Sequence and expression of Drosophila Antigen 5-related 2, a new member of the CAP gene family.

A cDNA synthesized and cloned from Drosophila melanogaster mRNA was shown to encode a protein with significant identity to the protein encoded by the Drosophila Antigen 5-related (Agr) gene. Because of its similarity to Agr, this novel cDNA was termed Antigen 5-related 2 (Agr2). The proteins encoded by Agr and Agr2 both belong to the CAP family of proteins, which include the mammalian Cysteine-rich secretory proteins, wasp venom Antigen 5 proteins, and plant group 1 Pathogenesis-related proteins. CAP family proteins share a common core sequence, and most appear to function extracellularly. Agr2 encodes a protein that is 254 amino acids in length. The cDNA contains a short 5' untranslated region (UTR) 36bp in length, and a 3' UTR of 46bp. The protein encoded by Agr2 has 48% identity and 61% similarity to that encoded by Agr. The Agr2 gene was localized to region 12F of the X chromosome and probably lies near the Agr gene. Agr2 RNA was approx. 950nt in length and was most abundant during the larval period, although transcripts could also be detected at other developmental stages. Transcription of Agr2 is initiated in the embryo within the midgut epithelium near the site where the third midgut constriction will form. As embryogenesis proceeds, Agr2 expression expands to form a domain located just posterior to the third midgut constriction. This domain corresponds to a single loop of the midgut in late-stage embryos. Based on its expression pattern and its similarity to other CAP family proteins, the Agr2 protein is hypothesized to function either as a novel type of protease inhibitor or as an antimicrobial protein.

Structure and expression of the antigen 5-related gene of Drosophila melanogaster.

The Agr gene of Drosophila melanogaster was localized to the X chromosome 12F region between the Netrin B and rutabaga genes. A P1 clone containing this area was identified, and restriction sites within this clone were mapped. The entire Agr gene and some flanking region were sequenced, and the transcription start site was determined. The gene was 938 bp in length and included a 108 bp intron, dividing the coding region into a 140 bp 5' exon and a 690 bp 3' exon. The 5' untranslated region (UTR) was 23 bp in length and the 3' UTR 36 bp. Gene expression was initiated during late embryogenesis and was also observed during the larval, prepupal, and adult stages. Expression was found only in the outer layer of the proventriculus, a structure located at the fore- and midgut junction. Deletion of the Agr gene had no effect on proventricular morphogenesis in embryos. The Agr expression pattern within the proventriculus suggests that it encodes a component of the peritrophic matrix, a tubular envelope produced by the proventriculus and which lines the midgut. Sequences similar to the Agr coding region were detected in other dipteran species. suggesting that the function of the Agr gene product is conserved.

Calmodulin point mutations affect Drosophila development and behavior.

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations.

A novel cDNA from Drosophila encoding a protein with similarity to mammalian cysteine-rich secretory proteins, wasp venom antigen 5, and plant group 1 pathogenesis-related proteins.

The CAP protein family is made up of a group of secreted proteins that share sequence similarity. Members of this family are found in animals, plants, and fungi, and their shared sequence similarity suggests that members share a common, but as yet unknown, molecular function. As a first step in defining the function of CAP family proteins, an 878 bp partial cDNA encoding a novel member of the CAP family was cloned by the polymerase chain reaction (PCR) from total RNA of adult Drosophila. The cDNA contained the complete coding sequence for a protein 256 amino acids in length, as well as the complete 3' untranslated region (UTR) and a portion of the 5' UTR. The protein, named Antigen 5-related (Agr), was most similar in sequence to antigen 5 (Ag5), a CAP family member found in social wasps and ants. The corresponding Agr RNA is about 1 kb in length and is present at all stages of development, with highest levels observed in adults. Agr RNA is transcribed from a single gene that is located within region 12F of the X chromosome. The identification of Agr in Drosophila expands the number of known CAP family members to well over four dozen. Further studies of Agr and the gene which encodes this protein using the Drosophila model system may help provide important insight into the molecular functioning of this little known, but increasingly significant protein family.

