Isolation of a cDNA encoding a 31-kDa, pathogenesis-related 5/thaumatin-like (PR5/TL) protein abundantly expressed in apple fruit (Nalus domestica cv. Fuji)

A fruit-specific and pathogenesis-related 5/thaumatin-like (PR5/TL), 31-kDa protein was isolated by 2D-PAGE from fully-grown apples (Malus domestica cv. Fuji) and named Mdtl1 (Malus domestica thaumatin-like protein 1). Using the N-terminal sequence of the protein, the full-length cDNA encoding Mdtll was isolated. The cDNA clone (Mdtl1) consists of 944 bp with an open reading frame (ORF) of 744 bp encoding a protein of 247 amino acids. The deduced amino acid sequence of Mdtl1 shows high similarity to the sequences of PR5/TL proteins. Mdtl1 is a slightly acidic protein with a putative signal peptide and a putative N-glycosylation site, and lacks a C-terminal extension. This suggests that Mdtl1 is an apoplastic glycoprotein. Results of northern blotting indicated that expressions of Mdtl1 are developmentally regulated. Southern blot analysis showed that Mdtl1 may be present as a single copy, and there exist other genes closely related to Mdtl1 in the apple genome.     

Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

Isolation and characterization of a novel RING-H2 finger gene induced in response to cold and drought in the interfertile Citrus relative Poncirus trifoliata

Many genes in different organisms encode proteins with really interesting gene (RING) finger domain(s). The RING zinc finger domain is involved in a wide variety of functions in diverse organisms. A cDNA clone showing homology with RING zinc finger genes and nine-fold induction in response to cold was previously identified during a gene expression study in the interfertile Citrus relative Poncirus trifoliata (L.) Raf. In this study, the full-length cDNA of this clone was isolated from 2-day cold-acclimated P. trifoliata by a rapid amplification of cDNA ends method using gene-specific primers. The full-length cDNA was 956 bp containing a complete open reading frame of 474 bp encoding a polypeptide of 158 amino acids. The full-length cDNA showed a high level of homology with genes encoding putative RING zinc finger proteins in plants. The deduced amino acid sequence of this gene contained a signature sequence motif for a RING zinc finger close to the C terminus of the protein. The RING zinc finger domain was significantly similar to previously characterized RING zinc finger proteins from different organisms. Additionally, it had a histidine residue at the fifth co-ordination site, indicating that this gene encodes a RING-H2 finger protein. Northern blot hybridization showed that the expression of the RING finger gene was induced in response to cold in cold-hardy P. trifoliata but not to the same extent in cold-sensitive Citrus grandis L. Osb. (pummelo). However, the gene was induced by drought stress similarly in both the species. To our knowledge, this study presents the first isolation of the full-length sequence of a RING zinc finger gene induced in response to abiotic stress in plants and the initial characterization of this gene in Citrus .     

Identification of a novel unconventional myosin from scallop mantle tissue.

We isolated a cDNA encoding a novel unconventional myosin from scallop mantle tissue (scallop unconventional myosin: ScunM) and determined the nucleotide sequence. It comprises 2,739 bp with 5' and 3'-noncoding sequences and has an open reading frame of 2,334 bp that encodes 778 amino acids. While ScunM has a motor domain and a short tail domain without having light chain-binding IQ motifs like myosin XIV, the deduced amino acid sequence exhibits low homology, 30-36%, to known myosins. Phylogenetic analysis of the motor domain suggested that ScunM belongs to a novel unconventional myosin class. ScunM has an insertion of 67 amino acids in the putative actin-binding site (loop2 site). Western blot analysis with an antibody produced against the N-terminal region revealed that ScunM was strongly expressed in the mantle and mantle pallial cell layer of scallop.     

Isolation and characterization of the gene encoding a manganese peroxidase from <Emphasis Type="Italic">Lentinula edodes</Emphasis>

A genomic DNA sequence and cDNA encoding a putative manganese peroxidase were isolated from the white-rot basidiomycete Lentinula edodes. The gene, called lemnp1, consists of a 1985-bp open reading frame interrupted by 16 introns and was flanked by an upstream region having putative CAAT, TATA, and heat shock elements and by a downstream region having polyadenylation signals. The lemnp1 gene encodes a protein of 364 amino acids that shows high sequence homology to manganese peroxidases of other basidiomycetes. The deduced N-terminal amino acid sequence is different from the L. edodes manganese peroxidase reported previously.     

