loci was followed by matching them to gene models derived from genome sequence data: this was done by using molecular mapping plus sequence analysis of mutant alleles, thereby aligning genetic and physical maps of the region. We also identified putative essential gene sequences in 3L Het by using RNA interference to target candidate gene sequences. We report that at least 25, or just under 2/3 of loci in 3L Het, are essential for viability and/or fertility. This work contributes to the functional annotation of centric heterochromatin in Drosophila , and the genetic and molecular tools generated should help to provide important insights into the organization and functions of gene sequences in 3L Het.

Translesion DNA synthesis-driven mutagenesis in very early embryogenesis of fast cleaving embryos

“…Because approximately two third of the genes located on 3L centromeric heterochromatin are required for developmental viability and/or adult fertility ( 38 ) we evaluated the predicted effects of mutations in dpolh −/− and dpolh −/+ adults by attributing variant effect predictor (VEP) score to each variation. As a comparison, we performed the same analysis on the 3R arm of the same chromosome.…”

“…Because approximately two third of the genes located on 3L pericentromeric heterochromatin are required for developmental viability and/or adult fertility ( 46 ) we evaluated the predicted effects of mutations in either maternally-depleted or maternally-provided adults by attributing variant effect predictor (VEP) score to each variation. VEP score determines the effect of variants (SNVs, insertions, deletions, CNVs or structural variants) on genes, transcripts, and protein sequence, as well as regulatory regions.…”

“…A great majority of SNVs in this region are predicted to generate mutations with low or moderate impact on genes functions. Accumulating mutations with low impact could represent a winning strategy to guarantee embryo development as opposed to apoptosis ( 38 ). Notwithstanding, we have also observed a small proportion of SNVs accumulating on this chromosome region that are predicted to generate mutations with high impact on gene functions.…”

Insulator proteins contribute to expression of gene loci repositioned into heterochromatin in the course ofDrosophilaevolution

“…By surveying factors that contribute to the normal functioning of the relocated genes in distant Drosophila species, i.e. D. melanogaster and D. virilis, we identify certain insulator proteins (e.g.BEAF-32) that facilitate the expression of heterochromatic genes in spite of the repressive environment.Despite the repressive environment, dozens of essential genes were identified in the pericentric heterochromatin of D. melanogaster (13)(14)(15)(16). Interestingly, genes embedded in pericentric heterochromatin in D. melanogaster may occupy distinct genomic loci, euchromatic and heterochromatic, in other Drosophila species (17).…”

“…Despite the repressive environment, dozens of essential genes were identified in the pericentric heterochromatin of D. melanogaster (13)(14)(15)(16). Interestingly, genes embedded in pericentric heterochromatin in D. melanogaster may occupy distinct genomic loci, euchromatic and heterochromatic, in other Drosophila species (17).…”