Spontaneous avoidance behavior in Drosophila null for calmodulin expression.

The regulatory protein calmodulin is a major mediator of calcium-induced changes in cellular activity. To analyze the roles of calmodulin in an intact animal, we have generated a calmodulin null mutation in Drosophila melanogaster. Maternal calmodulin supports calmodulin null individuals throughout embryogenesis, but they die within 2 days of hatching as first instar larvae. We have detected two pronounced behavioral abnormalities specific to the loss of calmodulin in these larvae. Swinging of the head and anterior body, which occurs in the presence of food, is three times more frequent in the null animals. More strikingly, most locomotion in calmodulin null larvae is spontaneous backward movement. This is in marked contrast to the wild-type situation where backward locomotion is seen only as a stimulus-elicited avoidance response. Our finding of spontaneous avoidance behavior has striking similarities to the enhanced avoidance responses produced by some calmodulin mutations in Paramecium. Thus our results suggest evolutionary conservation of a role for calmodulin in membrane excitability and linked behavioral responses.

Calmodulin transcription is limited to the nervous system during Drosophila embryogenesis.

We have examined the RNA expression pattern for the Drosophila calmodulin gene during embryogenesis by in situ hybridization to transcripts in whole embryos. Our results indicate that maternally derived calmodulin mRNA is homogeneously distributed throughout the early embryo, but that these maternal transcripts are lost by maximal germ band extension. Zygotic transcription of the gene in mid- to late-stage embryos is restricted to neural cell precursors and their progeny in both the central and peripheral nervous systems. Thus, activation of calmodulin transcription during embryonic development appears to mark a commitment to a neural fate. Northern blot analysis revealed that the two transcripts from the calmodulin gene are differentially expressed during embryogenesis. Comparison of Northern blot and in situ hybridization data indicates that the longer calmodulin mRNA is a nervous tissue-specific transcript. This suggests that neural-specific regulation of polyadenylation site usage occurs. We have also examined calmodulin expression in embryos homozygous for mutations in four loci which are known to affect nervous system development: numb, the achaete-scute complex, daughterless, and mastermind. The calmodulin transcription pattern is altered in embryos mutant for each of these loci, suggesting that regulation by these genes, either directly or indirectly, is taking place.

Drosophila melanogaster contains a single calmodulin gene. Further structure and expression studies.

We have previously characterized a calmodulin gene from the organism Drosophila melanogaster. In the higher vertebrates a multi-gene system for encoding calmodulin is present and, in at least one invertebrate species, genes encoding highly related calmodulin isotypes exist. We have therefore searched for additional calmodulin genes within D. melanogaster. Although our searches were sensitive enough to detect a relatively divergent gene encoding a calmodulin family protein, we were unable to detect any additional genes for calmodulin per se. Further studies of the structure and expression of the single calmodulin gene of D. melanogaster have established that the gene contains a tiny additional 5' exon encoding only 50 residues of the 5' leader. Sequencing at the 3' terminus has established that the two transcript size classes derived from the gene are produced as a result of alternative polyadenylation site usage. The relative abundance of the two size classes of mRNAs differs throughout the life cycle, indicating developmental regulation of polyadenylation site usage.

Photoaffinity labeling of the hemolymph juvenile hormone binding protein of Manduca sexta.

