Drosophila Immunity: Analysis of Larval Hemocytes by P-Element-Mediated Enhancer Trap

Our aim was to identify new genes involved in the cellular aspects of defense mechanisms of Drosophila, as well as in melanotic tumor formation processes that are linked to blood cell disregulation. We have screened 1341 enhancer detector fly lines for expression of the lacZ reporter gene in larval hemocytes at the end of the third instar. We have selected 21 lines in which we observed a reproducible lacZ expression in blood cells. These lines were classified according to the subsets of hemocytes in which lacZ was expressed, and we identified five lines that can be used as lamellocyte markers. Three lines were selected for further analysis. The first exhibited strong lacZ expression in all lamellocytes. The second expressed lacZ in plasmatocytes and lamellocytes, and exhibited a melanotic tumor phenotype in larvae homozygous for the insertion. A third line showed a striking insertion-linked phenotype of melanized lymph glands (the hematopoietic organ), which resulted in the total absence of circulating hemocytes in the mutant larvae. We anticipate that this mutation, which we named domino, will prove a useful tool in the analysis of the role of hemocytes during the various aspects of immune response and melanotic tumor formation.     

Regulation of Hemocytes in Drosophila Requires dappled Cytochrome b5

A major category of mutant hematopoietic phenotypes in Drosophila is melanotic tumors or nodules, which consist of abnormal and overproliferated blood cells, similar to granulomas. Our analyses of the melanotic mutant dappled have revealed a novel type of gene involved in blood cell regulation. The dappled gene is an essential gene that encodes cytochrome b5, a conserved hemoprotein that participates in electron transfer in multiple biochemical reactions and pathways. Viable mutations of dappled cause melanotic nodules and hemocyte misregulation during both hematopoietic waves of development. The sexes are similarly affected, but hemocyte number is different in females and males of both mutants and wild type. Additionally, initial tests show that curcumin enhances the dappled melanotic phenotype and establish screening of endogenous and xenobiotic compounds as a route for analysis of cytochrome b5 function. Overall, dappled provides a tractable genetic model for cytochrome b5, which has been difficult to study in higher organisms.     

An auto-inducible vector conferring high glucocorticoid inducibility upon stable transformant cells

A new gene expression system in mammalian cells was developed by using the glucocorticoid receptor (GR) as an inducible positive feedback factor. Mouse Ltk- cells were transfected with plasmids carrying the GR-encoding gene and the lacZ reporter gene, both of which were fused with the glucocorticoid-inducible enhancer/promotor of the mouse mammary tumor virus (MTV). The GR gene was first induced to supply the receptor protein, which further induced the expression of both GR and reporter genes. Stable transformants induced with dexamethasone, a synthetic glucocorticoid hormone, demonstrated beta-galactosidase activity 60-140-fold higher than uninduced controls. Similarly, the human alpha-interferon-encoding gene fused with the MTV enhancer/promoter was induced more than 12,000-fold. This system allowed us to increase the expression of the reporter or target genes without augmenting basal levels of expression significantly, and may be useful to investigate the unknown function of a cloned gene, particularly when the gene product of interest is cytotoxic or growth-inhibiting.     

Genes expressed in the ring gland,the major endocrine organ of Drosophila melanogaster.

