Collagen type III alpha I is a gastro-oesophageal reflux disease susceptibility gene and a male risk factor for hiatus hernia.

BACKGROUND AND OBJECTIVES: Gastro-oesophageal reflux disease (GORD) is a common gastrointestinal disorder with a genetic component. Our aim was to identify genetic factors associated with GORD. PATIENTS AND METHODS: Four separate patient cohorts were analysed using a step-wise approach. (1) Whole genome linkage analysis was performed in 36 families. (2) Candidate genes were tested for GORD association in a trio cohort. (3) Genetic association was replicated in a case-control cohort. We also investigated genetic association to hiatus hernia (HH). (4) Protein expression was analysed in oesophageal biopsies. RESULTS: A region on chromosome 2, containing collagen type III alpha 1 (COL3A1), was identified (LOD = 3.3) in families with dominant transmission of GORD, stratified for hiatus hernia (HH). COL3A1 showed significant association with GORD in an independent paediatric trio cohort (p(corr) = 0.003). The association was male specific (p(corr) = 0.018). The COL3A1 association was replicated in an independent adult case control cohort (p(corr) = 0.022). Moreover, male specific association to HH (p(corr) = 0.019) was found for a SNP not associated to GORD. Collagen type III protein was more abundant in oesophageal biopsies from male patients with GORD (p = 0.03). CONCLUSION: COL3A1 is a disease-associated gene in both paediatric and adult GORD. Furthermore, we show that COL3A1 is genetically associated with HH in adult males. The GORD- and HH-associated alleles are different, indicating two separate mechanisms leading to disease. Our data provides new insight into GORD aetiology, identifying a connective tissue component and indicating a tissue remodelling mechanism in GORD. Our results implicate gender differences in the genetic risk for both for GORD and HH.

Trio combines with dock to regulate Pak activity during photoreceptor axon pathfinding in Drosophila.

Correct pathfinding by Drosophila photoreceptor axons requires recruitment of p21-activated kinase (Pak) to the membrane by the SH2-SH3 adaptor Dock. Here, we identify the guanine nucleotide exchange factor (GEF) Trio as another essential component in photoreceptor axon guidance. Regulated exchange activity of one of the two Trio GEF domains is critical for accurate pathfinding. This GEF domain activates Rac, which in turn activates Pak. Mutations in trio result in projection defects similar to those observed in both Pak and dock mutants, and trio interacts genetically with Rac, Pak, and dock. These data define a signaling pathway from Trio to Rac to Pak that links guidance receptors to the growth cone cytoskeleton. We propose that distinct signals transduced via Trio and Dock act combinatorially to activate Pak in spatially restricted domains within the growth cone, thereby controlling the direction of axon extension.

Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics.

During development of the adult Drosophila visual system, axons of the eight photoreceptors in each ommatidium fasciculate together and project as a single bundle towards the optic lobes of the brain. Within the brain, individual photoreceptor axons from each bundle then seek specific targets in distinct layers of the optic lobes. The axons of photoreceptors R1-R6 terminate in the lamina, while R7 and R8 axons pass through the lamina to terminate in separate layers of the medulla. To identify genes required for photoreceptor axon guidance, including those with essential functions during early development, we have devised a strategy for the simple and efficient generation of genetic mosaics in which mutant photoreceptor axons innervate a predominantly wild-type brain. In a large-scale saturation mutagenesis performed using this system, we recovered new alleles of the gene encoding the receptor tyrosine phosphatase PTP69D. PTP69D has previously been shown to function in the correct targeting of motor axons in the embryo and R1-R6 axons in the visual system. Here, we show that PTP69D is also required for correct targeting of R7 axons. Whereas mutant R1-R6 axons occasionally extend beyond their normal targets in the lamina, mutant R7 axons often fail to reach their targets in the medulla, stopping instead at the same level as the R8 axon. These targeting errors are difficult to reconcile with models in which PTP69D plays an instructive role in photoreceptor axon targeting, as previously proposed. Rather, we suggest that PTP69D plays a permissive role, perhaps reducing the adhesion of R1-R6 and R7 growth cones to the pioneer R8 axon so that they can respond independently to their specific targeting cues.

Relish, a central factor in the control of humoral but not cellular immunity in Drosophila.

The NF-kappa B-like Relish gene is complex, with four transcripts that are all located within an intron of the Nmdmc gene. Using deletion mutants, we show that Relish is specifically required for the induction of the humoral immune response, including both antibacterial and antifungal peptides. As a result, the Relish mutants are very sensitive to infection. A single cell of E. cloacae is sufficient to kill a mutant fly, and the mutants show increased susceptibility to fungal infection. In contrast, the blood cell population, the hematopoietic organs, and the phagocytic, encapsulation, and melanization responses are normal. Our results illustrate the importance of the humoral response in Drosophila immunity and demonstrate that Relish plays a key role in this response.