The PR-1a promoter contains a number of elements that bind GT-1-like nuclear factors with different affinity

A novel cDNA encoding for a peptidyl-prolyl-cis-trans-isomerase (PPIase) belonging to the FK506-binding protein (FKBP) family was isolated from wheat. It contains an open reading frame of 559 amino acids and it represents the first plant FKBP-PPIase to be cloned. It possesses a unique sequence which is composed of three FKPB-like domains, in addition to a putative tetratricopeptide repeat (TPR) motif and a calmodulin-binding site. The recombinant FKBP-PPIase expressed in and purified from Escherichia coli exhibits PPIase activity that is efficiently inhibited by the immunosuppressive drugs FK506 and rapamycin. Northern blot analysis showed that wheat FKBP was found mainly in young tissues. Polyclonal antibodies revealed the presence of cross-reacting proteins in embryos, roots and shoots. The unique structural features, the enzymatic activity and the presence of putative isoforms in wheat tissues indicate the possibility of the involvement of wheat PPIase in essential biological functions, similar to other members of the FKBP gene family.     

Molecular cloning and biochemical characterization of a lipoxygenase in almond (Prunus dulcis) seed.

We have characterized an almond (Prunus dulcis) lipoxygenase (LOX) that is expressed early in seed development. The presence of an active lipoxygenase was confirmed by western blot analysis and by measuring the enzymatic activity in microsomal and soluble protein samples purified from almond seeds at this stage of development. The almond lipoxygenase, which had a pH optimum around 6, was identified as a 9-LOX on the basis of the isomers of linoleic acid hydroperoxides produced in the enzymatic reaction. A genomic clone containing a complete lipoxygenase gene was isolated from an almond DNA library. The 6776-bp sequence reported includes an open reading frame of 4667 bp encoding a putative polypeptide of 862 amino acids with a calculated molecular mass of 98.0 kDa and a predicted pI of 5.61. Almond seed lipoxygenase shows 71% identity with an Arabidopsis LOX1 gene and is closely related to tomato fruit and potato tuber lipoxygenases. The sequence of the active site was consistent with the isolated gene encoding a 9-LOX.     

A cDNA encoding a putative lipid transfer protein expressed in sunflower seeds

Based on the N-terminal sequence of a sunflower antifungal protein, a full length cDNA (Ha-LTP5) encoding a putative lipid transfer protein from sunflower seeds was cloned using a RT-PCR based strategy. However, the sequence of the deduced protein is not identical to that of the antifungal protein previously isolated. The nucleotide sequence presents an ORF of 116 amino acids with a putative signal peptide, thus encoding a mature protein of 90 amino acids that is basic and hydrophobic. In contrast to the pattern of expression described for most LTP-like genes from dicots, Northern blot analyses detected constitutive expression of Ha-LTP5 in seeds, but not in aerial parts of sunflower plants.     

水稻中一个人类肿瘤抑制基因同源物的克隆与表达分析

用同源筛选方法,从水稻 (Oryza sativa L.) 基因组文库中分离到一个与人类肿瘤抑制基因QM具有同源性的基因,命名为OSQM1.该基因包括4个外显子和3个内含子,编码219个氨基酸,其中有46个碱性氨基酸,其等电点高达11.02.同源性搜寻发现此基因存在于真核生物中而且保守性较强,表明它可能具有重要的作用.Northern分析结果表明,它在不同的水稻器官中都有表达,但在花和愈伤组织中的表达水平明显低于其他营养器官.它在根和叶中的表达水平受环境因素的影响.     

