Molecular cloning of the prothoracicotropic hormone from the tobacco hornworm,Manduca sexta

A cDNA encoding a putative precursor of prothoracicotropic hormone (PTTH) from the tobacco hornworm, Manduca sexta, was isolated and sequenced. This clone contains an open reading frame encoding a 226-amino acid prepropeptide hormone. The deduced amino acid sequence is composed of a signal sequence, a precursor domain and a mature hormone and shows similarities to the other PTTHs that have been cloned from closely related lepidopteran species, Bombyx mori, Samia cynthia ricini, Antheraea peryni, and Hyalophora cecropia. Although these cDNAs showed slightly less similarities in predicted amino acid sequences, seven cysteine residues and the hydrophobic regions within those mature peptides were conserved. In situ hybridization using a cDNA probe encoding the Manduca PTTH showed that PTTH mRNA was in two pairs of neurosecretory cells in the Manduca brain. The recombinant putative Manduca PTTH produced in E. coli was biologically active, both causing a larval molt in neck-ligated Manduca 4th instar larvae (ED(50)=50 pM) and the adult molt of diapausing Manduca pupae (ED(50)=79 pM), but was unable to stimulate molting of debrained Bombyx pupae.     

Molecular cloning and characterization of a cDNA encoding proline transporter in rice

A cDNA encoding a proline (Pro) transporter (ProT) was isolated and characterized from a cDNA library prepared from 14-d-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the rice ProT protein (OsProT) had 68.8% homology to the ProT protein 1 from Arabidopsis thaliana and 59.6% homology to that from Lycopersicon esculentum. Northern blot analysis revealed that the gene for OsProT (OsProT) was expressed in all organs examined, comparatively strongly in leaf sheath and stem. Salt treatment did not induce expression of OsProT but strongly induced expression of the gene for delta1-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme in Pro biosynthesis. Southern blot analysis revealed that OsProT has a gene family. OsProT specifically transported L-Pro in a transport assay using Xenopus laevis oocytes.     

Molecular cloning and functional expression of the human Golgi UDP-N-acetylglucosamine transporter.

We have cloned the human UDP-N-acetylglucosamine (UDP-GlcNAc) transporter cDNA, which was recognized through a homology search in the expressed sequence tags database (dbEST) based on its similarity to the human UDP-galactose transporter. The chromosomal location of the UDP-GlcNAc transporter gene was assigned to chromosome 1p21 by fluorescence in situ hybridization (FISH). The transporter was expressed ubiquitously in every tissue so far examined. Expression of the transporter cDNA in CHO-K1 cells in its native and in a C-terminally HA-tagged form indicated that the human UDP-GlcNAc transporter was localized in the Golgi apparatus. The membrane vesicles prepared from yeast cells expressing the cDNA product exhibited UDP-GlcNAc-specific transporting activity. Comparison among UDP-galactose, CMP-sialic acid, and UDP-GlcNAc transporters from several organisms enabled us to identify residues highly conserved among the transporters and residues specific for each group of transporters.     

Molecular cloning and functional characterization of a neuronal choline transporter from Trichoplusia ni

A cDNA encoding a high-affinity Na(+)-dependent choline transporter (TrnCHT) was isolated from the CNS of the cabbage looper Trichoplusia ni using an RT-PCR-based approach. The deduced amino acid sequence of the CHT cDNA predicts a 594 amino acid protein of 64.74 kDa prior to glycosylation. TrnCHT has 80%, 79%, 76%, and 58% amino acid identity to putative CHTs from Anopheles gambiae, Drosophila melanogaster and Apis mellifera, and a cloned CHT from Limulus polyphemus, respectively. In situ hybridization of TrnCHT cRNA in whole-mount preparations of caterpillar CNS revealed that TrnCHT mRNA is expressed by hundreds of presumably cholinergic neurons present in both the brain and cortex of all segmental ganglia. Na(+)-dependent [(3)H]-choline uptake was induced in Sf9 cells in vitro following infection with a TrnCHT-expressing recombinant baculovirus. Virally induced [(3)H]-choline uptake was found to approximately equal the endogenous rate of choline uptake in insect cells, seen either after infection with a control virus or in TrnCHT-infected cells exposed to [(3)H]-choline in the absence of Na(+). The Na(+)-dependent component of [(3)H]-choline uptake by TrnCHT-infected cells was saturable with a K(m) for choline transport of 8.4 microM. Several compounds reported to be potent blockers of [(3)H]-choline uptake by cloned vertebrate choline transporters proved to be relatively weak inhibitors of choline uptake by Sf9 cells expressing TrnCHT. Hemicholinium-3 (K(i)=4.1 microM) and two oxoquinuclidium analogues of choline, quireston-A (K(i) approximately 10 microM) and quireston (K(i) approximately 100 microM) inhibited 50% of control uptake only at micromolar concentrations. The endogenous low-affinity Na(+)-independent uptake of [(3)H]-choline was also inhibited by high micromolar concentrations of hemicholinium-3.     

