Deduced Peptide Sequence Research Articles

Suppression of the dwarf phenotype of an Arabidopsis mutant defective in thermospermine biosynthesis by a synonymous codon change in the SAC51 uORF.

Thermospermine plays a critical role in negatively regulating xylem development in angiosperms. A mutant of Arabidopsis thaliana that is defective in thermospermine biosynthesis, acaulis5 (acl5), exhibits a dwarf phenotype with excessive xylem formation. Mechanistically thermospermine acts in attenuating the inhibitory effect of an evolutionarily conserved upstream open reading frame (uORF) on the main ORF of SAC51, which encodes a basic helix-loop-helix protein involved in xylem repression. Here, we revealed that a semidominant suppressor of acl5, sac503, which partially restores the acl5 phenotype, has a point mutation in the conserved uORF of SAC51 with no amino acid substitution in the deduced peptide sequence. In transgenic lines carrying the β-glucuronidase (GUS) reporter gene fused with the SAC51 5' region containing the uORF, the mutant construct was shown to confer higher GUS activity than does the wild-type SAC51 construct. We confirmed that sac503 mRNA was more stable than SAC51 mRNA in acl5. These results suggest that the single-base change in sac503 positively affects the translation of its main ORF instead of thermospermine. We further found that the uORF-GUS fusion protein could be synthesized in planta from the wild-type and sac503 translational fusion constructs.

Cloning, tissue distribution and functional characterization of the donkey (Equus asinus) oligopeptide transporter 2.

Oligopeptide transporter 2 (PepT2) is an important transporter of oligopeptides. In the present study, we describe the molecular cloning, tissue distribution and functional characterization of a donkey (Equus asinus) PepT2. The cloned cDNA sequence was 2202bp at full length, encoding a 733 amino acid peptide with a molecular weight of 81.9kDa and a theoretical pI of 8.92. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of PepT2. The expression of PepT2 in the kidney and lung was significantly higher than that observed in the ileum, duodenum, jejunum, spleen, liver, heart and stomach. Functional characterization by heterologous expression in Chinese hamster ovary cells showed that the uptake of β-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA) by donkey PepT2-Chinese hamster ovary cells was dependent on time, pH and substrate concentration, with a low Km value of 91.51±14.14μM and a maximum velocity of 41.37±2.193pmol/min/mg protein. In the present study, for the first time, the expression and functional characteristics of donkey PepT2 were evaluated, the results of which provide new insights and a better understanding of its crucial role in oligopeptide transport in donkeys.

Cloning, characterization and expression of a phenylalanine ammonia-lyase gene (CcPAL) from cumin (Cuminum cyminum L.)

Phenylalanine ammonia-lyase (PAL) that functions a substantial role in defense mechanisms, growth, and development of the plant, is situated at branching point from primary to secondary metabolism. In this study, using highly conserved sequences of plant PALs registered in GenBank, a partial cDNA clone of cumin (Cuminum cyminum) PAL gene (designated as CcPAL) was isolated. The relative transcript levels of CcPAL gene, its enzyme activity, and accumulation of total phenolics were also analyzed in shoots in response to different stresses. The isolated CcPAL had 414 bp in length with a maximum open reading frame (ORF) of 351 bp and encodes a peptide with 116 amino acids. The deduced peptide sequence of CcPAL has classical PAL domains and is a stable protein. Results of multiple sequence alignment indicate that CcPAL had high similarity with other plant PALs available in the GenBank. Analysis of the phylogenetic tree indicated that CcPAL categorized in the angiosperm-type PAL family and dicotyledon subfamily and clustered together with other PAL from Apiaceae species. Transcription analysis indicated that CcPAL expressed in all tested tissues, but more highly in shoots. Moreever, the expression of CcPAL was found to be induced by cadmium, wounding, salicylic acid (SA) and cold treatments. A positive correlation was found between transcript levels of CcPAL and total phenolics accumulation of cumin shoots in response to different stresses. These results suggest that the CcPAL and phenylpropanoid pathway may play a great role in the biosynthesis of the bioactive compounds and counteracting various stresses in C. cyminum.

The DNA Sequence Encoding Actin (ACT1) of Pandan (Benstonea sp.)

