Mature Polypeptide Research Articles

A Substrate Specificity-determining Unit of Three Lin12-Notch Repeat Modules Is Formed in Trans within the Pappalysin-1 Dimer and Requires a Sequence Stretch C-terminal to the Third Module

Members of the pappalysin family of metzincin metalloproteinases, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) and PAPP-A2 (pappalysin-2), regulate the bioavailability of insulin-like growth factors (IGFs) by specific proteolytic inactivation of IGF-binding proteins (IGFBPs). PAPP-A cleaves IGFBP-4 and IGFBP-5, whereas PAPP-A2 cleaves only IGFBP-5. The pappalysins contain three Lin12-Notch repeat (LNR1-3) modules, previously considered unique to the Notch receptor family in which they function to regulate receptor cleavage. In contrast to the Notch receptor where three LNR modules are tandemly arranged, LNR3 is separated by more than 1000 residues from LNR1-2 in the pappalysin sequence. Each of the three LNR modules of PAPP-A is required for proteolysis of IGFBP-4, but not IGFBP-5. However, we here find that a C-terminal truncated variant of PAPP-A, which lacks LNR3 and therefore activity against IGFBP-4, cleaves IGFBP-4 when co-expressed with a PAPP-A variant, which is mutated in the active site. This suggests that LNR3 from the inactive subunit interacts in trans with LNR1-2 of the truncated PAPP-A subunit to form a functional trimeric LNR unit. We also show that formation of such a functional LNR unit depends on dimerization, as dissociation of a mutated non-covalent PAPP-A dimer results in reduced activity against IGFBP-4, but not IGFBP-5. Using PAPP-A/PAPP-A2 chimeras, we demonstrate that PAPP-A2 LNR1-2, but not LNR3, are functionally conserved with respect to IGFBP proteolysis. Additionally, we find that a sequence stretch C-terminal to LNR3 and single residues (Asp1521, Arg1529, and Asp1530) within this are required for LNR functionality.

Molecular characterization of a novel 32-kDa merozoite antigen ofBabesia gibsoniwith a better diagnostic performance by enzyme-linked immunosorbent assay

We cloned and expressed a novel gene encoding a 32-kDa merozoite protein of Babesia gibsoni (BgP32). The length of nucleotide sequence of the cDNA was 1464 bp with an open reading frame of 969 bp. The truncated recombinant BgP32 (rBgP32) without a signal peptide and C-terminal hydrophobic sequence was expressed in Escherichia coli as a soluble glutathione-S-transferase (GST) fusion protein. Western blotting demonstrated that the native protein was 32-kDa, consistent with molecular weight of the predicted mature polypeptide. Enzyme-linked immunosorbent assay (ELISA) using rBgP32 detected specific antibodies from 8 days to 541 days post-infection in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with B. canis subspecies and closely related protozoan parasites, indicating that rBgP32 is a specific diagnostic antigen. Analysis of 47 sera taken from dogs with anaemic signs revealed that rBgP32 detected a higher proportion of B. gibsoni seropositive samples (77%) than its previously identified rBgP50 (68%) homologue. These results indicate that the BgP32 is a novel immunodominant antigen of B. gibsoni, and rBgP32 might be useful for diagnosis of B. gibsoni infection.

A novel tetrameric lectin from Lycoris aurea with four mannose binding sites per monomer.

The mannose-binding agglutinin from bulbs of Lycoris aurea (LAA) agglutinates rabbit but not human erythrocytes. The molecular mass of the monomer in SDS/PAGE is 12 kDa while the apparent molecular mass in gel filtration is 48 kDa, indicating that LAA is a homotetramer. The full-length cDNA of LAA contains 683 bp with an open reading frame encoding a protomer of 162 amino-acid residues. Hydrophobic Cluster Analysis and molecular modeling of the 109-residue mature polypeptide suggested a similar secondary and tertiary structure to those of Narcissus pseudonarcissus agglutinin (NPA). Molecular docking revealed that, besides the three mannose-binding sites common among Amaryllidaceae lectins, LAA also contains a fourth unique mannose-binding site formed by a tryptophan cluster. The existence of four mannose-binding sites in each monomer of LAA is very unusual and has only been reported for NPA earlier.

