The use of suppression subtractive hybridization in the identification of a novel gene encoding a protein containing a BTB-POZ domain in the Mediterranean fruit fly, Ceratitis capitata

Abstract

Differential gene expression plays a key role in developmental pathways within an organism. Examples of such pathways include primary sex determination signaling and the formation of secondary sexual characteristics. This dissertation is focused on the use of suppression subtractive hybridization (SSH) to identify genes that are differentially expressed and involved in some aspect of sexual development in the Mediterranean fruit fly (medfly), Ceratitis capitata. In the course ofthis project, a method for sexing individual specimens from pre-adult stages was developed. This method was used to collect sex-specific RNAs at different developmental stages for use in SSH. A total of25 subtraction products were obtained across all the stages examined. Analysis of these products revealed that approximately half were similar to cytoplasmic ribosomal proteins and mitochondrial ribosomal RNA The remaining products represent putative medfly homologs of other previously identified genes or potentially novel genes One ofthe subtraction products, representing a potentially novel gene, was characterized in detail. This gene, named mapotge', represents a novel medfly gene that appears to encode a polypeptide of 299 amino acids. The N-terminus of this polypeptide contains a BTB-POZ domain. This domain functions as a protein-protein interaction motif found in a wide range of organisms from humans to Drosophila that mediates protein dimerization and oligomerization. The temporal expression pattern of mapotge' was determined using RT-PCR and Northern blot analysis. These revealed that the transcript is expressed throughout embryogenesis in both females and males, and in adult females that are > 0.5 days post-eclosion. Minimal expression is observed in female and male third instar larvae, early pupae, and in adult males. Studies were also initiated to characterize the representation of additioual sequences containing a BTB-POZ domain in the medfly genome. This was performed using Southern blot analysis and degenerate primers for the polymerase chain reaction (PCR). These results indicate the presence of at least three sequences in the medfly, in addition to 'mapotge', that contain a BTB-POZ domain. Potential evolutionary relationships ofthe BTB-POZ domain sequences from the medfly and other insect species were also analyzed.