One of housekeeping genes is actin gene. This gene is frequently used in gene expression studies as an internal control. The DNA sequence encoding actin from Pandan ( Benstonea sp.) clone Riau has not been reported, therefore, this study investigated the DNA sequence encoding actin isolated from Benstonea sp. clone Riau. Total DNA isolation was performed in fresh leaves, total RNA isolation from stem, total cDNA synthesis, polymerase chain reaction using degenerate actin primer, electrophoresis, cloning, transformation, blue white colony selection, colony PCR, sequencing, data analysis using BioEdit and MEGA6 softwares and BLASTn program. The partial DNA sequence encoding actin from Benstonea sp. clone Riau obtained was 1,403 bp. The sequence was grouped as part of actin1 (ACT1) and it was consisted of two exons and one intron. The predicted coding and peptide sequences were 616 bp and 205 amino acids, respectively. The predicted coding sequence had 90% similarity to some ACT1 mRNA from some plants species but none of which belongs to Benstonea genus or Pandanaceae family. The deduced peptide sequence had similarity to some ACT1 peptide from some plant species of up to 99% and also none of them belongs to Benstonea genus or Pandanaceae family. Thus, the partial ACT1 gene obtained in this study was the first sequence reported from Benstonea genus. Furthermore, this sequence can be used as a reference to isolate actin genes from other species within Benstonea genus for gene expression analysis purposes.

Gene-Wide Analysis of Aquaporin Gene Family in Malus domestica and Heterologous Expression of the Gene MpPIP2;1 Confers Drought and Salinity Tolerance in Arabidposis thaliana.

The aquaporins (AQPs) are a family of integral membrane proteins involved in the transcellular membrane transport of water and other small molecules. A scan of the apple (Malus domestica) genome revealed the presence of 42 genes encoding putative AQPs. Based on a phylogenetic analysis of the deduced peptide sequences of the AQPs generated by Arabidopsis thaliana, poplar (Populus trichocarpa), and rubber (Hevea brasiliensis), the apple AQPs were each assigned membership of the five established AQP subfamilies, namely the PIPs (eleven members), the TIPs (thirteen members), the NIPs (eleven members), the SIPs (five members), and the XIPs (two members). The apple AQPs included asparagine-proline-alanine (NPA) motifs, an aromatic/arginine (ar/R) selectivity filter, and the Froger’s positions. The heterologous expression of MpPIP2;1 in A. thaliana was shown to enhance the level of tolerance exhibited against both drought and salinity.

Molecular Characterisation and Expression of Heat Shock Protein Gene (HSP70) inTribolium castaneumAdults Under Different Environmental Stressors

Insects react to temperature variation, chemical and physical stressors by synthesis of a set of proteins known as heat shock proteins (HSPs). In this study, a cDNA gene coding a heat shock protein70 (HSP70) from Tribolium castaneum has been sequenced and characterised from adult beetles. The deduced peptide sequence ORF (Open Reading Frame) of HSP70 had 220 amino acid residues, had a calculated molecular mass of 24.30 kDa and an isoelectric point (Ip) of 6.85. T. castaneum HSP70 showed high identities with other coleopteran HSP70 by BLAST search in NCBI. Multiple sequence alignment at both nucleotide and amino acid levels were performed showing conserved regions between HSP70 and other heat shock proteins isolated from insects. Phylogenetic analysis was constructed by neighbour-joining distance analysis showing that HSP70 putative peptide was clustered with other heat shock protein from the same species. 3-D structure modelling of HSP70 was employed. Transcriptional profiles of HSP70 were determined by quantitative RT-PCR analysis after adult beetles were exposed to 45 °C, 4 °C and novaluron (insect growth regulator) for 60 min as physical and chemical stressors. HSP70 transcript was up-regulated at the highest level when adults were exposed to 45 °C for 60 min. In contrast, HSP70 transcript was down-regulated when adults were exposed to 4 °C and novaluron for 60 min.

Purification of an alpha amylase from Aspergillus flavus NSH9 and molecular characterization of its nucleotide gene sequence.

In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50°C. The enzyme was also thermally stable at 50°C, with 87% residual activity after 60min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca2+-binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.

