The imd gene is required for local Cecropin expression in Drosophila barrier epithelia

Surfaces of higher eukaryotes are normally covered with microorganisms but are usually not infected by them. Innate immunity and the expression of gene-encoded antimicrobial peptides play important roles in the first line of defence in higher animals. The immune response in Drosophila promotes systemic expression of antimicrobial peptides in response to microbial infection. We now demonstrate that the epidermal cells underlying the cuticle of larvae respond to infected wounds by local expression of the genes for the antimicrobial peptide cecropin A. Thus, the Drosophila epidermis plays an active role in the innate defence against microorganisms. The immune deficiency (imd) gene was found to be a crucial component of the signal-induced epidermal expression in both embryos and larvae. In contrast, melanization, which is part of the wound healing process, is not dependent on the imd gene, indicating that the signalling pathways promoting melanization and antimicrobial peptide gene expression can be uncoupled.     

Evolutionary history and mechanism of the Drosophila Cecropin gene family

 Upon bacterial infection, insects secrete a set of synthesized antibacterial proteins into the hemolymph and initiate synergistic destruction of invaders. Cecropin is one such antibacterial protein which is also found in vertebrates. To study the evolutionary history and mechanism of the Cecropin gene family, we determined DNA sequences of one isogenic In(3R)C and six isofemale lines of Drosophila melanogaster as well as one line of D. simulans and of D. yakuba. The phylogenetic analysis of these sequences together with those published for D. virilis and Sarcophaga peregrina reveals frequent gene re-organization. It was also found that silent nucleotide differences within D. melanogaster are quite heterogeneous across the gene region of approximately 3 kilobases and the extent of polymorphism is unusually usually high. These data suggest that the Cecropin gene region of D. melanogaster underwent intragenic recombination as well as introgression from a closely related sibling species, D. simulans. Received: 31 July 1997 / Revised: 24 October 1997     

Purification and properties of yolk protein factor I, a sequence-specific DNA-binding protein from Drosophila melanogaster

We report the purification and some of the biochemical properties of yolk protein factor I (YPF1). This protein binds to a specific site in the yolk protein 1 gene (yp1) of Drosophila melanogaster. YPF1 has been purified to 95% homogeneity and consists of a heterodimer of two subunits with molecular weights 85,000 and 69,000. The protein is highly asymmetric with a frictional ratio of 1.56 which leads to calculated dimensions of 510 x 51 A when modeled as a prolate ellipsoid of revolution. It binds the yp1 DNA site with a protein/DNA stoichiometry of 1:1. Binding to that site is essentially irreversible with a dissociation rate constant of koff less than or equal to 2 x 10(-7) s-1, which gives the complex a dissociation half-life of approximately 55 days. The measured apparent second order association rate constant is 4 x 10(8) M-1 s-1 resulting in a calculated equilibrium dissociation constant of KD less than or equal to 5 x 10(-16) M. YPF1 also has a 10(8) selectivity for the yp1 site over poly(dA).poly(dT) (KDapp = 2 x 10(-8) M(nucleotide].     

The Drosophila STAM gene homolog is in a tight gene cluster, and its expression correlates to that of the adjacent gene ial

Drosophila STAM is a homolog of mammalian STAM genes, which encode Jak associated signal-transducing adapter molecules. A 20-kilobase stretch of genomic DNA at 32B on chromosome arm 2L, which contains Drosophila STAM, has been sequenced. By comparison to cDNAs isolated and characterized, this region contains four tightly clustered genes: ial, mitochondrial porin, and the two newly discovered genes, STAM and DNZ1. Like its mouse and human homologs, STAM bears SH3 and ITAM domains. DNZ1 is a founding member of a sub-family of proteins bearing a DHHC/NEW1 zinc finger domain. Although these four genes are contained in a defined Deficiency overlap interval, no available P-element mutations in the region disrupt any of the genes, and no other discrete mutations in the genes have been identified. Among the four genes, ial and STAM share a common 5' control region, suggesting coordinate expression. Developmental Northern data and embryonic and ovariole expression data show that STAM and ial expression are correlated. The other two genes in the cluster appear to be expressed at constitutive levels throughout development.     