We have used an enhancer-trap approach to begin characterizing the function of the Drosophila endocrine system during larval development. Five hundred and ten different lethal PZ element insertions were screened to identify those in which a reporter gene within the P element showed strong expression in part or all of the ring gland, the major site of production and release of developmental hormones, and which had a mutant phenotype consistent with an endocrine defect. Nine strong candidate genes were identified in this screen, and eight of these are expressed in the lateral cells of the ring gland that produce ecdysteroid molting hormone (EC). We have confirmed that the genes detected by these enhancer traps are expressed in patterns similar to those detected by the reporter gene. Two of the genes encode proteins, protein kinase A and calmodulin, that have previously been implicated in the signaling pathway leading to EC synthesis and release in other insects. A third gene product, the translational elongation factor EF-1alpha F1, could play a role in the translational regulation of EC production. The screen also identified the genes couch potato and tramtrack, previously known from their roles in peripheral nervous system development, as being expressed in the ring gland. One enhancer trap revealed expression of the gene encoding the C subunit of vacuolar ATPase (V-ATPase) in the medial cells of the ring gland, which produce the juvenile hormone that controls progression through developmental stages. This could reveal a function of V-ATPase in the response of this part of the ring gland to adenotropic neuropeptides. However, the gene identified by this enhancer trap is ubiquitously expressed, suggesting that the enhancer trap is detecting only a subset of its control elements. The results show that the enhancer trap approach can be a productive way of exploring tissue-specific genetic functions in Drosophila.     

基因捕捉及其在植物基因分离和功能基因组学上的应用

基因捕捉是一种报告基因的随机整合技术。基因捕捉系统已成为分离基因、鉴定基因功能的重要手段。基因捕捉(gene traps)包括增强子捕捉(enhancer trap)、启动子捕捉(promoter trap)和基因捕捉(gene trap),通称为基因捕捉(gcne traps)。在增强子捕捉中,报告基因与一个基本启动子融合,这个启动子不能使报告基因表达,但可被临近的增强子激活。在启动子捕捉和基因捕捉中,报告基因的启动子被去除,融合基因只有以正确的方向插入到转录单元内才能表达。对基因捕捉系统的结构特征、构建方法、应用范围、研究现状和应用前景等作了系统论述,并对有关问题进行了讨论。     

人乳头瘤病毒6b型(HPV-6b)的上游调控区在大肠杆菌中也具有增强子样的效应

采用痘苗病毒7.5k、11k及N2启动子,以大肠杆菌β-半乳糖苷酶(β-lac)、氯霉素乙酰基转移酶(CAT)作为标记基因,构建了大肠杆菌增强子样序列的系列检测载体。采用上述检测载体发现人乳头瘤病毒6b(Human papillomavirus type 6b,HPV-6b)上游调控区(Upstream regulating region,URR)中540bp的Sau3A-Nar Ⅰ片段,在大肠杆菌中对痘苗病毒启动子控制的CAT基因的表达有明显的增强作用,可使基因表达提高4～5倍;对β-lac基因的表达可提高3～6倍。根据这一片段的插入方向增强基因表达水平有所不同。     

New genetic approaches to craniofacial growth and malformation in the mouse.

New genetic tools that have been developed in the mouse for the study of genetic variation and molecular biology can be applied to the study of craniofacial growth and malformation. D.W. Bailey (Journal of Heredity, 76:107-114, 1985, 77:17-25, 1986) has used congenic and recombinant inbred strains to identify a large number of genes that result in growth variation in local regions of the mandible. The function of "morphogenes" have not been characterized, but they may act as switches that regulate already recognized structural genes. We have studied the cartilage matrix deficiency (cmd/cmd) mutant mouse and found that their chondrocytes fail to synthesize the cartilage-specific proteoglycan at both protein and mRNA levels. In vitro experiments demonstrate biochemical feedback control in the formation of the cartilage extracellular matrix. Abnormalities of cartilage matrix result in facial clefts and in dental malocclusion in mice. A recombinant plasmid containing the type II collagen promoter and enhancer fused to a reporter gene, chloramphenicol acetyl transferase, has been used to produce transgenic mice. These mice reveal that the type II collagen enhancer controls the high level of tissue-specific expression of the gene. The transgenic mice provide a potential test system for study of development and teratogenesis at the gene level.     

Activation of human papillomavirus type 18 gene expression by herpes simplex virus type 1 viral transactivators and a phorbol ester.