Cloning, tissue distribution, functional characterization and nutritional regulation of a fatty acyl Elovl5 elongase in chu's croaker Nibea coibor

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids (PUFA) are key to the biosynthesis of the long-chain polyunsaturated fatty acids (LC-PUFA). Here we report on the molecular cloning, tissue distribution, functional characterization and nutritional regulation of a elovl5 gene from Nibea coibor. The full-length cDNA was 1315 bp, including a 5-untranslated region (UTR) of 134 bp, a 3-UTR of 296 bp and an open reading frame of 885 bp, which specified a peptide of 294 amino acids. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of microsomal fatty acyl elongases, including the so-called histidine box (HXXHH), the canonical C-terminal endoplasmic reticulum retention signal, several predicted transmembrane regions and other highly conserved motifs. Expression of elovl5 was strongly observed in stomach, and more weakly in kidney, spleen, intestine, brain, eye, liver, gill, muscle and heart. Functional characterization revealed that the chu's croaker Elovl5 was able to elongate both C18 and C20 PUFA substrates. Nutritional study indicated that the hepatic expression of elovl5 could be up-regulated by low dietary n−3 LC-PUFA. These results may contribute to better understanding the LC-PUFA biosynthetic pathway and regulation mechanism in chu's croaker.

Characterization of a novel y-type high molecular weight glutenin subunit at Glu-D1 locus

Glu-D1y12.K as a novel y-type subunit was found in HMW-GSs encoded at the Glu-D1 locus in the JB20, which a Korean wheat line from F9 lines crossed by Keumkang with Glu-D1d and Chinese Spring (CS) with Glu-D1a alleles. This novel subunit shows faster electrophoretic mobility and lower molecular weight than Dy12 subunit on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The result of linear ion-trap and Fourier-transform mass spectrometry (LTQ-FT-MS) based on two-dimensional electrophoresis (2-DE) showed that the Dy12.K subunit has high similarity against protein ID: P08488 (GLT3_WHEAT) as ‘Glutenin, high molecular weight subunit 12’ form UniProtKB. The gene of the Glu-1Dy12.K subunit is composed of 1962 nucleotide base pairs containing open reading frame (ORF) as 652 amino acids corresponding to about 70.1 kDa. It has four indels (36 bp insertions: two repeated 18 and 24 bp deletion: two deletions with 6 + 18 bp) and 21 SNPs compared to Glu-1Dy10 (GI: 164457872 in NCBI), and one deletion (18 bp) and three SNPs compared to Glu-1Dy12 (GI: 1036031968) by DNA markers. Consequentially, in comparison with Dy10, 13 SNPs were non-synonymous SNPs and eight SNPs were synonymous SNPs of 21 SNPs. In comparison with Dy12, only one SNP was non-synonymous SNP of three SNPs. Furthermore, the deduced peptide sequences as ‘TGQGQQ’ corresponding to ‘AACAGGACAAGGGCAACA’ are deleted only in the Dy12.K subunit.

Distribution of Bexsero® Antigen Sequence Types (BASTs) in invasive meningococcal disease isolates: Implications for immunisation

Serogroup B is the only major disease-associated capsular group of Neisseria meningitidis for which no protein-polysaccharide conjugate vaccine is available. This has led to the development of multi-component protein-based vaccines that target serogroup B invasive meningococcal disease (IMD), including Bexsero®, which was implemented for UK infants in 2015, and Trumenba®. Given the diversity of meningococcal protein antigens, post-implementation surveillance of IMD isolates, including characterisation of vaccine antigens, is essential for assessing the effectiveness of such vaccines. Whole genome sequencing (WGS), as realised in the Meningitis Research Foundation Meningococcus Genome Library (MRF-MGL), provides a rapid, comprehensive, and cost-effective approach to this. To facilitate the surveillance of the antigen targets included in Bexsero® (fHbp, PorA, NHBA and NadA) for protective immunity, a Bexsero® Antigen Sequence Type (BAST) scheme, based on deduced peptide sequence variants, was implemented in the PubMLST.org/neisseria database, which includes the MRF-MGL and other isolate collections. This scheme enabled the characterisation of vaccine antigen variants and here the invasive meningococci isolated in Great Britain and Ireland in the epidemiological years 2010/11 to 2013/14 are analysed. Many unique BASTs (647) were present, but nine of these accounted for 39% (775/1966) of isolates, with some temporal and geographic differences in BAST distribution. BASTs were strongly associated with other characteristics, such as serogroup and clonal complex (cc), and a significant increase in BAST-2 was associated with increased prevalence of serogroup W clonal complex 11 meningococci. Potential coverage was assessed by the examination of the antigen peptide sequences present in the vaccine and epidemiological dataset. There were 22.8–30.8% exact peptide matches to Bexsero® components and predicted coverage of 66.1%, based on genotype-phenotype modelling for 63.7% of serogroup B isolates from 2010/14 in UK and Ireland. While there are many caveats to this estimate, it lies within the range of other published estimates.

