Molecular cloning, genomic organization, and expression of a C-type (Manduca sexta-type) allatostatin preprohormone from Drosophila melanogaster

The insect allatostatins are a diverse group of neuropeptides that obtained their names by their inhibitory actions on the corpora allata (two endocrine glands near the insect brain), where they block the biosynthesis of juvenile hormone (a terpenoid important for development and reproduction). Chemically, the allatostatins can be subdivided into three different peptide groups: the large group of A-type (cockroach-type) allatostatins, which have the common C-terminal sequence Y/FXFGLamide; the B-type (cricket-type) allatostatins, which have the C-terminal sequence W(X(6))Wamide in common; and a single allatostatin that we now call C-type allatostatin that was first discovered in the moth Manduca sexta, and which has a nonamidated C terminus, and a structure unrelated to the A- and B-type allatostatins. We have previously cloned the preprohormones for the A- and B-type allatostatins from Drosophila melanogaster. Here we report on the cloning of a Drosophila C-type allatostatin preprohormone (DAP-C). DAP-C is 121 amino acid residues long and contains one copy of a peptide sequence that in its processed form has the sequence Y in position 4) from the Manduca sexta C-type allatostatin. The DAP-C gene has three introns and four exons and is located at position 32D2-3 on the left arm of the second chromosome. Northern blots show that the gene is strongly expressed in larvae and adult flies, but less in pupae and embryos. In situ hybridizations of larvae show that the gene is expressed in various neurons of the brain and abdominal ganglia and in endocrine cells of the midgut. This is the first publication on the structure of a C-type allatostatin from insects other than moths and the first report on the presence of all three types of allatostatins in a representative of the insect order Diptera (flies).     

Molecular cloning and genomic organization of an allatostatin preprohormone from Drosophila melanogaster

The insect allatostatins are neurohormones, acting on the corpora allata (where they block the release of juvenile hormone) and on the insect gut (where they block smooth muscle contraction). We screened the "Drosophila Genome Project" database with electronic sequences corresponding to various insect allatostatins. This resulted in alignment with a DNA sequence coding for some Drosophila allatostatins (drostatins). Using PCR with oligonucleotide primers directed against the presumed exons of this Drosophila allatostatin gene and subsequent 3'- and 5'-RACE, we were able to clone its cDNA. The Drosophila allatostatin preprohormone contains four amino acid sequences that after processing would give rise to four Drosophila allatostatins: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH(2) (drostatin-1), Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH(2) (drostatin-2), Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH(2) (drostatin-3), and Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2) (drostatin-4). Drostatin-2 is identical to helicostatin-2 (11-18) and drostatin-3 to helicostatin-3, two neurohormones previously isolated from the moth Helicoverpa armigera. Furthermore, drostatin-3 has previously been isolated from Drosophila itself. Drostatins-1 and -4 are novel members of the insect allatostatin neuropeptide family. The Drosophila allatostatin preprohormone gene contains two introns and three exons. The gene is located on the right arm of the third chromosome, position 96A-B. The existence of at least four different Drosophila allatostatins opens the possibility of a differential action of some of these hormones on the two recently cloned Drosophila allatostatin receptors, DAR-1 and -2. This is the first report on an allatostatin preprohormone from Drosophila.     

Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster

The insect allatostatins obtained their names because they block the biosynthesis of juvenile hormone (a terpenoid) in the corpora allata (two endocrine organs near the insect brain). Chemically, the allatostatins can be subdivided into three different peptide groups: the A-type allatostatins, first discovered in cockroaches, which have the C-terminal sequence Y/FXFGLamide in common; the B-type allatostatins, first discovered in crickets, which all have the C-terminal sequence W(X)(6)Wamide; and the C-type allatostatins, first discovered in the moth Manduca sexta, which have an unrelated and nonamidated C terminus. We have previously reported the structure of an A-type allatostatin preprohormone from the fruitfly Drosophila melanogaster. Here we describe the molecular cloning of a B-type prepro-allatostatin from Drosophila (DAP-B). DAP-B is 211 amino acid residues long and contains one copy each of the following putative allatostatins: AWQSLQSSWamide (drostatin-B1), AWKSMNVAWamide (drostatin-B2), 相似文献   

Chromosomal localization of FLT4, a novel receptor-type tyrosine kinase gene.

