The transformer gene in Bactrocera oleae: the genetic switch that determines its sex fate

Abstract

Transformer (tra) is the second gene of a regulatory cascade based on RNA splicing that determines sex in Drosophila melanogaster. Splicing of tra transcripts is regulated by the master gene Sex lethal and tra itself regulates splicing of the transcriptional regulator doublesex (dsx). We present the isolation and characterization of Botra, the olive fruit fly Bactrocera oleae orthologue to the Drosophila gene transformer. As in Drosophila, Botra transcripts are spliced in a sex-specific manner so that only females encode a functional polypeptide of 422 amino acids, whereas males encode presumably nonfunctional peptide(s). The identification of multiple TRA/TRA-2 binding sites within the Botra male-specific exons, suggests an autoregulation mechanism of tra, through TRA/TRA2 activities. The fundamental role of the TRA protein in sex determination of Bactrocera was investigated by RNA interference, where the introduction of Botra dsRNA into embryos resulted in complete transformation of XX flies into fertile males.

About females and males: continuity and discontinuity in flies

“…Furthermore, the Ceratitis homologue of dsx was identified as one of its downstream targets. Similar findings in other Tephriditae suggest the cascade tra→dsx is widely used among Acalyptratae to transmit the sex-determining signal (Lagos et al 2007;Ruiz et al 2007). More recently, we were able to isolate the tra orthologue in the Calyptratea species, M. domestica.…”

Sexual Back Talk With Evolutionary Implications: Stimulation of the Drosophila Sex-Determination Gene Sex-lethal by Its Target transformer

“…Very recent work suggests that the honeybee tra ortholog may function like tra in the Tephritids (Hasselmann et al 2008). In flies for which tra is the master sex switch, tra is expressed in the female germline and that germline expression has been proposed to be an important element in the sex-determination mechanism by which the tra feedback loop is initiated (Lagos et al 2007). We suggest that both the functionally redundant, tra-mediated circuit for Sxl-positive feedback in D. melanogaster and female germline expression of tra in melanogaster are vestiges of the evolutionary transition between tra and Sxl as the autoregulated master sex switch.…”

“…Even allowing up to a 3-bp degeneracy in the established 13-bp TRA-F consensus binding site, we found only two overlapping candidate sites in S. cynipsea tra, neither of which seemed likely to be functional: gaATCAATCAACA and TCAA CAATCgcCA (degenerate sites lower case and overlap underlined). TRA-F from D. melanogaster had been shown to not utilize the first sequence (Inoue et al 1992), and the second sequence lacks the TCAAxx 39 sequence common to every predicted TRA-F binding site for tra, dsx, and fru orthologs all the way to mosquitos (Pane et al 2002;Scali et al 2005;Lagos et al 2007). Moreover, we sequenced a 722-bp fragment of tra from the sister species S. neocynipsea (EU636098) centered on homology to the S. cynipsea degenerate TRA-F sites and found that neither site was conserved, despite 85% overall sequence identity for the two corresponding regions.…”

“…The female-specific SXL-F protein thereby generated acts on various regulatory gene targets, including the feminizing switch gene transformer (tra) to elicit female differentiation. In some fly species that do not use an X-chromosome dose to determine sex (Pane et al 2002;Lagos et al 2007), and possibly even in the honeybee (Hasselmann et al 2008), tra occupies the position of Sxl as the master developmental switch gene that epigenetically maintains the female sexual fate commitment by direct positive autoregulation of its own pre-mRNA splicing. In those species, Sxl itself does not appear to be sexspecifically regulated and its function is unknown.…”

“…Cloning of Sepsid tra: Advantage was taken of synteny between tra and l(3)73Ah (Pane et al 2002;Lagos et al 2007). We PCR amplified l(3)73Ah using the primers (forward) 59-CAAGAGTTGCCTGGTGAAGCAC-39 and (reverse) 59-GT ACTTTTCGATGCCGTTGAGG-39.…”