Cloning and characterization of the lectin cDNA clones from onion,shallot and leek

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells.cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5–13 kDa) after post-translational modifications.Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.     

Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from Chlamydomonas reinhardtii

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges. The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively.Abbreviations OEE1, 2 and 3 oxygen evolution enhancer proteins 1, 2 and 3 - Rubisco ribulose bisphosphate carboxylase/oxygenase - PS photosystem - P21 and P30 C. reinhardtii thylakoid polypeptides 21 and 30     

Novel antimicrobial peptides identified from an endoparasitic wasp cDNA library

We screened an endoparasitic wasp (Pteromalus puparum) cDNA library for DNA sequences having antimicrobial activity using a vital dye exclusion assay. Two dozens of clones were isolated that inhibited the growth of host Escherichia coli cells due to expression of the cloned genes. Three peptides (PP13, PP102 and PP113) were synthesized chemically based on the amino acid sequences deduced from these clones and assayed for their antimicrobial activity. These peptides have net positive charges and are active against both Gram‐negative and ‐positive bacteria, but are not active against fungi tested. Their hemolytic activity on human red blood cells was measured, and no hemolytic activity was observed after 1‐h incubation at a concentration of 62.5 µM or below. A Blast search indicated that the three peptides have not been previously characterized as antimicrobial peptides (AMPs). Salt‐dependency studies revealed that the biocidal activity of these peptides against E. coli decreased with increasing concentration of NaCl. Transmission electron microscopic (TEM) examination of PP13‐treated E. coli cells showed extensive damage of cell membranes. The CD spectroscopy studies noted that the enhanced α‐helical characteristics of PP13 strongly contribute to its higher antimicrobial properties. These results demonstrate the feasibility to identify novel AMPs by screening the expressional cDNA library. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L.

Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L. (proso millet), an NAD-malic enzyme-type C₄ plant. The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns. The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150. The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane -helices that is a common property of members in the mitochondrial transporter family. The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria. An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity-purified. The antibody against the recombinant protein cross-reacted with proteins of 31–32 kDa in the membrane fraction from P. miliaceum mitochondria, but not with the chloroplast fraction. The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate.     

Nucleotide sequence and characterization of a cDNA encoding the acetohydroxy acid isomeroreductase from Arabidopsis thaliana

The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site.     

Isolation and sequencing of cDNA clones encoding ethylene-induced putative peroxidases from cucumber cotyledons

A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1–5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55% sequence identity), a horseradish (53%), a turnip (45%), and a potato (41%) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative peroxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours.     

Isolation and characterization of a novel class of plant antimicrobial peptides form Mirabilis jalapa L. seeds.

We have isolated from seeds of Mirabilis jalapa L. two antimicrobial peptides, designated Mj-AMP1 and Mj-AMP2, respectively. These peptides are highly basic and consist of 37 and 36 residues for Mj-AMP1 and Mj-AMP2, respectively. Both peptides contain three disulfide bridges and differ from one another only by 4 amino acids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the reduced and unreduced peptides suggests that the peptides associate into dimers in their native form. The Mj-AMPs exhibit a broad spectrum of antifungal activity since they are active against all 13 tested plant pathogenic fungi. Concentrations required for 50% inhibition of fungal growth vary from 6 to 300 micrograms/ml for Mj-AMP1 and from 0.5 to 20 micrograms/ml for Mj-AMP2. These peptides were also active on two tested Gram-positive bacteria but were apparently nontoxic for Gram-negative bacteria and cultured human cells. Although the Mj-AMPs show sequence similarity to mu-agatoxins, a class of insecticidal neurotoxic peptides isolated from the venom of spiders, they do not affect pulse transmission in insect nerves.     

Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.     

家蚕吡哆醛激酶cDNA的克隆、表达和基因结构分析

吡哆醛激酶（pyridoxal kinase,PLK, EC2.7.1.35）是维生素B₆关键代谢酶,其cDNA的克隆在昆虫类还未见报道。利用生物信息学原理和使用PCR方法,克隆出编码家蚕Bombyx mori吡哆醛激酶的cDNA (GenBank登录号DQ452397),体外原核表达成功,并对表达粗提产物进行了酶活检测。克隆到的cDNA含有一894 bp的完整可读框,编码一条分子量为33.1 kD,含298个氨基酸残基的蛋白质。序列比对显示此蛋白质与人类吡哆醛激酶具有52.84%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但比哺乳动物和植物克隆到的吡哆醛激酶均少10多个氨基酸残基,几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换。依据家蚕基因组数据库信息和PLK的cDNA,家蚕PLK基因包含5个外显子和4个内含子,跨越10 kb DNA序列,所有外显子/内含子交接点都遵从gt/ag剪接规则,基因的5′端启动子调控区发现有TATA-box和CAAT-box保守基序。     

