Cloning and heterologous expression of nitrate reductase genes from <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.     

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca²⁺-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.     

A barley cDNA clone encoding a type III chlorophyll a/b-binding polypeptide of the light-harvesting complex II

The nucleotide sequence of a leaf cDNA clone encoding a Type III chlorophyll a/b-binding (CAB) protein of light-harvesting complex II (LHCII) in barley is reported. Sequence comparisons and results from in vitro import into chloroplasts demonstrate that the cDNA clone encodes a functional transit peptide of 45 amino acid residues and a mature polypeptide of 223 residues with a predicted molecular mass of 24.3 kDa. After insertion into thylakoids, the mature protein is resistant to protease attack. Hybridization analysis using a gene-specific probe shows that the gene is expressed in dark-grown seedlings and that the amount of mRNA increases during illumination.     

Molecular characterization of the sheep <Emphasis Type="Italic">CIB1</Emphasis> gene

The calcium and integrin binding protein 1(CIB1), is an EF-hand-containing protein that binds many effector proteins including the platelet αIIbβ3 integrin and potentially regulates their functions. Here we report the cloning and characterization of the sheep CIB1 gene. The CIB1 cDNA is 885-bp in size, containing a 45-bp of 5′ untranslated region (UTR), a 264-bp long 3′-UTR and a 576-bp open reading frame that encodes 191 amino acids. The sheep CIB1 cDNA shows 98.3, 92.0, 91.8, 91.3, 90.5 and 90.1% of similarity, at the nucleotide level, to its equivalents in cattle, pigs, rhesus monkey, humans, rats and mice, respectively at the deduced protein level, the corresponding values are more than 94%. The sheep CIB1 gene consisted of seven exons. Quantitative PCR (Q-PCR) showed that CIB1 was widely expressed in different tissues with the highest level in the testis, suggesting that it may play a role in ram fertility. We cloned the sheep CIB2, CIB3 and CIB4 genes and detected their expression patterns in different tissues.     

Characterization of an Extracellular Serine Protease Gene from the Nematophagous Fungus <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis>

The gene encoding a cuticle-degrading serine protease was cloned from three isolates of Lecanicillium psalliotae (syn. Verticillium psalliotae) by 3′ and 5′ RACE (rapid amplification of cDNA ends) method. The gene encodes for 382 amino acids and the protein shares conserved motifs with subtilisin N and peptidase S8. Comparison of translated cDNA sequences of three isolates revealed one amino acid polymorphism at position 230. The deduced protease sequence shared high degree of similarities to other cuticle-degrading proteases from other nematophagous fungi.     

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, <Emphasis Type="Italic">Gloeophyllum</Emphasis><Emphasis Type="Italic">trabeum</Emphasis>

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5′UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.     

Molecular cloning and daily variations of the <Emphasis Type="Italic">Period</Emphasis> gene in a reef fish <Emphasis Type="Italic">Siganus guttatus</Emphasis>

As the first step in understanding the molecular oscillation of the circa rhythms in the golden rabbitfish Siganus guttatus—a reef fish with a definite lunar-related rhythmicity—we cloned and sequenced a Period gene (rfPer). The rfPer gene contained an open reading frame that encodes a protein consisting of 1,452 amino acids; this protein is highly homologous to PER proteins of vertebrates including zebrafish. Phylogenetic analyses indicated that the rfPER protein is related to the zebrafish PER1 and PER4. The expression of rfPer mRNA in the whole brain, retina, and liver under light/dark (LD) conditions increased at 06:00 h and decreased at 18:00 h, suggesting that its robust circadian rhythm occurs in neural and peripheral tissues. When daily variation in the expression in rfPer mRNA in the whole brain and cultured pineal gland were examined under LD conditions, similar expression patterns of the gene were observed with an increase around dawn. Under constant light condition, the increased expression of rfPer mRNA in the whole brain disappeared around dawn. The present results demonstrate that rfPer is related to zPer4 and possibly zPer1. The present study is the first report on the Period gene from a marine fish.     