A new human gene encoding a putative receptor-type tyrosine kinase (RTK) was isolated by screening a placenta cDNA library with a mouse Flt3 probe. The deduced amino acid sequence of the intracellular region of the molecule showed that it was strongly related to the FLT1 and KDR/FLK1 gene products and to a lesser degree to members of the class III RTKs: FMS/CSF1R, PDGFRA/B, KIT, and FLT3. The gene was named FLT4. Cosmid clones of the mouse Flt4 gene were isolated. The human gene was localized to bands q34-q35 of chromosome 5, i.e., slightly telomeric to the CSF1R/PDGRFB tandem of genes, and the mouse homolog to chromosome 11, region A5-B1.     

Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster

We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgül et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide belonging to the insect allatostatin neuropeptide family. In the present paper, we screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to the conserved regions of the four rat (delta, kappa, mu, nociceptin/orphanin FQ) opioid receptors. This yielded alignment with a Drosophila genomic database clone that contained a DNA sequence coding for a protein having, again, structural similarities with the rat galanin receptors. Using PCR with primers coding for the presumed exons of this second Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as template, we subsequently cloned the cDNA of this receptor. The receptor cDNA codes for a protein that is strongly related to the first Drosophila receptor (60% amino acid sequence identity in the transmembrane region; 47% identity in the overall sequence) and that is, therefore, most likely to be a second Drosophila allatostatin receptor (named DAR-2). The DAR-2 gene has three introns and four exons. Two of these introns coincide with two introns in the first Drosophila receptor (DAR-1) gene, and have the same intron phasing, showing that the two receptor genes are clearly evolutionarily related. The DAR-2 gene is located at the right arm of the third chromosome, position 98 D-E. This is the first report on the existence of two different allatostatin receptors in an animal.     

Allatostatin C and its paralog allatostatin double C: The arthropod somatostatins

Arthropods do not have one, but two genes encoding an allatostatin C-like peptide. The newly discovered paralog gene was called Ast-CC, and the peptide which it is predicted to make was called allatostatin double C (ASTCC). Genes for both allatostatin C (ASTC) and its paralog were found in the tick Ixodes scapularis as well as dipteran, lepidopteran, coleopteran, aphidoidean and phthirapteran insect species. In addition partial or complete cDNAs derived from Ast-CCs were found in a number of species, including Drosophila melanogaster, Bombyx mori and Rhodnius prolixus. The ASTCC precursors have a second conserved peptide sequence suggesting that they may produce two biologically active peptides. The predicted precursors encoded by the Ast-CCs have some unusual features, particularly in Drosophila, where they lack a signal peptide, and have instead a peptide anchor. These unusual structural features suggest that they are perhaps expressed by cells that are not specialized in neuropeptide synthesis and that in Drosophila ASTCC may be a juxtacrine. Data from the Fly Atlas project show that in Drosophila Ast-CC is little expressed. Nevertheless a P-element insertion in this gene is embryonic lethal, suggesting that it is an essential gene. Similarity between the precursors and receptors of ASTC/ASTCC and somatostatin suggests that ASTC/ASTCC and somatostatin have a common ancestor.     

Structural, functional, and evolutionary characterization of novel members of the allatostatin receptor family from insects

By using degenerate primers based on known mammalian somatostatin receptors and the recently identified Drosophila allatostatin receptors (AlstR), we have cloned a novel receptor for the neuropeptide, allatostatin, from the cockroach Periplaneta americana. The receptor exhibits about 60% amino acid identity in the transmembrane regions when compared to the two known AlstRs from Drosophila melanogaster. In addition, two cDNA fragments were obtained from the stick insect Carausius morosus, one of which is similar to Drosophila AlstR, whereas the other is more similar to mammalian somatostatin receptors. Functional expression in Xenopus oocytes shows that the Periplaneta-AlstR exhibits high affinity to endogenous cockroach allatostatin peptides. Studies with synthetic peptides demonstrate that agonistic activity is mediated by the conserved C-terminal pentapeptide YXFGL-amide; in this sequence, amidation of the C-terminus is obligatory to maintain affinity. Thus, our studies provide a molecular basis for understanding the widespread biological activities of the allatostatin peptides.     