Cell Surface Detachment of Pregnancy-associated Plasma Protein-A Requires the Formation of Intermolecular Proteinase-Inhibitor Disulfide Bonds and Glycosaminoglycan Covalently Bound to the Inhibitor

The metzincin metalloproteinase pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) promotes cell growth by proteolytic cleavage of insulin-like growth factor-binding proteins 4 and 5, causing the release of bound insulin-like growth factors. PAPP-A binds an unknown cell-surface heparan sulfate proteoglycan, suggesting that it controls insulin-like growth factor signaling spatially. In human pregnancy, the majority of PAPP-A circulates as a disulfide-bonded complex with its inhibitor, the proform of eosinophil major basic protein (proMBP). Interestingly, Ser-62 of proMBP is substituted with a glycosaminoglycan (GAG) chain, possibly a heparan sulfate type, and the PAPP-A.proMBP complex is unable to bind to the cell surface. We show here that proMBP detaches surface-bound PAPP-A in a process that depends on the proMBP GAG and also on the formation of intermolecular disulfide bonds between PAPP-A and proMBP. Unlike what was expected, we demonstrate that the GAG of proMBP is not required for PAPP-A.proMBP complex formation and that proMBP residues His-137, Ser-178, Arg-179, and Asn-181 are important for the recognition of PAPP-A. Using a mouse model, we find that the half-life of circulating PAPP-A and proMBP in complex is severalfold higher than both of the uncomplexed proteins, further suggesting that the PAPP-A.proMBP complex is formed at the cell surface in vivo rather than in the circulation. Further supporting this, we show that formation of the PAPP-A.proMBP complex at the cell surface proceeds rapidly compared with the slow rate of complex formation in solution. Because both PAPP-A and proMBP are expressed ubiquitously, this model may be applicable to many tissues in which insulin-like growth factor bioavailability is locally regulated.

Chinese-scorpion (Buthus martensi Karsch) toxin BmK αIV, a novel modulator of sodium channels: from genomic organization to functional analysis

In the present study, BmK alphaIV, a novel modulator of sodium channels, was cloned from venomous glands of the Chinese scorpion (Buthus martensi Karsch) and expressed successfully in Escherichia coli. The BmK alphaIV gene is composed of two exons separated by a 503 bp intron. The mature polypeptide contains 66 amino acids. BmK alphaIV has potent toxicity in mice and cockroaches. Surface-plasmon-resonance analysis found that BmK alphaIV could bind to both rat cerebrocortical synaptosomes and cockroach neuronal membranes, and shared similar binding sites on sodium channels with classical AaH II (alpha-mammal neurotoxin from the scorpion Androctonus australis Hector), BmK AS (beta-like neurotoxin), BmK IT2 (the depressant insect-selective neurotoxin) and BmK abT (transitional neurotoxin), but not with BmK I (alpha-like neurotoxin). Two-electrode voltage clamp recordings on rNav1.2 channels expressed in Xenopus laevis oocytes revealed that BmK alphaIV increased the peak amplitude and prolonged the inactivation phase of Na+ currents. The structural and pharmacological properties compared with those of other scorpion alpha-toxins suggests that BmK alphaIV represents a novel subgroup or functional hybrid of alpha-toxins and might be an evolutionary intermediate neurotoxin for alpha-toxins.

Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli

A cDNA of fatty acyl- acyl carrier protein (ACP) thioesterase (Fat) from developing seed of Madhuca butyracea has been cloned. The deduced amino acid sequence of the cDNA corresponding to the mature polypeptide showed 30–40% and 60–75% identity to the reported FatA and FatB class of plant thioesterases, respectively. This gene, MbFatB, is present as a single copy in M. butyracea genome and the MbFatB protein was detected clearly in seed tissues of this plant but not in that of Indian mustard ( Brassica juncea). Heterologous expression of the MbFatB gene driven by different promoters in E. coli wild type and fatty acid β-oxidation mutant ( fadD88) strains resulted production of the recombinant protein with various fusion tags either as biologically inactive (insoluble) or functionally active forms. Expression of functionally active recombinant MbFatB in E. coli affected bacterial growth and cell morphology as well as changed the fatty acid profiles of the membrane lipid and the culture supernatant. Alteration of the fatty acid composition was directed predominantly towards palmitate and to a lesser extent myristate and oleate due to acyl chain termination activity of plant thioesterase in bacteria. Thus, this new MbFatB gene isolated from a non-traditional oil-seed tree can be used in future for transgenic development of oil-seed Brassica, a widely cultivated crop that expresses predominantly oleoyl-ACP thioesterase (FatA) in its seed tissue and has high amount of unwanted erucic acid in edible oil in order to alter the fatty acid profile in a desirable way.