Several viral trans-activators and a tumor promoter were examined for the ability to activate human papillomavirus type 18 (HPV-18) gene expression. A plasmid containing the HPV-18 noncoding region placed upstream of the chloramphenicol acetyltransferase reporter gene was cotransfected with different herpes simplex virus type 1 (HSV-1) genes into several cell lines. Both HSV-1 TIF and ICP0 activated HPV-18 expression; however, activation by TIF was observed only in epithelial cells, while ICP0 stimulated expression in a wide variety of cells. The element activated by both TIF and ICP0 was mapped to a 229-base-pair fragment which also contains an HPV-18 epithelial cell-preferred enhancer. The inclusion of a papillomavirus E2 trans-activator with TIF and ICP0 further increased HPV-18 expression. In contrast, the HSV-1 ICP4 and ICP27 genes, as well as the human T-cell lymphotropic virus type I and human immunodeficiency virus type 1 tat genes, were found to have no effect on HPV-18 expression. In transient assays, the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated HPV-18 expression. The region of HPV-18 activated by TPA was localized to a sequence which is homologous to other TPA-responsive elements.     

Lethal(1) aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster

Using P element-mediated mutagenesis we have isolated 20 X-linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tuml and tu(1)Szts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from "autoimmune responses" or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.     

Phenotype and genotype of Clostridium sp. strains producing botulism neurotoxin

Neurotoxins produced by strains of Clostridium sp. are belonging to the most toxic biological substances. In the study phenotypes and genotypes of C. botulinum strains in animal studies in vivo and on the DNA level were evaluated, respectively. Additionally, the presence of genes encoding BoNT toxins of A, B, and E types among strains of Clostridium sp. were identified. In case of C. botulinum DNA was isolated from vegetative bacterial cells and from spores. Two different genes encoding two different neurotoxins harboured by three strains of Ae biotype/ae genotype, and by two strains of B biotype/be genotype were detected. Additionally, above E type C. botulinum strains, the presence of gene encoding E type neurotoxin, was found in genome of two C. baratii, two C. butyricum, and C. bifidobacterium, and C. oedematicum strains. C. bifidobacterium and C. oedematicum strains positive for presence of gene encoding E type neurotoxin, were found negative for E neurotoxin production in vivo in TN test. The study indicates that genes encoding BoNT/E neurotoxins are very common among Clostridium species. Phenotype and genotype analysis indicated co-presence of B phenotype together with be genotype and A phenotype together with ae genotype among C. botulinum strains.     

Melanotic mutants in Drosophila: pathways and phenotypes

Mutations in >30 genes that regulate different pathways and developmental processes are reported to cause a melanotic phenotype in larvae. The observed melanotic masses were generally linked to the hemocyte-mediated immune response. To investigate whether all black masses are associated with the cellular immune response, we characterized melanotic masses from mutants in 14 genes. We found that the melanotic masses can be subdivided into melanotic nodules engaging the hemocyte-mediated encapsulation and into melanizations that are not encapsulated by hemocytes. With rare exception, the encapsulation is carried out by lamellocytes. Encapsulated nodules are found in the hemocoel or in association with the lymph gland, while melanizations are located in the gut, salivary gland, and tracheae. In cactus mutants we found an additional kind of melanized mass containing various tissues. The development of these tissue agglomerates is dependent on the function of the dorsal gene. Our results show that the phenotype of each mutant not only reflects its connection to a particular genetic pathway but also points to the tissue-specific role of the individual gene.     

Brucella melitensis cyclic di-GMP phosphodiesterase BpdA controls expression of flagellar genes

Brucella melitensis encounters a variety of conditions and stimuli during its life cycle--including environmental growth, intracellular infection, and extracellular dissemination--which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in B. melitensis, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the bpdA and bpdB genes resulted in attenuation of virulence of the bacterium, while deletion of the cgsB gene produced a hypervirulent strain. In a Vibrio reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of bpdA resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the bpdA and bpdB mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in Brucella melitensis via cyclic-di-GMP.     

Lethal(1)aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster

Using P element-mediated mutagenesis we have isolated 20 X-linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tum^l and tu(1)Sz^ts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from “autoimmune responses” or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.