Molecular cloning,expression analysis and chromosome localization of the <Emphasis Type="Italic">Tpt1</Emphasis> gene coding for the pig translationally controlled tumor protein (TCTP)

This work describes the cloning and structural analysis of a Tpt1 cDNA coding for the porcine translationally controlled tumor protein (TCTP) molecule and its expression in porcine cells and tissues. Pig Tpt1 cDNA is 842-pb long that displays typical features of translationally controlled mRNAs, including a 5′-UTR containing a 5′-terminal oligopyrimidine tract (5′-TOP), and a 3′-UTR with a high CG-content and one AU rich element (ARE). Both 5′-UTR and 3′-UTR are highly conserved when they are compared with those of other mammals. The pig Tpt1 cDNA contains a 516-b open reading frame that encodes a predicted TCTP protein composed of 172 amino acids that exhibits extensive conservation compared with TCTP sequences from other species and a common structural feature with all the other TCTP proteins analyzed in mammals. Expression analysis demonstrated that Tpt1 mRNA is ubiquitously expressed in normal porcine tissues and cells, showing a higher expression in spleen, lymph nodes and lung, and a lower one in skin and heart. The pig Tpt1 gene localizes on the porcine chromosome 11, region p11.     

Coordinate expression of light-harvesting chlorophyll a/b gene family of photosystem II and chlorophyll a oxygenase gene regulated by salt-induced phosphorylation in Dunaliella salina

As a stress factor, salt induces the phosphorylation of light-harvesting chlorophyll (Chl) a/b proteins (LHCII) in Dunaliella salina. In this study, we found that the salt-induced phosphorylation of LHCII was not affected by phosphatase, and that salt simultaneously regulated both the phosphorylation of LHCII and the expression of genes encoding light-harvesting Chl a/b proteins of photosystem II (lhcb) and the gene encoding Chl a oxygenase (cao) in dark-adapted D. salina. The mRNA accumulation patterns of lhcb and cao were similar, which further affected the size of LHCII and the ratio of Chl a to Chl b. Therefore, we inferred this simultaneous regulation is one of the mechanisms of D. salina to adapt to the high-salinity environment.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

<Emphasis Type="Italic">APHO1</Emphasis> from the yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis> encodes an acid phosphatase of broad substrate specificity

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36–39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60°C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.     

Expression of a phenylcoumaran benzylic ether reductase-like protein in the ovules of <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to identify differentially expressed proteins in wild-type (DP 5690) and fiberless (SL 1-7-1) cotton ovules. One protein, designated V2 was unique to ovules of the fiber producing DP 5690 line. The protein was purified from 2D-PAGE of 4 d post anthesis DP 5690 ovules and partially sequenced. The short amino acid sequence was nearly identical to the deduced amino acid sequence for cotton phenylcoumaran benzylic ether reductase (PCBER) protein. A consensus sequence was assembled from ESTs encoding cotton PCBER genes, primers were designed, and a full length gene was amplified from plasmid DNA from a 72 h etiolated cotton cotyledon library. The polymerase chain reaction generated a 950 bp product with unique EcoRI (5′) and (3′) KpnI restriction sites for directional insertion into the expression vector pPICZA. Nucleotide sequencing was performed, and the full length coding region was 924 bp encoding a protein of 308 amino acids. The molecular mass and pI measured (2D PAGE) were similar to the theoretical protein.     

Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5′ untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3′ UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37°C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage.     

Molecular cloning and characterization of novel heat shock protein 90 gene from a wild <Emphasis Type="Italic">Vitis pseduoreticulata</Emphasis> native to China

Heat shock protein 90 (Hsp90), known as molecular chaperone, is involved in protein folding and assembly in the cell. In the present study, a full-length cDNA named Vitis pseduoreticulta heat shock protein 90 (VpHsp90) (GenBank accession Number:EU239815), encoding a heat shock protein 90, was obtained by degenerated primers and 3′-and 5′-RACE from Vitis pseudoreticulata according to our previously obtained EST sequence (GenBnak accession number:DV182112), putatively known as Hsp90. Comparison of VpHsp90 sequence has revealed that an open reading frame (ORF) consists of 2,100 bp nucleotides and the translated proteins of 699 amino acid residues. The molecular mass of VpHsp90 calculated from the deduced amino acid sequence was 80.2 kDa, Isolectric Point was 4.893, which is in close proximity of Hsp90. The maximum similarity of VpHsp90 at nucleotides level (85%) and protein level (96%) was found to be with Nicotiana tabacum. Phylogenetic tree analysis at both the nucleotides and amino acids levels indicates that Vitis pseduoreticulata, Nicotiana tabacum, and Arabidopsis thaliana Hsp90 sequences comprise one clade, which is closely related to Oryza sativa, Hordeum vulgare and Triticum aestivum Hsp90s. It may be reasonably concluded that VpHsp90 possesses the ancestral gene of Hsp90 similar to that of higher plant species.     