Aberrant expression of HLA-B*3565Q is associated with a disrupted disulfide bond

The identification of expression variants is a challenge in HLA diagnostics. We here describe the identification of the novel allele HLA-B*3565Q. The serological HLA class I type, as determined by a lymphocytotoxicity test, was A11,24; B38; Bw4; Cw-; whereas PCR-sequence-specific primers resulted in A*11,*24, B*35,*38; Cw*12, thus suggesting the presence of a nonexpressed B*35 allele. To clarify the lack of serological HLA-B35 reactivity, exons 2 and 3 were sequenced following haplotype-specific amplification. At position 564 from the beginning of the coding region (exon 3), a transversion (C-->G) was observed, which, at the amino acid level, results in a substitution from cysteine to tryptophane at position 164 of the mature polypeptide. Because this position is essential for the formation of a disulfide bond linking the cysteine residues at positions 101 and 164, which is strongly conserved in functional class I molecules of vertebrates, the disruption of this bond is very likely to be the reason for the lack of serological detectability. We later found the same novel allele in a second unrelated individual, of whom we were able to establish a lymphoblastoid cell line (B-LCL). Serological testing of this B-LCL indicated a very low aberrant expression of HLA-B*3565Q, which cannot be expected to be detected by standard serology techniques.

Molecular characterization of the hypothetical 66.3-kDa protein in mouse: Lysosomal targeting, glycosylation, processing and tissue distribution

Recently, we and others identified the 66.3-kDa protein as one of several putative novel lysosomal matrix proteins by analyzing mannose 6-phosphate receptors binding proteins [Kollmann K., Mutenda K.E., Balleininger M., Eckermann E., von Figura K., Schmidt B., Lübke T. (2005) Identification of novel lysosomal matrix proteins by proteome analysis. Proteomics 5(15), 3966–3678, Sleat D.E., Lackland H., Wang Y., Sohar I., Xiao G., Li H., Lobel P. (2005) The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins. Proteomics. 5(6), 1520–1532]. Here, we describe the expression of the mouse 66.3-kDa protein in HT1080 cells in which it is synthesized as a precursor of about 75 kDa and subsequently processed by limited proteolysis to mature polypeptides accumulating in the lysosomal compartment. The lysosomal localisation of the endogenous 66.3-kDa protein was verified by indirect immunofluorescence in mouse embryonic fibroblasts and by subcellular fractionation of tyloxapol-filled mouse liver lysosomes. Northern blot analysis reveals high transcriptional levels in testis, liver and kidney, whereas Western blot analysis shows high protein levels in brain, heart, lung and spleen. Interestingly, in mouse the endogenous 66.3-kDa protein is processed in a highly tissue-dependent manner to mature forms.

A Male Reproduction-Related Kazal-Type Peptidase Inhibitor Gene in the Prawn, Macrobrachium rosenbergii: Molecular Characterization and Expression Patterns

Peptidase inhibitors in the male reproductive tract are well known in mammals, in which they play roles in protecting the tract epithelium against proteolytic damage or in regulating the fertilization process. By screening the subtracted cDNA clones enriched for male reproductive tract-specific transcripts, one clone encoding a putative protein that showed significant similarity to Kazal-type peptidase inhibitor (KPI) was obtained. This is the first report of an invertebrate in which a male reproductive tract-specific KPI gene has been identified and characterized. The gene contains a 405-bp open reading frame (ORF), a 72 bp 5' untranslated region (UTR), and a 259 bp 3' UTR. The conceptually translated protein consisted of a 21-amino-acid signal peptide and a 113-amino-acid mature polypeptide with two Kazal-type domains (named after the discoverer). Significant levels of the mRNA were observed only in the male reproductive tract, while mRNA expression was not detected in any other tissues tested. The transcription of the gene remained constant during maturation, although not in the postlarval stage. In situ hybridization demonstrated the presence of the mRNA in the secretory epithelial cells of vas deferens and terminal ampullae.

