OB-R is a high affinity receptor for leptin, an important circulating signal for the regulation of body weight. We identified an alternatively spliced transcript that encodes a form of mouse OB-R with a long intracellular domain. db/db mice also produce this alternatively spliced transcript, but with a 106 nt insertion that prematurely terminates the intracellular domain. We further identified G --> T point mutation in the genomic OB-R sequence in db/db mice. This mutation generates a donor splice site that converts the 106 nt region to a novel exon retained in the OB-R transcript. We predict that the long intracellular domain form of OB-R is crucial for initiating intracellular signal transduction, and as a corollary, the inability to produce this form of OB-R leads to the severe obese phenotype found in db/db mice.

No abstract

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.

The antimicrobial defence of Drosophila relies largely on the challenge-induced synthesis of an array of potent antimicrobial peptides by the fat body. The defence against Gram-positive bacteria and natural fungal infections is mediated by the Toll signalling pathway, whereas defence against Gram-negative bacteria is dependent on the Immune deficiency (IMD) pathway. Loss-of-function mutations in either pathway reduce the resistance to corresponding infections. The link between microbial infections and activation of these two pathways has remained elusive. The Toll pathway is activated by Gram-positive bacteria through a circulating Peptidoglycan recognition protein (PGRP-SA). PGRPs appear to be highly conserved from insects to mammals, and the Drosophila genome contains 13 members. Here we report a mutation in a gene coding for a putative transmembrane protein, PGRP-LC, which reduces survival to Gram-negative sepsis but has no effect on the response to Gram-positive bacteria or natural fungal infections. By genetic epistasis, we demonstrate that PGRP-LC acts upstream of the imd gene. The data on PGRP-SA with respect to the response to Gram-positive infections, together with the present report, indicate that the PGRP family has a principal role in sensing microbial infections in Drosophila.

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.