Investigation of <Emphasis Type="Italic">Lpin1</Emphasis> as a candidate gene for fat deposition in pigs

Lpin1 deficiency prevents normal adipose tissue development and remarkably reduces adipose tissue mass, while overexpression of the Lpin1 gene in either skeletal muscle or adipose tissue promotes adiposity in mice. However, little is known about the porcine Lpin1 gene. In the present study, a 5,559-bp cDNA sequence of the porcine Lpin1 gene was obtained by RT-PCR and 3′RACE. The sequence consisted of a 111-bp 5′UTR, a 2,685-bp open reading frame encoding a protein of 894 amino acids and a 2,763-bp 3′UTR. Semi-quantitative RT-PCR analysis revealed that Lpin1 had a high level of expression in the liver, spleen, skeletal muscle and fat, a low level of expression in the heart, lung and kidney. The porcine Lpin1 gene was assigned to 3q21-27 by using the somatic cell hybrid panel (SCHP) and the radiation hybrid (IMpRH) panel. One C93T single nucleotide polymorphism (SNP) was identified and genotyped using the TaqI PCR-RFLP method. Association analysis between the genotypes and fat deposition traits suggested that different genotypes of the Lpin1 gene were associated with percentage of leaf fat and intramuscular fat.     

Analysis of odorant-binding proteins in antennae of a geometrid species, Ascotis selenaria cretacea, which produces lepidopteran Type II sex pheromone components

Information on the olfactory system in antennae of Geometridae moths is very limited, and odorant-binding proteins (OBPs) working as transporters of lipophilic odors have not been identified. In the first investigation on this family of insects, we examined antennal OBPs of the Japanese giant looper, Ascotis selenaria cretacea. RT-PCR experiments using several pairs of degenerate primers designed from known cDNA sequences encoding lepidopteran OBPs successfully amplified partial sequences of two pheromone-binding proteins (PBPs), named AscrPBP1 and AscrPBP2 in reference to their corresponding nucleotide sequence homologies with other PBPs. Using 5′- and 3′-rapid amplification of cDNA end strategies, a cDNA clone for AscrPBP1 encoding a protein of 141 amino acids was isolated. Western blotting with the antiserum against recombinant AscrPBP1 overexpressed in Escherichia coli showed that the AscrPBP1 gene was more strongly expressed in male antennae than in female antennae. Furthermore, natural AscrPBP1was isolated by immunoprecipitation with the antiserum, and its binding ability was evaluated by using synthetic sex pheromonal compounds with a C₁₉ chain. The result indicated that AscrPBP1 bound not only the pheromone components, 3,6,9-nonadecatriene and its 3,4-epoxy derivative, but also unnatural 6,7- and 9,10-epoxy derivatives. While no general odorant-binding proteins (GOBPs) were amplified in the RT-PCR experiments, two antisera prepared from GOBP1 and GOBP2 of Bombyx mori suggested the occurrence of at least two GOBPs in the A. s. cretacea antennae.     

Isolation and characterization of a gene encoding cinnamate 4-hydroxylase from <Emphasis Type="Italic">Parthenocissus henryana</Emphasis>

Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) plays an important role in the phenylpropanoid pathway, which produces many economically important secondary metabolites. A gene coding for C4H, designated as PhC4H (GenBank accession no. DQ211885) was isolated from Parthenocissus henryana. The full-length PhC4H cDNA is 1,747 bp long with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 40-bp 5′ non-coding region and a 189-bp 3′-untranslated region. Secondary structure of the deduced PhC4H protein consists of 41.78% alpha helix, 15.64% extended strand and 42.57% random coil. The genomic DNA of PhC4H is 2,895 bp long and contains two introns; intron I is 205-bp and intron II is 1,172-bp (GenBank accession no. EU440734). DNA gel blot analysis revealed that there might be a single copy of PhC4H in Parthenocissus henryana genome. By using anchored PCR, a 963-bp promoter sequence was isolated and it contains many responsive elements conserved in the upstream region of PAL, C4H and 4CL including the P-, A-, L- and H-boxes.