Pregnancy-associated plasma protein-E (PAPP-E)

A full-length cDNA encoding a novel human protein was cloned from placenta cDNA. The corresponding 1542 amino acid protein sequence was termed 'pregnancy-associated plasma protein-E' (PAPP-E) as it shows a 62% homology to the human pregnancy-associated plasma protein-A (PAPP-A) that is a diagnostic marker for trisomies, especially Down syndrome. The conserved domain structure contains five motifs related to the short consensus repeats of complement proteins and selectins, three motifs related to the lin-notch motifs of proteins regulating early tissue differentiation, and a putative zinc-binding motif and active site of the metzincin-superfamily of metalloproteases. The PAPP-E gene was localized to chromosome 1q23-25. Northern blot analysis showed that PAPP-E is predominantly expressed in placenta.     

斑马鱼DrSpin-1基因的克隆和特征分析

Spin蛋白家族是具有Spin/Ssty保守结构域并在配子发生过程中发挥关键作用的一类分子。研究利用简并引物PCR,从斑马鱼成熟卵母细胞SMART cDNA文库中筛选到260 bp的DrSpin-1和DrSpin-2部分序列,经序列同源性比对,斑马鱼DrSpin-1的部分氨基酸序列与银鲫CagSpin一致性高达81%。利用RACEPCR从该cDNA文库中获得斑马鱼DrSpin-1的全长cDNA序列。序列分析表明,DrSpin-1全长cDNA为1082 bp,开放阅读框771 bp,编码257个氨基酸,具有三个Spin/Ssty保守域,8个可能的磷酸化位点,初步确定斑马鱼DrSpin-1是Spin基因家族成员。斑马鱼DrSpin-1蛋白与已报道的鱼类Spin蛋白多重序列比对表明,DrSpin-1蛋白与银鲫CagSpin蛋白同源性最高。可以推测克隆得到的斑马鱼DrSpin-1与已知功能的银鲫CagSpin具有相近的表达谱和生物学功能,可能在配子发生和受精过程中发挥重要作用。     

Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.     

Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli

A genomic clone encoding a thiohydroximate S -glucosyltransferase ( S -GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S -GT gene were present in B. napus . The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was expressed in Escherichia coli , and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S -GT activity detected from the recombinant protein confirmed that this clone was indeed a S -glucosyltransferase.     

Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean

Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian δ-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian δ-type PI-PLCs and yeast PI-PLC1, the putative Ca²⁺-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane.     

Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues

Eleven cDNA clones encoding UDP-glucose pyrophosphorylase (UGPase) have been isolated from cDNA libraries prepared from seed embryo, seed endosperm and leaves of barley (Hordeum vulgare L.). The sequences were identical, with the exception of positioning of the poly(A) tail; at least five clones with different polyadenylation sites were found. For a putative full-length cDNA [1775 nucleotides (nt) plus polyadenylation tail], isolated from an embryo cDNA library, an open reading frame of 1419 nt encodes a protein of 473 amino acids (aa) of 51.6 kDa. An alignment of the derived aa sequence with other UGPases has revealed high identity to UGPases from eukaryotic tissues, but not from bacteria. Within the aa sequence, no homology was found to a UDP-glucose-binding motif that has been postulated for a family of glucosyl transferases. The derived aa sequence of UGPase contains three putative N-glycosylation sites and has a highly conserved positioning of five Lys residues, previously shown to be critical for catalysis and substrate binding of potato tuber UGPase. A possible role for N-glycosylation in the intracellular targeting of UGPase is discussed.     

Molecular cloning and characterization of a putative protein kinase gene from the thermophilic fungus Thermomyces lanuginosus.

Based on the conserved amino acid sequence (DLKPEN) of serine-threonine protein kinase from several fungi, a degenerate primer was designed and synthesized. Total RNA was isolated from the thermophilic fungus Thermomyces lanuginosus. Using RACE-PCR, full-length cDNA of a putative serine-threonine protein kinase gene was cloned from T. lanuginosus. The full-length cDNA of T. lanuginosus protein kinase was 2551 bp and contained an 1806 bp open reading frame encoding a putative protein kinase precursor of 601 amino acid residues. Sequencing analysis showed that the cloned cDNA of T. lanuginosus had consensus protein kinase sequences. Conservative amino acid subdomains which most serine-threonine kinases contain can be found in the deduced amino acid sequence of T. lanuginosus putative protein kinase. Comparison results showed that the deduced amino acid sequence of T. lanuginosus putative protein kinase was highly homologous to that of Neurospora crassa dis1-suppressing protein kinase Dsk1. The putative protein kinase contained three arginine/serine-rich (SR) regions and two transmembrane domains. These showed that it might be a novel putative serine-threonine protein kinase.     

Molecular cloning and characterization of a cDNA encoding a novel antibacterial peptide, defensin, from the mulberry longicorn beetle, Apriona germari.