PRIMARY STRUCTURE OF ALLATOSTATIN 4, A NEVROPEPTIDE INHIBITOR OF JUVENILE HORMONE SYNTHESIS

Abstract Isolation and identification of allatostatin 4 (AST 4) from crude brain extracts of female Diploptera punctata are described. Synthetic analogues of allatostatin 4 truncated from the N-terminal were evaluated to gain some knowledge of structure-activity correlation. AST 4 reversibly inhibits the biosynthesis of JH in vitro . AST 4 has the following sequence: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH₂. This neurohormone (AST 4) has no sequence similarity with any known neuropeptide from other organisms. Synthetic AST 4 as well as truncation fragments, including two with the five and six amino acids terminal residues deleted showed in vitro activity distinguishable from that of the native allatostatin.     

Identification of four Drosophila allatostatins as the cognate ligands for the Drosophila orphan receptor DAR-2

The allatostatins are generally inhibitory insect neuropeptides. The Drosophila orphan receptor DAR-2 is a G-protein-coupled receptor, having 47% amino acid residue identity with another Drosophila receptor, DAR-1 (which is also called dros. GPCR, or DGR) that was previously shown to be the receptor for an intrinsic Drosophila A-type (cockroach-type) allatostatin. Here, we have permanently expressed DAR-2 in CHO cells and found that it is the cognate receptor for four Drosophila A-type allatostatins, the drostatins-A1 to -A4. Of all the drostatins, drostatin-A4 (Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2)) is the most effective in causing a second messenger cascade (measured as bioluminescence; threshold, 10(-9) M; EC(50), 10(-8) M), whereas the others are less effective and about equally potent (EC(50), 8 x 10(-8) M). Northern blots showed that the DAR-2 gene is expressed in embryos, larvae, pupae, and adult flies. In adult flies, the receptor is more strongly expressed in the thorax/abdomen than in the head parts, suggesting that DAR-2 is a gut receptor. This is confirmed by Northern blots from 3rd instar larvae, showing that the DAR-2 gene is mainly expressed in the gut and only very weakly in the brain. The Drosophila larval gut also contains about 20-30 endocrine cells, expressing the gene for the drostatins-A1 to -A4. We suggest, therefore, that DAR-2 mediates an allatostatin (drostatin)-induced inhibition of gut motility. This is the first report on the permanent and functional expression of a Drosophila gut neurohormone receptor.     

调控保幼激素合成的神经肽抑制素Allatostatin 4的初级结构分析

由太平洋折翅蠊Diploptera punctata脑提取,纯化出脑神经肽Allatostatin 4(AST 4),并合成了AST 4及两种类似物。这种神经肽可有效地抑制体外咽侧体的保幼激素生物合成。测定了这种肽的初级结构,有8个氨基酸残基。它与已知的AST 1—3的氨基酸序列有某些相似性,即靠近C-末端的三个氨基酸和从C-末端数第五和第六位氨基酸相同。AST 4的氨基酸序列为: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH_2 合成的肽和由N末端截断的片段(末端残基为5或6个氨基酸),其体外活性明显。     

Modulation of cardiac rhythm by allatostatins in the cockroach Blattella germanica (L.) (Dictyoptera,Blattellidae)