Antibacterial activities of peptides designed as hybrids of antimicrobial peptides

Hybrid peptides (HP-MA, HP-ME), each of 20 residues and incorporating 2–9 residues of Helicobacter pylori ribosomal protein L1 (HP) and 1–12 residues of magainin 2 and melittin, were designed. The antibiotic activities of these peptides were evaluated using bacterial, tumor and human erythrocyte cells. HP-MA had a stronger antibacterial activity against Gram-positive bacteria and Gram-negative bacteria than HP (2-20) and magainin 2, and HP-ME was similar to melittin. None of the hybrids had anti-tumor or hemolytic activity. These peptides were further investigated using an artificial liposomal vesicle and 1,6-diphenyl-1,3,5-hexatriene as a membrane probe, and confirmed to have similar antibacterial activities. The antibacterial effect of these hybrids is probably caused by their ability to damage the bacterial plasma membrane. Additional circular dichroism spectra suggested that the -helical structure of these peptides plays an important role in their antibiotic effect but that -helical property is less connected with the enhanced antibiotic activity.     

An anionic antimicrobial peptide from toad Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides like maximins and maximins H from toad Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises maximin 3 and a novel peptide, named maximin H5, was isolated from a skin cDNA library of B. maxima. The predicted primary structure of maximin H5 is ILGPVLGLVSDTLDDVLGIL-NH2. Containing three aspartate residues and no basic amino acid residues, maximin H5 is characterized by an anionic property. Different from cationic maximin H peptides, only Gram-positive strain Staphylococcus aureus was sensitive to maximin H5, while the other bacterial and fungal strains tested were resistant to it. The presence of metal ions, like Zn2+ and Mg2+, did not increase its antimicrobial potency. Maximin H5 represents the first example of potential anionic antimicrobial peptides from amphibians. The results provide the first evidence that, together with cationic antimicrobial peptides, anionic antimicrobial peptides may also exist naturally as part of the innate defense system.     

Isolation and characterization of cDNA clones encoding photosystem I subunits with molecular masses 11.0, 10.0 and 8.4 kDa from Chlamydomonas reinhardtii

Summary cDNA clones encoding three photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses 13, 5 and 3 kDa (thylakoid polypeptides 28, 35 and 37; P28, P35 and P37, respectively) were isolated using gene specific oligonucleotides as probes. The sequences of these oligonucleotides were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that the proteins are encoded by single-copy genes. The mRNA sizes of the three components are 960 (P28), 1120 (P35) and 790 (P37) nucleotides. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the nascent polypeptides possess N-terminal transit sequences that are removed to give mature proteins of 11.0 (P28), 10.0 (P35) and 8.4 (P37) kDa. Analysis of the deduced protein sequences suggests that P28 and P35 are extrinsic membrane proteins and that P37 spans the thylakoid membrane. All three proteins have short transit peptides that probably route them to the stromal side of the thylakoid membrane.Abbreviations OEE1, 2 and 3 oxygen evolution enhancer proteins 1, 2 and 3 - RuBisCO ribulose bisphosphate carboxylase/oxygenase - PS photosystem - P28, P35 and P37 Chlamydomonas reinhardtii thylakoid polypeptides 28, 35 and 37 The nucleotide sequences presented here will appear in the EMBL/Genbank/DDBJ Nucleotide Sequence Databases under the accession numbers X15164 (11.0 kDa subunit; P28), X15165 (10.0 kDa subunit; P35) and X15166 (8.4 kDa subunit; P37)     

Cloning and sequencing of cDNAs encoding two SSS

A defective S-allele, S ₀, and a functional S-allele, S _x, have previously been found to be retained in an F₁ hybrid of a self-compatible commercial cultivar of Petunia hybrida. Pistil proteins associated with these two alleles have also been identified. Their amino-terminal sequences have been found to share a high degree of similarity with those of S-proteins characterized from self-incompatible solanaceous species. Here we report the isolation and sequencing of cDNAs encoding S ₀- and S _x-proteins. Their deduced amino acid sequences contain all the consensus primary structural features of S-proteins from self-incompatible solanaceous species. Both proteins also have ribonuclease activity. The implications of these findings are discussed in relation to the presumed function of the S-protein in the self-incompatibility interaction.