A full-length cDNA clone with high homology (62% mature peptide sequence identity) to an Acalolepta luxuriosa antibacterial gene, possessing a conserved cysteine-stabilized alphabeta motif, was cloned by screening an Apriona germari cDNA library. This gene (AgCRP) had a total length of 360 bp with an open reading frame of 207 bp, and encoded a predicted peptide of 69 amino acid residues. The mature AgCRP peptide was 27 amino acid residues long and had a cysteine-stabilized alphabeta motif of C...CXXXC...C...CXC consensus sequence, similar to insect defensins. Northern blot analysis revealed that the AgCRP exhibited fat body-specific expression and was up-regulated by wounding, bacterial or fungal challenge.     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules.

A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.     

Rat pancreatic colipase mRNA: nucleotide sequence of a cDNA clone and nutritional regulation by a lipidic diet

A cDNA clone encoding rat pancreatic colipase was isolated using as a probe a synthetic deoxyoligonucleotide corresponding to a highly conserved amino acid sequence region in colipases from other species. The cloned messenger codes for a protein of 95 amino acids plus a signal peptide of 17 amino acids. The structure of the full-length cDNA was also determined and the corresponding amino acid sequence showed a high degree of homology with those of other known colipases. Quantification of the homologous mRNA in the pancreas of animals fed a high-lipid diet was consistent with a specific though moderate induction of colipase messenger by the nutritional manipulation.     

OsHT, a Rice Gene Encoding for a Plasma-Membrane Localized Histidine Transporter

Using a degenerative probe designed according to the most conservative region of a known Lys- and His-specific amino acid transporter (LHT1) from Arabidopsis, we isolated a full-length cDNA named OsHT (histidine transporter of Oryza sativa L.) by screening the rice cDNA library. The cDNA is 1.3kb in length and the open reading frame encodes for a 441 amino acid protein with a calculated molecular mass of 49 kDa. Multiple sequence alignments showed that OsHT shares a high degree of sequence conservation at the deduced amino acid level with the Arabidopsis LHT1 and six putative lysine and histidine transporters. Computational analysis indicated that OsHT is an integral membrane protein with 11 putative transmembrane helices. This was confirmed by the transient expression assay because the OsHT-GFP fusion protein was, indeed, localized mainly in the plasma membrane of onion epidermal cells. Functional complementation experiments demonstrated that OsHT was able to work as a histidine transporter in Saccharomyces cerevisiae, suggesting that OsHT is a gene that encodes for a histidine transporter from rice.This is the first time that an LHT-type amino acid transporter gene has been cloned from higher plants other than A rabidopsis.     

一个陆地棉bZIP蛋白cDNA的克隆及表达分析

利用PCR筛选方法从陆地棉纤维cDNA文库中筛选到一个全长cDNA序列,命名为GhbZIP。其编码产物长度为645个氨基酸残基,序列中含有两个未知功能的保守区域DUF630和DUF632,而DUF632区中有一个类似碱性亮氨酸拉链基元;此外氨基酸序列中还存在一个富脯氨酸区和一个富苯丙氨酸区,因此该蛋白具有植物碱性亮氨酸拉链蛋白的结构特征。亲水性分析表明,GhbZIP为一个典型的膜蛋白。GhbZIP基因主要是在开花3d之后在胚珠和纤维细胞中表达,这表明该基因可能与棉纤维伸长过程中的基因表达调控有关。     

高山离子芥MAP激酶基因CbMAPK3的克隆

从高山离子芥中克隆得到了一种新的MAPK基因的cDNACbMAPK3,全长1419bp,其中包括1110bp的开放阅读框、91bp的5＇非翻译区（5＇UTR）和218bp的3＇非翻译区（3＇UTR）。CbMAPK3基因编码369个氨基酸的蛋白质,分子量为42.5kDa,等电点为5.79,含有MAP激酶所具有的11个保守序列区以及MAP激酶的磷酸化位点TEY基序。CbMAPK3蛋白与响应环境胁迫有关的植物MAPK亚类有很高的同源性。半定量分析表明,该基因表达受低温胁迫诱导。     

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii.

Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degrees C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.     

Cloning and sequencing of bullfrog growth hormone complementary DNA

Total mRNA was isolated from the pituitary glands of bullfrog (Rana catesbeiana), purified by affinity chromatography with oligo(dT)-cellulose columns. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The cDNA library was screened by hybridization with 32P-labeled duck growth hormone (GH) cDNA. A positive clone was selected and sequenced. The full-length bullfrog GH cDNA contains 950 nucleotide pairs with an open reading frame coding for the precursor GH of 215 amino-acid residues. The partial amino-acid sequence from the protein confirms that derived from the cDNA, with Phe as the first residue in the mature bullfrog GH preceded by a 25-residue hydrophobic signal peptide. The bullfrog GH shares sequence homology with those of other vertebrate species in the following order: duck (61% protein sequence homology; 67% cDNA homology), rat (56%; 61%), human (47%; 57%) and salmon (42%; 50%).