A humoral stress response in Drosophila.

The ability to react to unfavorable environmental changes is crucial for survival and reproduction, and several adaptive responses to stress have been conserved during evolution [1-3]. Specific immune and heat shock responses mediate the elimination of invading pathogens and of damaged proteins or cells [4-6]. Furthermore, MAP kinases and other signaling factors mediate cellular responses to a very broad range of environmental insults [7-9]. Here we describe a novel systemic response to stress in Drosophila. The Turandot A (TotA) gene encodes a humoral factor, which is secreted from the fat body and accumulates in the body fluids. TotA is strongly induced upon bacterial challenge, as well as by other types of stress such as high temperature, mechanical pressure, dehydration, UV irradiation, and oxidative agents. It is also upregulated during metamorphosis and at high age. Strikingly, flies that overexpress TotA show prolonged survival and retain normal activity at otherwise lethal temperatures. Although TotA is only induced by severe stress, it responds to a much wider range of stimuli than heat shock genes such as hsp70 or immune genes such as Cecropin A1.

Genomic organization of the leukotriene B(4) receptor locus of human chromosome 14.

The genomic region containing the genes encoding the first leukotriene B(4) receptor, BLTR, as well as the recently cloned second leukotriene B(4)-activated receptor, BLTR2, was mapped by (a) sequence analysis of a human bacterial artificial chromosome (BAC) library containing a 15-kb segment corresponding to chromosome 14q11. 2-12 where the BLTR/BLTR2 genes were previously shown to be located, together with (b) sequence analysis of 83 expressed sequence tags (ESTs) from this region. The BLTR gene includes four different 5' untranslated regions (UTRs) and a mutual acceptor site for the exon containing the intronless open reading frame. The BLTR2 gene is intronless and overlapped by a 5' UTR splice version of BLTR and, on the reverse strand, of the apoptosis-related CIDE-B gene. This indicates a complex posttranscriptional gene regulation. Further adding to the complexity of the region is evidence of a fourth putative and novel gene, most homologous to the rat adenylyl cyclase IV gene.

Cloning and characterization of cDNA encoding a novel human leukotriene B(4) receptor.

By homology screening using BLAST searches of expressed sequence tags (ESTs), we have found a previously unidentified cDNA encoding a putative seven-transmembrane receptor with highest similarity to the leukotriene B(4) receptor, BLTR. Analysis of calcium flow in transfected cells, along with sequence analysis, revealed that the EST encoded a functionally inactive protein, lacking the segment corresponding to the C-terminal part of the putative receptor protein. The missing segment was obtained by PCR amplification of a human leukocyte cDNA library and ligated to the truncated EST cDNA. The novel cDNA encodes a full-length receptor with 39% identity to the previously cloned BLTR. Studies of intracellular calcium flow of transfected HeLa cells exposed to various leukotrienes showed that also the novel BLTR-like receptor can be activated by leukotriene B(4), and it is therefore tentatively named BLTR2.

Cysteine proteinase 1 (CP1), a cathepsin L-like enzyme expressed in the Drosophila melanogaster haemocyte cell line mbn-2.

We have isolated cDNA clones encoding the full-length Drosophila melanogaster cysteine proteinase 1 (CP1). The clones were isolated from the Drosophila melanogaster haemocytic mbn-2 cell line, where the gene is relatively strongly expressed, giving a transcript of 1.6 kb in size. We present the sequence encoding the full-length protein, and deduced the genomic organization of the gene by comparison to previously published genomic partial sequence data. Immunofluorescence shows that CP1 is localized in small granules, probably lysosomes, in mbn-2 cells. The data presented suggest a role for cysteine proteinase in immune functions in insects. It is likely to participate in the degradation of internalized material in phagocytic cells.

CalpA, a Drosophila calpain homolog specifically expressed in a small set of nerve, midgut, and blood cells.

Calpains are calcium-dependent proteases believed to participate in calcium-regulated signal pathways in cells. Ubiquitous calpains as well as tissue-specific calpains have been found in vertebrates. We isolated cDNA clones for a highly tissue-specific calpain gene from Drosophila melanogaster, CalpA, at 56C-D on the second chromosome. The expression of the CalpA gene product was monitored by using a specific antiserum directed against the product expressed by one cDNA clone. The encoded protein is found in a few neurons in the central nervous system, in scattered endocrine cells in the midgut, and in blood cells. In the blood cell line mbn-2, calpain is associated with a granular component in the cytoplasm. The expression of this protein is more restricted than that of the corresponding transcripts, which are widely distributed in the central nervous system, digestive tract, and other tissues. The sequence of CalpA is closely related to that of vertebrate calpains, but an additional segment is inserted in the calmodulin-like carboxy-terminal domain. This insert contains a hydrophobic region that may be involved in membrane attachment of the enzyme. Differential splicing also gives rise to a minor transcript that lacks the calmodulin-like domain.

CecC, a cecropin gene expressed during metamorphosis in Drosophila pupae.

Cecropins are antibacterial peptides, induced in insects in response to bacterial infections. In Drosophila, three cecropin genes have previously been characterized, CecA1, CecA2, and CecB, in a dense cluster at 99E on the third chromosome. From the same locus, we now describe a fourth member of the cecropin gene family, CecC, which is mainly expressed at the early pupal stage. In situ hybridization to immunized pupae show that CecC is induced in the anterior end of the larval hindgut and in other larval tissues that are undergoing histolysis. Within these other tissues it is often expressed in distinct foci that may correspond to hemocytes. A similar pattern of expression in the metamorphosing pupa is also observed for the CecA and CecB genes. Comparing the DNA sequences of the cecropin genes, a conserved region is observed about 30 bp upstream of the TATA box. It consists of three shorter motifs, two of which are reminiscent of a putative promoter element in immune protein genes from the cecropia moth.