An androgenic gland hormone (AGH) is synthesized in the male-specific androgenic gland (AG) and is responsible for sex differentiation in crustaceans. To date, AGH cDNAs have been cloned from only three isopod species, but not decapod crustaceans. Recently, however, cDNAs encoding the AG specific polypeptide, which was designated as an insulin-like AG factor (IAG), have been cloned from two decapods, the red-claw crayfish Cherax quadricarinatus and the giant freshwater prawn Macrobrachium rosenbergii. These studies suggested that IAG may be involved in controlling sex differentiation in decapod crustaceans, but this point remains unclear. In order to accumulate knowledge on IAG, here we cloned IAG cDNAs from three palaemonid species (Crustacea, Decapoda), M. lar, Palaemon paucidens and P. pacificus. The three IAG precursors consisted of a signal peptide, a B chain, a C peptide and an A chain. This organization corresponds to that of the known isopod AGH and decapod IAG precursors. The three IAGs showed higher sequence identities to M. rosenbergii IAG (49~72%) than to C. quadricarinatus IAG (29~32%). These results are consistent with their phylogenetic relationship. In the analysis of the tissue-specific gene expression of the cloned three IAGs by RT-PCR, each IAG was detected only in the AG. Our data obtained in this study will facilitate further investigation of sex differentiation controlled by the AG in decapods.
The impact of the Drosophila experimental system on studies of modern biology cannot be understated. The ability to tag endogenously expressed proteins is essential to maximize the use of this model organism. Here, we describe a method for labeling endogenous proteins with self-complementing split fluorescent proteins (split FPs) in a cell-type–specific manner in Drosophila. A short fragment of an FP coding sequence is inserted into a specific genomic locus while the remainder of the FP is expressed using an available GAL4 driver line. In consequence, complementation fluorescence allows examination of protein localization in particular cells. Besides, when inserting tandem repeats of the short FP fragment at the same genomic locus, we can substantially enhance the fluorescence signal. The enhanced signal is of great value in live-cell imaging at the subcellular level. We can also accomplish a multicolor labeling system with orthogonal split FPs. However, other orthogonal split FPs do not function for in vivo imaging besides split GFP. Through protein engineering and in vivo functional studies, we report a red split FP that we can use for duplexed visualization of endogenous proteins in intricate Drosophila tissues. Using the two orthogonal split FP systems, we have simultaneously imaged proteins that reside in distinct subsynaptic compartments. Our approach allows us to study the proximity between and localization of multiple proteins endogenously expressed in essentially any cell type in Drosophila.
Androgenic gland hormone (AGH), which is produced in the male-specific androgenic gland (AG) and controls sex differentiation in crustaceans, has been characterized only from isopod species. To date, complementary DNA (cDNA) encoding an androgenic gland-specific polypeptide, which was designated as an insulin-like androgenic gland factor (IAG), has been cloned from three decapod species. IAG has been thought to be a candidate for AGH in decapod crustaceans; however, there has been no clear evidence. To accumulate sequence information of additional IAGs, we cloned cDNA encoding an IAG precursor from the kuruma prawn Marsupenaeus japonicus by reverse-transcription polymerase chain reaction (RT-PCR) coupled with 5 0 -and 3 0
Evolutionarily conserved insulin/insulin-like growth factor (IGF) signaling (IIS) correlates nutrient levels to metabolism and growth, thereby playing crucial roles in development and adult fitness. In the fruit fly Drosophila, ImpL2, an ortholog of IGFBP7, binds to and inhibits the function of Drosophila insulin-like peptides. In this study, we isolated a temperature-sensitive mutation in the insulin receptor (InR) gene as a spontaneous revertant in ImpL2 null mutants. The p.Y902C missense mutation is located at the functionally conserved amino acid residue of the first fibronectin type III domain of InR. The hypomorphic InR mutant animals showed a temperature-dependent reduction in IIS and body size. The mutant animals also exhibited metabolic defects, such as increased triglyceride and carbohydrate levels. Metabolomic analysis further revealed that defects in InR caused dysregulation of amino acid and ribonucleotide metabolism. We further observed that InR mutant females produced tiny irregular-shaped embryos with reduced fecundity. In summary, this novel allele of InR is a valuable tool for the Drosophila genetic model of insulin resistance and type 2 diabetes.
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