Carboxypeptidase yscS: gene structure and function of the vacuolar enzyme

The gene encoding carboxypeptidase yscS in Saccharomyces cerevisiae, CPS1, was cloned by complementation of the cps1-3 mutation. The cloned CPS1 gene, which again enabled a leucine auxotrophic cps1-3 mutant to grow on the modified dipeptide Cbz-Gly-Leu (Cbz, benzyloxycarbonyl) as sole leucine source, was sequenced and found to consist of an open reading frame of 1728 bp encoding a protein of 576 amino acids. The putative protein contains a hydrophobic stretch of 20 amino acids and a putative signal sequence cleavage site. Five putative N-glycosylation sites are also in the protein sequence. This data is consistent with the previous finding of carboxypeptidase yscS being a vacuolar peptidase. Chromosomal disruption of the CPS1 gene completely abolishes carboxypeptidase yscS activity. This protein is yet another member of the peptidases in S. cerevisiae involved in nitrogen metabolism.     

Molecular cloning of a cDNA encoding diacylglycerol kinase (DGK) in Arabidopsis thaliana

Diacylglycerol kinase (DGK) synthesizes phosphatidic acid from diacylglycerol, an activator of protein kinase C (PKC), to resynthesize phosphatidylinositols. The structure of DGK has not been characterized in plants. We report the cloning of a cDNA, cATDGK1, encoding DGK from Arabidopsis thaliana. The cATDGK1 cDNA contains an open reading frame of 2184 bp, and encodes a putative protein of 728 amino acids with a predicted molecular mass of 79.4 kDa. The deduced ATDGK1 amino acid sequence exhibits significant similarity to that of rat, pig, and Drosophila DGKs. The ATDGK1 mRNA was detected in roots, shoots, and leaves. Southern blot analysis suggests that the ATDGK1 gene is a single-copy gene. The existence of DGK as well as phospholipase C suggests the existence of PKC in plants.     

Cloning and characterization of a novel human leptin receptor overlapping transcript-like 1 gene (LEPROTL1)

A new full-length cDNA encoding a novel protein was isolated from our human fetal brain cDNA library. The cDNA consists of 2701 bp and has a putative open reading frame encoding 131 amino acids which possesses a JAK binding site (Pro(46)-Ile-Pro(48) which is preceded by a cluster of hydrophobic residues) and is highly homologous to the leptin receptor gene-related protein (OB-RGRP). Northern blot analysis showed that this new gene is widely expressed in human tissues and radiation hybrid mapping placed the gene to human chromosome 8p21.1-8p21.2.     

Cloning of a putative γ-aminobutyric acid (GABA) receptor subunit ϱ3 cDNA

Cloned cDNA encoding a putative member of GABA receptor ϱ-subunit class was isolated from rat-retina-mRNA-derived libraries. The cDNA encodes a signal peptide of 21 amino acids followed by the mature ϱ3 subunit sequence of 443 amino acids. The proposed amino acid sequence exhibits 63 and 61% homology to the previously-reported human ϱ1 and rat ϱ2 sequences, respectively. Northern blot analysis demonstrated the expression of mRNA for ϱ3 subunit in retina.     

A cryptic plasmid, pAO1, from a compost bacterium, Bacillus sp

The complete nucleotide sequence of a new cryptic plasmid, pAO1 isolated from a compost bacterium Bacillus sp., has been analyzed. Analysis of the PCR-based 16S rRNA sequence showed the bacterium harboring pAO1 was closely related to Bacillus pallidus. The plasmid pAO1 was 3,325 bp in size. Two open reading frames, ORF1 and ORF2, encoding putative polypeptides of 248 and 290 amino acids, respectively, were identified within the sequence. The ORF1 has a limited sequence similarity to an integrase/recombinase, while the ORF2 has high similarity with the replication protein of pBC1 from Bacillus coagulans. A putative origin sequence for a plus-strand was located between ORFs. Southern blot analysis indicates this plasmid replicates via a rolling circle-type mechanism.     

Cloning and functional expression of a human heparanase gene.

We have cloned a gene (HSE1) from a human placental cDNA library that encodes a novel protein exhibiting heparanase activity. The cDNA was identified through peptide sequences derived from purified heparanase isolated from human SK-HEP-1 hepatoma cells. HSE1 contains an open reading frame encoding a predicted polypeptide of 543 amino acids and possesses a putative signal sequence at its amino terminus. Northern blot analysis suggested strong expression of HSE1 in placenta and spleen. Transient transfection of HSE1 in COS7 cells resulted in the expression of a protein with an apparent molecular mass of 67-72 kDa. HSE1 protein was detectable in conditioned media but was also associated with the membrane fraction following cell lysis. The HSE1 gene product was shown to exhibit heparanase activity by specifically cleaving a labeled heparan sulfate substrate in a similar manner as purified native protein.     