Origins of immunity: Relish, a compound Rel-like gene in the antibacterial defense of Drosophila.

NF-kappa B/Rel transcription factors are central regulators of mammalian immunity and are also implicated in the induction of cecropins and other antibacterial peptides in insects. We identified the gene for Relish, a compound Drosophila protein that, like mammalian p105 and p100, contains both a Rel homology domain and an I kappa B-like domain. Relish is strongly induced in infected flies, and it can activate transcription from the Cecropin A1 promoter. A Relish transcript is also detected in early embryos, suggesting that it acts in both immunity and embryogenesis. The presence of a compound Rel protein in Drosophila indicates that similar proteins were likely present in primordial immune systems and may serve unique signaling functions.

Identification of early genes in the Drosophila immune response by PCR-based differential display: the Attacin A gene and the evolution of attacin-like proteins.

We are using the PCR-based differential display technique to isolate genes which are induced during the immune response in Drosophila. In this way, a cDNA clone for a member of the attacin family of antibacterial proteins was isolated. The corresponding Attacin A (Att A) gene is localized at 51A-B on the second chromosome, and it is closely linked to at least one more cross-hybridizing gene. Injection of bacteria induces a 0.8 kb transcript, with expression kinetics similar to that of cecropin. Drosophila attacin is most closely related to sarcotoxin II of Sarcophaga peregrina, but it lacks the extra domains that are unique to this protein, and the overall domain structure of the Att A gene product is identical to that of the attacins from Hyalophora cecropia.

In vitro induction of cecropin genes--an immune response in a Drosophila blood cell line.

The Drosophila melanogaster cell line mbn-2 was explored as a model system to study insect immune responses in vitro. This cell line is of blood cell origin, derived from larval hemocytes of the mutant lethal (2) malignant blood neoplasm (1(2)mbn). The mbn-2 cells respond to microbial substances by the activation of cecropin genes, coding for bactericidal peptides. The response is stronger than that previously described for SL2 cells, and four other tested Drosophila cell lines were totally unresponsive. Bacterial lipopolysaccharide, algal laminarin (a beta-1,3-glucan), and bacterial flagellin were strong inducers, bacterial peptidoglycan fragments gave a weaker response, whereas a formyl-methionine-containing peptide had no effect. Experiments with different drugs indicate that the response may be mediated by a G protein, but not by protein kinase C or eicosanoids, and that it requires a protein factor with a high rate of turnover.

The cecropin locus. Cloning and expression of a gene cluster encoding three antibacterial peptides in Hyalophora cecropia.

Cecropins A, B, and D are antibacterial peptides of 35-37 amino acids that are synthesized in pupae of the Cecropia moth (Hyalophora cecropia) as a response to a bacterial infection. cDNA cloning has shown that the cecropins are made as preproproteins that are processed in four steps to the mature peptides. We have now cloned the genes for preprocecropins A and D, data that together with earlier work on the B gene has made it possible to deduce the arrangement of the cecropin locus. The genes for the three cecropins are organized in a large cluster spanning 20 kilobases of DNA and for each gene there is one copy/haploid genome. The size of the cluster is in part due to long distances between the genes and to the presence of insertion elements in the introns of the A and D genes. The cecropin genes are not expressed in parallel. Transcripts for cecropins A and B appear within 2 h after injection of live bacteria, they reach a maximum after 48 h, and they are continuously expressed at this level for several days. The D gene has a delayed pattern of expression where transcripts appear within 48-96 h and reach a maximum after 144 h. In consonance is also the production of the mature cecropins A, B, and D where the active cecropins A and B are detected in the hemolymph within 10-24 h while the D form is not detected until 48 h post infection. Control injections with sterile saline produced only a weak induction of the cecropin genes.

Organization and expression of the immunoresponsive lysozyme gene in the giant silk moth, Hyalophora cecropia.

Lysozyme is one of the antibacterial proteins that are produced by the giant silk moth Hyalophora cecropia in response to bacterial infection or injury. As an essential step toward the understanding of the mechanisms involved in the immune response, we have isolated and characterized the lysozyme gene from Cecropia. The complete nucleotide sequence of the gene as well as the immediate flanking sequences have been determined. The gene includes three exons. Its first intron contains a repetitive sequence. In the evolutionary aspect, the Cecropia lysozyme gene and two vertebrate lysozyme genes have been found to maintain a similar organization pattern of exons. The lysozyme gene has been found to be strongly induced by lipopolysaccharides and a phorbol ester as well as bacteria. In the induction by bacteria, the lysozyme transcript appears at about 2 h, reaches to the maximum level at about 24 h, and then declines. Comparison of the 5'-flanking sequences with several other genes involved in the immune response of H. cecropia and Drosophila melanogaster revealed a kappa B-like consensus sequence. This sequence is specifically recognized by a nuclear protein from the induced pupa.