Isolation and functional characterization of an allatotropin receptor from Manduca sexta

Manduca sexta allatotropin (Manse-AT) is a multifunctional neuropeptide whose actions include the stimulation of juvenile hormone biosynthesis, myotropic stimulation, cardioacceleratory functions, and inhibition of active ion transport. Manse-AT is a member of a structurally related peptide family that is widely found in insects and also in other invertebrates. Its precise role depends on the insect species and developmental stage. In some lepidopteran insects including M. sexta, structurally-related AT-like (ATL) peptides can be derived from alternatively spliced mRNAs transcribed from the AT gene. We have isolated a cDNA for an AT receptor (ATR) from M. sexta by a PCR-based approach using the sequence of the ATR from Bombyx mori. The sequence of the M. sexta ATR is similar to several G protein-coupled receptors from other insect species and to the mammalian orexin receptor. We demonstrate that the M. sexta ATR expressed in vertebrate cell lines is activated in a dose-responsive manner by Manse-AT and each Manse-ATL peptide in the rank order ATL-I > ATL-II > ATL-III > AT, and functional analysis in multiple cell lines suggest that the receptor is coupled through elevated levels of Ca(2+) and cAMP. In feeding larvae, Manse-ATR mRNA is present at highest levels in the Malpighian tubules, followed by the midgut, hindgut, testes, and corpora allata, consistent with its action on multiple target tissues. In the adult corpora cardiaca--corpora allata complex, Manse-ATR mRNA is present at relatively low levels in both sexes.     

Developmental expression of Manduca sexta hemolin.

Hemolin is hemolymph protein that is a member of the immunoglobulin superfamily. Its induced expression after bacterial infection suggests that it functions in the immune response. In this paper, we describe the expression of the Manduca sexta hemolin gene at certain developmental stages in the absence of microbial challenge. Hemolin was present at a very low level in hemolymph of naive larvae until the beginning of the wandering stage prior to pupation, when its concentration in hemolymph increased dramatically. At the same time, hemolin could be found in the fluid contained in the midgut lumen. The appearance of hemolin mRNA in fat body and midgut at the beginning of the wandering stage correlated with the presence of hemolin in the hemolymph and midgut lumen. Hemolin was present in hemolymph through the pupal and adult stages. Hemolin was also present in newly deposited eggs, and persisted in eggs throughout embryonic development. A hemolin cDNA isolated from an adult fat body library had the same sequence as those previously obtained from larval libraries. Hemolin purified from hemolymph of bacteria-injected larvae, from hemolymph of naive wandering stage larvae and adult moths, and from midgut fluid of wandering stage larvae had the same apparent mass, which was consistent with the mass predicted from the hemolin cDNA sequence. Hemolin from hemolymph of wandering stage larvae did not contain any detectable carbohydrate, but hemolin from the hemolymph of bacteria-injected larvae and from naive adult moths was associated with carbohydrate, although of different amounts and composition. These results suggest that a single hemolin gene is developmentally regulated and is also induced when insects are exposed to microbial infection. M. sexta hemolin apparently lacks post-translational covalent glycosylation, but instead is associated under some conditions with non-covalently bound carbohydrates. Arch.     