Cardiac rhythm was measured in Blattella germanica females during the reproductive cycle. The rate increased from day 0 to 1, remained constant during the vitellogenic period and fell by about 20% during the period of oothecal transport. The effects of allatostatins, allatostatin analogues and corazonin were tested on semi-isolated heart preparations. Allatostatins showed a rapid, reversible and dose-dependent cardioinhibitory activity. Blattella allatostatin 1 (BLAST-1: LYDFGL-NH(2)), was the most active, eliciting 76% inhibition at 10(-7) M and even 19% inhibition at 10(-9) M. BLAST-2 (DRLYSFGL-NH(2)), BLAST-3 (AGSDGRLYSFGL-NH(2)) and BLAST-4 (APSSAQRLYGFGL-NH(2)) were less active. An analogue of BLAST-2 with C-terminus in acid form and a pseudopeptide analogue of BLAST-2 with a methyleneamino Psi[CH(2)NH] peptide bond surrogate between residues L(3) and Y(4) were inactive. Corazonin elicited rapid, reversible and dose-dependent cardioacceleratory activity. When tested together with BLAST-1, corazonin overrode the cardioinhibitory effect of allatostatin. Our previous results had shown that high levels of allatostatin were maintained during the period of oothecal transport. This and the fact that physiological concentrations of allatostatins produce physiological levels of inhibition, suggest that allatostatins are involved in the modulation of cardiac rhythm in this cockroach.     

Neuropeptidergic control of the hindgut in the black-legged tick Ixodes scapularis

The hindgut, as a part of the tick excretory system, plays an important physiological role in maintaining homoeostases and waste elimination. Immunoreactive projections from the synganglion to the hindgut were found using antibodies against four different neuropeptides: FGLamide related allatostatin, myoinhibitory peptide, SIFamide, and orcokinin. The presence of FGLamide related allatostatin, myoinhibitory peptide and SIFamide in both synganglia (source) and hindgut (target organ) extracts was confirmed by MALDI-TOF. Tissue-specific PCR revealed the expression of four putative FGLamide related allatostatin receptors and an SIFamide receptor in the hindgut. An antibody against Ixodes scapularis SIFamide receptor detected immunoreactive spots in epithelial cells as well as the visceral muscles surrounding the rectal sac, while staining with the antibody against myoinhibitory peptide receptor 1 revealed that the immunoreactivity was only associated with the visceral muscles. In hindgut motility assays, SIFamide activated hindgut motility in a dose-dependent manner. None of other three neuropeptides (FGLamide related allatostatin, myoinhibitory peptide and orcokinin) activated hindgut motility when tested alone. Myoinhibitory peptide antagonised the SIFamide-stimulated hindgut mobility when it was tested in combination with SIFamide.     

Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells.

To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (4(6), Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH( 2,) and Hel 11-7, KLLK-LLLK-LWKK-LLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 4(6) had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 4(6) and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide-DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.     

An internal AUU codon initiates a smaller peptide encoded by bacteriophage T4 baseplate gene 26

Summary Bacteriophage T4 baseplate gene 26 codes for two in-frame overlapping peptides with identical C-terminal regions. By site-directed mutagenesis we have now determined that an internal AUU, codon 114 of gene 26, is used as the initiation codon for the synthesis of a smaller peptide (gp26^*). Thus gene 26^* gives rise to a peptide of 95 amino acid residues with an M_r of 10873, while the complete gene 26 encodes a peptide of 208 amino acid residues of M_r 23 880. Expression of gene 26^* is shown to depend on the RNA secondary structure in the translational initiation region of this gene.     

Biochemical and genetic characterization of coagulin, a new antilisterial bacteriocin in the pediocin family of bacteriocins, produced by Bacillus coagulans I(4)

A plasmid-linked antimicrobial peptide, named coagulin, produced by Bacillus coagulans I(4) has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42-50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I(4). Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI(4) DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI(4) when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus.     

Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae)

We previously purified and determined the partial amino acid sequence of a 4 kDa peptide having high homology with scorpion defensin from the hemolymph of adult fed female soft ticks, Ornithodoros moubata. In this study, the full length sequences of two defensin isoforms were obtained. Deduced amino acid sequences reveal a precursor protein of 73 amino acid residues with a mature portion consisting of 37 amino acid residues. This mature peptide contains six cysteine residues conserved in the same location as other invertebrate defensins. Phylogenetic analysis reveals that Ornithodoros defensin is most closely related to scorpion defensin and other more ancient arthropods. Ornithodoros defensin mRNA is constitutively expressed and up-regulated by blood-feeding and bacterial injection. Ornithodoros defensin gene expression occurs mainly in the midgut. This is the first report of the cloning and gene expression of an antibacterial peptide from the Acari.