A synthetic analogue of the insect juvenile hormone (JH) III, 10,11-epoxy[10-3H]farnesyl diazoacetate [( 3H]-EFDA), binds to several proteins in a partially purified preparation of hemolymph protein from fourth instar larvae of Manduca sexta when irradiated with UV light. Approximately 80% of this binding could be inhibited by the addition of excess unlabeled JH I. To compare the relative affinity of EFDA for the juvenile hormone binding protein (JHBP) with that of the various JH homologues, the ability of unlabeled EFDA and JH homologues to displace [3H]JH I from binding sites was measured. The relative affinities were EFDA greater than JH I greater than JH II greater than JH III. When Scatchard analysis of the binding of [3H]EFDA or [3H]JH I to the larval JHBP was performed, an estimated apparent KD of 4.5 X 10(-8)M was found for EFDA, whereas for JH I a slightly higher KD of 8.8 X 10(-8) M was calculated. To determine if [3H]EFDA bound at the JH I binding site, displacement of [3H]JH I from the JHBP complex with unlabeled JH I, JH II, and JH III was compared to the displacement of [3H]EFDA with the same homologues. The results demonstrated that the photoaffinity label bound covalently at the JH I binding site on the hemolymph binding protein of Manduca sexta. Fluorescence autoradiography of [3H]EFDA photoaffinity labeled proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that [3H]EFDA bound covalently to two major proteins in the absence of JH I.(ABSTRACT TRUNCATED AT 250 WORDS)

A specific photoaffinity label for hemolymph and ovarian juvenile hormone-binding proteins in Leucophaea maderae.

A tritium-labeled diazocarbonyl juvenile hormone (JH) analog, (10-[10,11-3H]epoxyfarnesyl diazoacetate, [3H]EFDA), covalently bound to proteins in both hemolymph and ovarian extracts when reaction mixtures were irradiated with UV light. The addition of various concentrations of unlabeled JH III selectively inhibited [3H]EFDA photoattachment to proteins. Using the Scatchard method of analysis, [3H]EFDA bound specifically and with relatively high affinity (KD = 1.5 X 10(-6) M) to a macromolecule in each extract, although nonspecific binding to other molecules was also present (20-50%). To determine if [3H]EFDA bound at the JH III-binding site on the binding proteins, radioactive [3H]JH III or [3H]EFDA was complexed with proteins in the presence of various concentrations of either unlabeled JH III or JH I under equilibrium conditions. The results demonstrated that the natural hormone, JH III, displaced both bound labeled ligands 4.1 +/- 0.5 times better than the homolog JH I. Thus, the photoaffinity label [3H]EFDA bound at the same site on the protein as [3H] JH III. Fluorescent autoradiography of [3H]EFDA-labeled proteins separated by sodium dodecyl sulfate electrophoresis revealed that several proteins in both hemolymph and ovarian extracts bound [3H]EFDA. To determine the specificity of binding, extracts were irradiated with UV light in the presence of unlabeled JH III and [3H]EFDA. The results demonstrated that JH III prevented photoattachment of [3H]EFDA to a major protein in each extract. The molecular weight of these proteins was estimated at approximately 200,000 for both the hemolymph protein and the ovarian protein.

Assay and identification of juvenile hormone binding proteins in Leucophaea maderae.

We modified a binding assay using polyethylene glycol (PEG) to precipitate bound hormone. Optimum precipitation occurred when reaction mixtures were incubated with 10-40% PEG and 1.25-2.5 mg/ml gamma-globulins for 2-90 min at 4 or 23 degrees C. Results from this assay and from the dextran-coated charcoal assay were similar. Addition of phenylmethylsulfonyl fluoride eliminated nonspecific esterase activity in extracts. JH III-binding macromolecules were identified in hemolymph and ovaries of Leucophaea maderae. These molecules were pronase- and heat-sensitive and saturable. Using Scatchard analysis an average KD of 2.04 (+/- 0.32) X 10(-8) M and 1.91 (+/- 0.80) X 10(-8) M was calculated for hemolymph and ovarian binding proteins. JH III had the highest affinity for binding sites, followed by JH I and JH 0. Various extraction procedures caused changes in JH affinity for both binding proteins. At high concentrations the (+) isomer and mixed isomer preparations of methoprene and hydroprene competed for binding sites. Binding proteins had no affinity for the (-) isomer or for the JH III acid.