cDNA cloning and expression of a hormone-regulated heat shock protein (hsc 70) from the prothoracic gland of Manduca sexta

The brain neuropeptide prothoracicotropic hormone (PTTH) stimulates a rapid increase in ecdysteroid hormone synthesis that is accompanied by general and specific increases in protein synthesis, including that of a 70 kDa cognate heat shock protein (hsc 70). To further understand the possible roles of hsc 70, hsc 70 cDNA clones were isolated from a tobacco hornworm (Manduca sexta) prothoracic gland cDNA library. All sequenced clones were highly homologous to the Drosophila hsc 70-4 isoform. Manduca hsc 70 mRNA levels during the last larval instar exhibited a peak at the onset of wandering and a peak that coincided with the major pre-metamorphic peak of ecdysteroid synthesis. Manipulations of the glands' hormonal milieu showed that hsc 70 mRNA levels respond to 20-hydroxyecdysone, dibutyryl cAMP, PTTH and the JH analogue hydroprene. The protein and mRNA data suggest that hsc 70 could be involved in a negative feedback loop regulating assembly of the ecdysone receptor complex.     

Molecular cloning and functional expression of a VIP-specific receptor

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC(1), with high affinity for PACAP, and VPAC(1) and VPAC(2) with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIP(s)) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH(2)-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC(2) receptor from GP gastric smooth muscle and VIP(s) from GP teniae coli smooth muscle. The cDNA sequence of the VIP(s) encodes a 437-amino acid protein (M(r) 49,560) that possesses 87% similarity to VPAC(2) receptors in rat and mouse and differs from the VPAC(2) receptor in GP gastric smooth muscle by only two amino-acid residues, F(40)F(41) in lieu of L(40)L(41). In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC(50) 1.4 nM) and stimulated cAMP formation with high potency (EC(50) 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC(50) 2.3 nM) and stimulated cAMP with equally high potency (EC(50) 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIP(s) distinct from VPAC(1) and VPAC(2) receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L(40)L(41)) in the NH(2)-terminal ligand-binding domain.     

Purification of juvenile hormone esterase and molecular cloning of the cDNA from Manduca sexta.

Juvenile hormone esterase (JHE) is a highly specific enzyme important for regulating the onset of metamorphosis in lepidopteran insects. After affinity chromatography of the hemolymph proteins of Manduca sexta, the pure JHE protein was digested with Lys-C and the resultant peptides were purified by microbore HPLC. Two peptides were selected for sequencing. Based upon these amino acid sequences, degenerate RT-PCR was performed in order to amplify a partial cDNA sequence from mRNA from the fat body of M. sexta. A 1512bp partial cDNA was generated and found to be highly homologous to the JHE from Heliothis virescens. 5' and 3' RACE were performed to obtain the full length cDNA sequence. The cDNA has a total length of 2220bp, with a 1749bp coding region. The deduced protein sequence contains 573 amino acids.     

Molecular cloning and functional expression of a Drosophila corazonin receptor

Here we report a novel method for selecting human antibody fragments from nonimmunized variable domain libraries. The antibody fragments are selected on the basis of stabilization of the variable domain fragment (F(v)) in the presence of target antigens ("open sandwich selection"). One variable domain is displayed on phages and another is prepared as soluble molecules. These two reagents are mixed with the biotinylated target molecule and ternary complexes are captured by using streptavidin-conjugated magnet beads. After extensive washing, enriched clones are eluted by using target antigen. Some of the clones selected after 3 rounds are prepared as soluble domains, which then undergo another selection process. We obtained several human antibody fragments specific for human soluble erythropoietin receptor by using this method. Our method minimizes several of the disadvantages associated with human antibody selection through a phage-display system, such as construction of a large-scale library, deletion of genes during selection, and nonspecific binding.     

Molecular cloning and functional characterization of an ATP-binding cassette transporter OtrC from Streptomyces rimosus

ABSTRACT: BACKGROUND: The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC) transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production. RESULTS: In order to investigate OtrC's function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC) assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877) and 1.4-fold in SR16 (P = 0.00973) duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively). CONCLUSIONS: The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.     