Altered gene expression patterns of innate and adaptive immunity pathways in transgenic rainbow trout harboring Cecropin P1 transgene

Background

We have recently developed several homozygous families of transgenic rainbow trout harbouring cecropin P1 transgene. These fish exhibit resistance characteristic to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). In our earlier studies we have reported that treatment of a rainbow trout macrophage cell line (RTS11) with a linear cationic α-helical antimicrobial peptide (e.g., cecropin B) resulted in elevated levels of expression of two pro-inflammatory relevant genes (e.g., IL-1β and COX-2). Therefore, we hypothesized that in addition to the direct antimicrobial activity of cecropin P1 in the disease resistant transgenic rainbow trout, this antimicrobial peptide may also affect the expression of immune relevant genes in the host. To confirm this hypothesis, we launched a study to determine the global gene expression profiles in three immune competent organs of cecropin P1 transgenic rainbow trout by using a 44k salmonid microarray.

Results

From the microarray data, a total of 2480 genes in the spleen, 3022 in the kidney, and 2102 in the liver were determined as differentially expressed genes (DEGs) in the cecropin P1 transgenic rainbow trout when compared to the non-transgenics. There were 478 DEGs in common among three tissues. Enrichment analyses conducted by two different bioinformatics tools revealed a tissue specific profile of functional pathway perturbation. Many of them were directly related to innate immune system such as phagocytosis, lysosomal processing, complement activation, antigen processing/presentation, and leukocyte migration. Perturbation of other biological functions that might contribute indirectly to host immunity was also observed.

Conclusions

The gene product of cecropin P1 transgene produced in the disease resistant transgenic rainbow trout not only can kill the pathogens directly but also exert multifaceted immunomodulatory properties to boost host immunity. The identified genes involved in different pathways related to immune function are valuable indicators associated with enhanced host immunity. These genes may serve as markers for selective breeding of rainbow trout or other aquaculture important fish species bearing traits of disease resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-887) contains supplementary material, which is available to authorized users.     

Actin gene expression is modulated by ecdysterone in a Drosophila cell line

The steroid hormone ecdysterone induced characteristic and specific changes of morphology, enzymatic activities and protein synthesis in a Kc 0% Drosophila melanogaster cell line. To study the ecdysterone action at a molecular level, a Drosophila genomic library was screened by differential hybridization to poly(A)+ RNA from control and ecdysterone-treated cells. Two recombinant phages were selected for hybridizing very intensively with poly(A)+ RNA of ecdysterone-treated cells and very weakly with poly(A)+ RNA of untreated ones. These two clones (lambda Dm 1632 and lambda Dm A5A1) mapped at the 5 C locus on polytene chromosomes; they overlap for a 9000 base-pair sequence that contains an abundantly transcribed region in ecdysterone-treated cells of about 2000 base-pairs. This region permits the selection of mRNA that gives, after translation in vitro, two polypeptides identified as cytoplasmic actin II and III. We demonstrated that these two recombinant phages, hybridizing preferentially with poly(A)+ RNA of ecdysterone-treated cells, contain the 5 C actin gene. Poly(A)+ RNA prepared from various times of treatment of cells were electrophoresed on agarose gels, transferred to nitrocellulose paper and then hybridized with the cloned actin probe. Results of these experiments indicate that there is a sharp increase in the level of RNA coding for actin after ecdysterone treatment of the cell, and that there are two forms of actin-specific RNA in the D. melanogaster cells. Using genomic blots with specific probes derived from lambda Dm 1632, we show that there are six actin genes per haploid Drosophila cell genome contained on six EcoRI fragments, as in Drosophila embryos, indicating that there is no rearrangement of these sequences in cultured cells. Our results suggest that the expression of actin genes in D. melanogaster Kc 0% cells is modulated by ecdysterone.