Identification and expression of a novel carbonic anhydrase isozyme in the pufferfish Takifugu vermicularis

Carbonic anhydrase (CA) is a key element for maintaining acid base balance in fish. In our present experiment, novel CA isozymes were identified from the pear puffer (Takifugu vermicularis). Based on the high homology of two predicted CA sequences of the tiger puffer (Takifugu rubripes), a 1715bp novel cDNA was obtained from T. vermicularis. The open reading frame showed a complete coding sequence of 552bp with a deduced peptide sequence of 183 amino acids that exhibited highest (97%) identity with pufferfish putative CA III and CA IV-like sequences. In addition, this translated protein sequence showed 36–37% identity with zebrafish CA IV-like, CA XVa, CA XVb, and CA XVc proteins. Phylogenetic analysis revealed that the pufferfish novel protein (pCAn) was a membrane-bound CA protein. Alignment of multiple CA sequences illustrated that most of the putative active site residues of the pCAn isozyme were situated at highly conserved regions of the CA sequences. Examination of motif distribution suggested that the pCAn isozyme was very similar to the puffer predicted CA IV-like isozyme. Reverse transcription-polymerase chain reaction (PCR) analysis showed highly differential expression in the brain, gills, kidney, and muscle, whereas CA mRNA expression was almost absent in heart, liver, and intestine. Quantitative PCR expression of CA mRNA abundance suggested several-fold higher expression of pCAn isozymes in the gills compared to other tissues tested. Our results suggest that the pCAn isozyme might be related to CA IV-like isozymes. Further functional studies are needed to investigate the function of the pCAn isozyme in T. vermicularis.

Letter to the editor: Importance of serum bactericidal activity for estimating the breadth of protection for new meningococcal vaccines

To the editor: Brehony and colleagues [1] recently reported their investigation of six Neisseria meningitidis serogroup B (NmB) vaccines by deducing the prevalence of the vaccine components in isolates represented in publicly accessible sequence repositories of NmB. Based on the frequency of exact match of deduced peptide sequences in the databases to at least one component of each vaccine, and by assessing published data, the authors estimated the breadth of strain coverage. Their findings led them to conclude that meningococcal vaccines with multiple antigens would provide greater breadth of coverage against meningococcal disease. However, their evaluation did not incorporate important information about the diversity of factor H binding protein (fHBP) variants and the demonstration of protective immune responses against antigenically diverse MnB invasive disease strains following vaccination with Trumenba. Consequently, the authors’ analysis underestimates the potential for vaccine benefit provided by this licensed vaccine. We provide here a description of published data, including results that use the recognised correlate of protection for NmB, serum bactericidal activity measured using serum bactericidal assays performed with human complement (hSBA) [2,3]. This correlation was first validated in large clinical studies using outer membrane vesicle (OMV) vaccines that confer protection via induction of immune response directed against the PorA antigen of NmB [4]. Protection is essentially restricted to strains expressing the matched PorA sequence in the vaccine [5], thus the approach used by the authors to estimate vaccine strain coverage is appropriate for PorA based vaccines, such as the experimental vaccine NonaMen, as described.

Molecular cloning and nutritional regulation of putative ∆6 desaturase mRNA from striped catfish (Pangasianodon hypophthalmus)