In vitro expression and biochemical characterization of juvenile hormone esterase from Manduca sexta

Juvenile hormone esterase (JHE) is a selective enzyme that hydrolyzes the methyl ester of juvenile hormone. This enzyme plays an important role in the regulation of metamorphosis in caterpillars, and is implicated in additional roles in development and reproduction in this and other orders of insect. The full length coding region of the JHE cDNA from Manduca sexta was subcloned into the baculovirus AcMNPV genome under the control of the p10 promoter. The recombinant virus demonstrated the expression of high levels of JHE activity when infected into Hi5 cells from Trichoplusia ni. The recombinant protein was partially purified by anion exchange chromatography and its biochemical characterization showed similar features to the wild type protein. The recombinant JHE has an estimated MW of 66500 Da. Some heterogeneity with the enzyme was observed when analyzed by isoelectric focusing, although the peak of JHE activity was observed at pI=6.0. It is highly sensitive to trifluoroketone inhibitors and certain phosphoramidothiolates, while relatively insensitive to other common esterase inhibitors. Incubating the enzyme with various organic solvents and detergents showed that the enzyme is activated at lower concentrations of solvents/detergents and remains significantly active even at high concentrations. The high tolerance of organic solvents may make this JHE enzyme useful in future applications as a synthetic catalyst.     

Molecular cloning and functional characterization of human vesicular glutamate transporter 3

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. It is loaded into synaptic vesicles by a proton gradient-dependent uptake system and is released by exocytosis upon stimulation. Recently, two mammalian isoforms of a vesicular glutamate transporter, VGLUT1 and VGLUT2, have been identified, the expression of which enables quantal release of glutamate from glutamatergic neurons. Here, we report a novel isoform of a human vesicular glutamate transporter (hVGLUT3). The predicted amino acid sequence of hVGLUT3 shows 72% identity to both hVGLUT1 and hVGLUT2. hVGLUT3 functions as a vesicular glutamate transporter with similar properties to the other isoforms when it is heterologously expressed in a neuroendocrine cell line. Although mammalian VGLUT1 and VGLUT2 exhibit a complementary expression pattern covering all glutamatergic pathways in the CNS, expression of hVGLUT3 overlaps with them in some brain areas, suggesting molecular diversity that may account for physiological heterogeneity in glutamatergic synapses.     

Characterization of tyrosine hydroxylase from Manduca sexta

In insects, 3,4-dihydroxyphenylalanine (DOPA) is required for tanning of newly formed cuticle and the production of melanin during some types of immune responses. DOPA is produced by the hydroxylation of tyrosine, and this reaction can be catalyzed by two types of enzymes: tyrosine hydroxylase (TH) and phenoloxidase (PO). TH is required for cuticle tanning in Drosophila melanogaster and for cuticle pigmentation in other insect species, but additional functions of TH have been uncertain. In contrast, an immune function for PO has been well documented. The goal of this study was to characterize TH from Manduca sexta with a focus on its possible contribution to cuticle tanning and immune-associated melanization. We cloned a full-length TH cDNA, purified recombinant TH, and confirmed that MsTH and MsPO have tyrosine hydroxylating activity. To determine possible functions, we analyzed TH expression profiles. TH mRNA and protein were present in eggs at the stage when the pharate larval cuticle begins to tan and also in the integument of molting larvae. The amount of TH in the integument was correlated with the degree of cuticle tanning. Unlike PO, which was found to be constitutively expressed by hemocytes and was present in plasma, TH was upregulated in hemocytes and the fat body in response to an immune challenge and remained intracellular. These data suggest that TH is required for cuticle tanning and immunity in M. sexta. Based on the collective information from many studies, we propose a model in which TH is a major producer of the DOPA required for both cuticle tanning and immune-associated melanization.     

Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem

A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable K(i) values of 1.18 x 10(-10) M and 9.53 x 10(-11) M, respectively. The recombinant cystatins were found to be thermally stable up to 60 degrees C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.