Characterization of Low Molecular Weight Glutenin Subunit Gene Representing Glu-B3 Locus of Indian Wheat Variety NP4

Low molecular weight (LMW) glutenin subunits represent major part (30%) of storage proteins in wheat endosperm and determine the quality of dough. Despite their importance few LMW glutenin genes have been characterized so far and none from Indian wheat variety. In the present investigation PCR technique was employed to characterize LMW-GS gene representing Glu-B3 locus from Indian bread wheat cultivar NP4. The deduced protein sequence coded by Glu-B3 locus of LMW-GS gene from NP4 showed the presence of regular structure of the repetitive domain with varying numbers of glutamine (Q) residues and the presence of 1st cysteine residue within the repetitive domain at 40th position in mature polypeptide. Such structure might increase and stabilize the gluten polymer through intermolecular interactions of the large numbers of glutamine side chains and cysteine residues for intermolecular disulphide bond formation leading to stronger dough quality of NP4. Moreover, Glu-B3 specific primers could also be used for identifying 1BL/1RS translocation in addition to amplifying LMW glutenin genes. There was no amplification in 1B/1R translocation lines as short arm of wheat was replaced by short arm of rye chromosome in these lines. Such information can be useful in wheat improvement for dough properties for better chapati and bread quality.

Interferon-α Genes from Bos and Bubalus bubalus

Interferon-α genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-α genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-α subtypes, the IFN-α gene of Holstein cow had only one point mutation with the BoIFN-αA subtype. The IFN-α gene of yellow cattle was similar to the BoIFN-αD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-αD1. The other four IFN-α genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-αI, BoIFN-αJ, BuIFN-α1, and BuIFN-α2, respectively. Each of the six clones was expressed in E. coli with molecular weight of ∼ 20kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-α (rIFN-α) all exhibited 1000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-αs could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-αs a potential agent for clinical application against virus diseases in cattle industry.

An Asymmetric Contribution to γ-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of α1, β2, and γ2 Subunits

An Asymmetric Contribution to γ-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of α1, β2, and γ2 Subunits

Molecular cloning of a cDNA encoding a crustacean hyperglycemic hormone from eyestalk ganglia of the blue crab, Callinectes sapidus

Crustacean hyperglycemic hormone (CHH), a polypeptide with multiple physiological effects, was first identified in the X-organ/sinus gland neurosecretory system of the eyestalks. In studies reported here, we used a PCR-based cloning strategy (RT-PCR followed by 5′- and 3′-RACE) to clone from blue crab ( Callinectes sapidus) eyestalk ganglia a cDNA ( CsCHH-1) encoding a putative CHH preprohormone. Sequence analysis revealed the preprohormone included all structural features previously reported for CHH preprohormones: a signal peptide, a CHH precursor-related peptide (CPRP), the CHH polypeptide, and a C-terminal basic processing site. Further, the deduced amino acid sequence of the mature polypeptide included all signature domains previously reported for CHH. The primary structure of blue crab CHH is most closely related to CHH from other brachyurans. RT-PCR revealed the CsCHH-1 transcript was present in eyestalk ganglia, but was undetectable in other tissues tested. A transcript encoding a similar CHH-like preprohormone was detected in thoracic ganglion, ventral nerve cord, and brain, but was not detected in eyestalk ganglia.

Granule-bound starch synthase cDNA in Chlorella kessleri 11 h: cloning and regulation of expression by CO2 concentration

The cDNA for the granule-bound starch synthase (GBSS; ADP-glucose-starch glucosyltransferase, EC 2.4.1.21) of Chlorella kessleri 11 h was isolated and characterized. CkGBSS encodes a 609-amino acid polypeptide (65,627 Da) that includes an N-terminal hydrophobic signal peptide of 55 amino acids. The deduced amino acid sequence of the mature CkGBSS polypeptide shares a greater identity (65%) to that of the GBSS protein of Chlamydomonas reinhardtii, than to those of vascular plant species, but does not have the extra-long C-terminal sequence found in C. reinhardtii. When CO(2 )concentration was decreased from 3 to 0.04% (air level) in light, the levels of CkGBSS mRNA, CkGBSS protein, and GBSS activity increased. Under this condition, pyrenoid and pyrenoid starch developed, and the relative amount of amylose in starch increased. These observations suggest that low CO(2) level up-regulates GBSS biosynthesis at the transcriptional level.