Molecular cloning and sequence analysis of the mouse protein C inhibitor gene

The gene encoding mouse protein C inhibitor (mPCI) was isolated and its nucleotide sequence determined. Alignment of the genomic sequence with that of a cDNA obtained from mouse testis revealed that the mPCI gene (like the human counterpart) is composed of five exons and four introns with highly conserved exon/intron boundaries. It encodes a pre-polypeptide of 405 amino acids, which shows 63% identity with human PCI (hPCI). The putative reactive site is identical to that of hPCI from P5 to P3′, suggesting a similar protease specificity. Also the putative heparin binding sites and `hinge' regions are highly homologous in mouse and hPCI.     

Molecular cloning and primary structure of rat testes metalloendopeptidase EC 3.4.24.15

The complete amino acid sequence of rat testes metalloendopeptidase (EC 3.4.24.15) was deduced from the nucleotide sequence of a cDNA clone isolated by screening a rat testes library with a polyclonal antibody raised against a homogeneous preparation of the rat testes enzyme. The correctness of the sequence was verified by N-terminal amino acid sequence analysis of the isolated enzyme and by partial amino acid sequence analysis of three tryptic peptides located near the N-terminus, the middle, and C-terminus of the native protein. The enzyme is composed of 645 amino acids with a molecular weight of 72,985. This value is close to that of the purified rat testes and brain enzyme as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. The enzyme contains the putative active-site sequence -H-E-F-G-H- that is homologous to the sequence in the active site of thermolysin and several other related bacterial enzymes, as well as to active-site sequences of several mammalian zinc metallopeptidases. No amino acid sequence homology, beyond this active site, was found with thermolysin, a bacterial zinc metalloendopeptidase, nor with several mammalian zinc metallopeptidases. Northern blot hybridization analyses showed the presence of mRNA encoding the enzyme in rat testes, but not in other rat tissues in spite of the finding that enzyme activity is widely distributed in all tissues and that relatively high activities are present in rat brain and pituitary.     

Cloning,sequence analysis,and prokaryotic expression of cDNA encoding a putative non-specific lipid-transfer protein from the bracts of dovetree (<Emphasis Type="Italic">Davidia involucrata</Emphasis> Baill)

The 967-bp cloneP1A5 was isolated from a suppression subtractive hybridization cDNA library of dovetree bracts (Davidia involucrata Baill.). A complete cDNA of 1047 bp was obtained via 5-RACE (5′Rapid Amplification of cDNA End) techniques, using the gene-specific primer P1A5-1. Northern blot analyses showed that the gene was predominantly expressed in the bracts, while the blotting signal from the leaves was weak. Its deduced amino acid sequence was most highly homologous to the lipid-transfer protein 3 precursor isolated from upland cotton, the lipid-transfer protein SDi-9 from the common sunflower, and the non-specific lipid-transfer protein precursor allergen from sweet cherry. It also had features in common with plant nsLTPs (non-specific lipid-transfer proteins), including eight conserved cysteine residues, a high isoelectric point (8.9), and a lack of tryptophans. The deduced amino acid sequence had two transmembrane helices -- the first from Position 5 (Gly) to Position 35 (Val); the second, from Position 28 (Ala) to Position 46 (Leu). A cleavage site for the putative signal peptide was predicted to occur between Positions 28 (Ala) and 29 (Ala). Therefore, the putative mature form of the protein would comprise 92 amino acids, with a molecular weight of 9.2 kD. All these results provide compelling evidence that theP1A5 clone belongs to the nsLTP1 gene family, thus being named theP1A5 putative nsLTPI gene. This is the first nsLTP gene reported from Davidiaceae. The nucleotide sequence data reported here has been released in the GenBank, EMBL, and DDBJ Nucleotide Sequence Databases, under the accession number AY059472.