Anti-HIV I/II Activity and Molecular Cloning of a Novel Mannose/Sialic Acid-binding Lectin from Rhizome of <italic>Polygonatum cyrtonema</italic> Hua

The anti-human immunodeficiency virus (HIV) I/II activity of a mannose and sialic acid binding lectin isolated from rhizomes of Polygonatum cyrtonema Hua was elucidated by comparing its HIV infection inhibitory activity in MT-4 and CEM cells with that of other mannose-binding lectins (MBLs). The anti-HIV activity of Polygonatum cyrtonema Hua lectin (PCL) was 10- to 100-fold more potent than other tested MBLs, but without significant cytotoxicity towards MT-4 or CEM cells. To amplify cDNA of PCL by 3'/5'-rapid amplification of cDNA ends (RACE), the 30 amino acids of N-terminal were determined by sequencing and the degenerate oligonucleotide primers were designed. The full-length cDNA of PCL contained 693 bp with an open reading frame encoding a precursor protein of 160 amino acid residues, consisting of a 28-residue signal peptide, a 22-residue C-terminal cleavage peptide and a 110-residue mature polypeptide which contained three tandemly arranged subdomains with an obvious sequence homology to the monocot MBL. However, only one active mannose-binding site (QDNVY) was found in subdomain I of PCL, that of subdomain II and III changed to HNNVY and PDNVY, respectively. There was no intron in PCL, which was in good agreement with other monocot MBLs. Molecular modeling of PCL indicated that its three-dimensional structure resembles that of the snowdrop agglutinin. By docking, an active sialic acid-binding site was found in PCL. The instabilization of translation initiation region (TIR) in mRNA of PCL benefits its high expression in rhizomes.

Molecular characterization of a tsetse fly midgut proteolytic lectin that mediates differentiation of African trypanosomes

Differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms is an important first step in the establishment of an infection within the tsetse fly. This complex process is mediated by a wide variety of factors, including those associated with the vector itself, the trypanosomes and the bloodmeal. As part of an on-going project in our laboratory, we recently isolated and characterized a bloodmeal-induced molecule with both lectin and trypsin activities from midguts of the tsetse fly, Glossina longipennis [Osir, E.O., Abubakar, L., Imbuga, M.O., 1995. Purification and characterization of a midgut lectin–trypsin complex from the tsetse fly, Glossina longipennis. Parasitol. Res. 81, 276–281]. The protein (lectin–trypsin complex) was found to be capable of stimulating differentiation of bloodstream trypanosomes in vitro. Using polyclonal antibodies to the complex, we screened a G. fuscipes fuscipes cDNA midgut expression library and identified a putative proteolytic lectin gene. The cDNA encodes a putative mature polypeptide with 274 amino acids (designated Glossina proteolytic lectin, Gpl). The deduced amino acid sequence includes a hydrophobic signal peptide and a highly conserved N-terminal sequence motif. The typical features of serine protease trypsin family of proteins found in the sequence include the His/Asp/Ser active site triad with the conserved residues surrounding it, three pairs of cysteine residues for disulfide bridges and an aspartate residue at the specificity pocket. Expression of the gene in a bacterial expression system yielded a protein ( M r ∼32,500). The recombinant protein ( Gpl) bound d(+) glucosamine and agglutinated bloodstream-form trypanosomes and rabbit red blood cells. In addition, the protein was found to be capable of inducing transformation of bloodstream-form trypanosomes into procyclic forms in vitro. Antibodies raised against the recombinant protein showed cross-reactivity with the α subunit of the lectin–trypsin complex. These results support our earlier hypothesis that this molecule is involved in the establishment of trypanosome infections in tsetse flies.

Highly efficient production of human interferon-.ALPHA. by transgenic cultured rice cells

Interferon-α (IFN-α) is an important antiviral pharmaceutical. A binary vector containing the first intron of the rice cytosolic SOD gene, the signal sequence of the 10 kDa rice prolamin, the amino-terminal region of β-glucuronidase, a thrombin recognition site, and the mature polypeptide region of human IFN-α was constructed, under the regulation of the cauliflower mosaic virus 35S promoter. Here, we report that transgenic rice cells transformed with this fusion protein vector produced a biologically active IFN-α. The vector was introduced into rice calli by Agrobacterium-mediated methods. Five lines of transgenic calli were obtained. IFN assay demonstrated that these calli expressed fusion proteins bearing biologically active IFN-α. Liquid-cultured cells exhibited stable growth and the production of active IFN-α during 10 successive generations, i.e. in 10 weeks. The expressed proteins were purified by immuno affinity chromatography and reverse-phase HPLC. Repeated selections of cultured cells that had been obtained by dividing calli into small cell aggregates considerably increased the production of IFN-α. Thrombin protease treatment of the fusion protein yielded the intact IFN-α polypeptide. Thus, transgenic suspension rice cells are expected to be useful for the production of large amounts of biologically active proteins at a low cost; moreover, such a system would be easier to employ than animal cell culture systems.

Production of biologically active human interferon-.ALPHA. in transgenic rice

Interferon-a (IFN-a) is a principle cytokine that plays a key regulatory role in mammalian immune systems. The recombinant DNA of IFN-a is composed of the signal sequence region for rice 10 kDa prolamin cDNA, the amino- terminal region of b-glucuronidase DNA, and the mature polypeptide region of human IFN-aDNA, and is expected to produce a biologically active form of IFN-a. This chimerical DNA for coding IFN-a under the control of a cauliflower mosaic virus 35S promoter and a 5� -intron of rice cytosolic superoxide dismutase gene (sodcC1), was transformed into dwarf rice by an Agrobacterium-mediated system. Three lines of transgenic rice plant expressing various levels of IFN-a polypeptide were finally generated. The expression level of the recombinant polypeptide in each line was analyzed by an IFN-a antiviral activity assay and enzyme immunoassay. Higher expression of IFN-a was achieved in developing seed endosperm in two of transgenic rice lines. The replacement of the native signal peptide of IFN-a with the prolamin signal peptide was effective for transporting the IFN-a polypeptide into the ER-derived protein body of developing seed endosperm. These results suggest that rice can be used to produce many biologically active mammalian proteins that accumulate in target organelles such as protein bodies.

Characterisation of disproportionating enzyme from wheat endosperm

Disproportionating enzyme or D-enzyme (EC 2.4.1.25) is an alpha-1,4 glucanotransferase which catalyses cleavage and transfer reactions involving alpha-1,4 linked glucans altering (disproportionating) the chain length distribution of pools of oligosaccharides. While D-enzyme has been well characterised in some plants, e.g. potato and Arabidopsis, very little is known about its abundance and function in cereals which constitute the major source of starch worldwide. To address this we have investigated D-enzyme in wheat (Triticum aestivum). Two putative D-enzyme cDNA clones have been isolated from tissue-specific cDNA libraries. TaDPE1-e, from an endosperm cDNA library, encodes a putative polypeptide of 575 amino acid residues including a predicted transit peptide of 41 amino acids. The second cDNA clone, TaDPE1-l, from an Aegilops taushii leaf cDNA library, encodes a putative polypeptide of 579 amino acids including a predicted transit peptide of 45 amino acids. The mature polypeptides TaDPE1-e and TaDPE1-l were calculated to be 59 and 60 kDa, respectively, and had 96% identity. The putative polypeptides had significant identity with deduced D-enzyme sequences from corn and rice, and all the expected conserved residues were present. Protein analysis revealed that D-enzyme is present in the amyloplast of developing endosperm and in the germinating seeds. D-enzyme was partially purified from wheat endosperm and shown to exhibit disproportionating activity in vitro by cleaving maltotriose to produce glucose as well as being able to use maltoheptaose as the donor for the addition of glucans to the outer chains of glycogen and amylopectin.

Molecular cloning, tissue distribution, and ontogeny of mRNA expression of growth hormone in orange-spotted grouper ( Epinephelus coioides)

A full-length cDNA encoding for growth hormone (GH) was cloned from orange-spotted grouper ( Epinephelus coioides) pituitary using reverse transcription and rapid amplification of cDNA ends (RACE). The GH precursor cDNA consists of 956 bp in size with a 85 bp 5′-untranslated region and 259 bp 3′-untranslated region. The 612 bp open reading frame encodes a 204 amino acid (aa) protein, which represents the precursor of grouper GH composed of a 17 aa signal peptide followed by a 187 aa mature GH polypeptide. The sequence of grouper GH shares 95% aa sequence homology with GH reported in gilthead sea bream ( Sparus aurata), and it also exhibits structural features highly homologous to GH reported in other fish species in the domains representing conserved motifs of GH polypeptides. A single GH transcript of 0.93 kb in size has been detected with Northern blot in the pituitary. Using semi-quantitative PCR approach, dominant PCR products were observed in grouper pituitary, while less PCR products were detected in the brain, spleen, and ovary. The expression of GH mRNA could be detected in 1 dph larvae, after that a significant increase in PCR products was found in 5-day-old fish larvae followed by a drop to very low levels in 15-day-old fish larvae. A second rise was then observed in 25-day-old grouper larvae. These findings suggest that in grouper GH mRNA expression can be detected in day 1 post-hatching larvae, and the GH present in eggs and larvae may play a key role in early development of grouper, especially during the process of metamorphosis of fish larva.