Here we report the full-length cDNA cloning and nutritional regulation of a putative Δ6 fatty acyl desaturase (Δ6 fads) gene from striped catfish, Pangasianodon hypophthalmus. The 5′ and 3′ RACE analysis revealed a cDNA sequence of 1896 base pairs (bp) comprising a 5′ UTR of 49bp, 3′ UTR of 509bp and an open reading frame (ORF) of 1338bp specifying a protein of 445 amino acids. Bioinformatics analysis showed that the deduced peptide sequence included all the characteristic features of microsomal fatty acyl desaturases, including N-terminal cytochrome b5 domain containing the haem binding motif H-P-G-G, fatty acyl (FA) desaturase domain, three histidine boxes and transmembrane regions. P. hypophthalmus ∆6 desaturase peptide sequence shares high similarity with Pangasius larnaudii (95%) and Clarias macrocephalus (90%). The predicted secondary and tertiary protein structures revealed that desaturase protein contains mostly α-helix followed by random coils and strands. Tissue distribution analysis showed that Δ6 fads is mainly expressed in brain followed by liver, heart, gill and muscle. Since α-linolenic acid is a substrate for the Δ6 FADS enzyme, various concentrations of this fatty acid (0.5, 1, 1.5 and 2gkg−1) were administered to experimental fish through feed to find out its effect on the expression of Δ6 fads. Results showed that the liver Δ6 fads gene expression is up-regulated in fish fed with experimental diets (p<0.05). Statement of relevanceThe fatty acid composition of fish depends on both dietary fatty acid input and endogenous HUFA biosynthesis capacity. Fatty acyl desaturases (fads) introduce double bonds in selected positions of the acyl chains. Particularly, Δ6 FADS are the first enzymes involved in the biosynthesis of long-chain HUFA from the C18 n-3 and n-6 precursors, which is a rate limiting step. The extent to which fish can desaturate/elongate C18 FAs to HUFAs varies with species. Hence it is important to study the relationships between LC-PUFA biosynthesis pathways and the habitat and feeding habit of fish species, which will provide the basis for understanding the regulatory mechanisms controlling LC-PUFA biosynthesis pathways. The present study reports the full-length cDNA cloning, tissue distribution and nutritional regulation of Δ6 fads-like gene from freshwater catfish, P. hypophthalmus. This is the first report on cloning and expression analysis of Δ6 fads-like gene from any freshwater catfish.

Fine Mapping and Candidate Gene Analysis of qSTL3, a Stigma Length-Conditioning Locus in Rice (Oryza sativa L.).

The efficiency of hybrid seed production can be improved by increasing the percentage of exserted stigma, which is closely related to the stigma length in rice. In the chromosome segment substitute line (CSSL) population derived from Nipponbare (recipient) and Kasalath (donor), a single CSSL (SSSL14) was found to show a longer stigma length than that of Nipponbare. The difference in stigma length between Nipponbare and SSSL14 was controlled by one locus (qSTL3). Using 7,917 individuals from the SSSL14/Nipponbare F2 population, the qSTL3 locus was delimited to a 19.8-kb region in the middle of the short arm of chromosome 3. Within the 19.8-kb chromosome region, three annotated genes (LOC_Os03g14850, LOC_Os03g14860 and LOC_Os03g14880) were found in the rice genome annotation database. According to gene sequence alignments in LOC_Os03g14850, a transition of G (Nipponbare) to A (Kasalath) was detected at the 474-bp site in CDS. The transition created a stop codon, leading to a deletion of 28 amino acids in the deduced peptide sequence in Kasalath. A T-DNA insertion mutant (05Z11CN28) of LOC_Os03g14850 showed a longer stigma length than that of wild type (Zhonghua 11), validating that LOC_Os03g14850 is the gene controlling stigma length. However, the Kasalath allele of LOC_Os03g14850 is unique because all of the alleles were the same as that of Nipponbare at the 474-bp site in the CDS of LOC_Os03g14850 among the investigated accessions with different stigma lengths. A gene-specific InDel marker LQ30 was developed for improving stigma length during rice hybrid breeding by marker-assisted selection.

Molecular cloning and expression of the calmodulin gene from guinea pig hearts.

The aim of the present study was to isolate and characterize a complementary DNA (cDNA) clone encoding the calmodulin (CaM; GenBank accession no. FJ012165) gene from guinea pig hearts. The CaM gene was amplified from cDNA collected from guinea pig hearts and inserted into a pGEM®-T Easy vector. Subsequently, CaM nucleotide and protein sequence similarity analysis was conducted between guinea pigs and other species. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to investigate the CaM 3 expression patterns in different guinea pig tissues. Sequence analysis revealed that the CaM gene isolated from the guinea pig heart had ∼90% sequence identity with the CaM 3 genes in humans, mice and rats. Furthermore, the deduced peptide sequences of CaM 3 in the guinea pig showed 100% homology to the CaM proteins from other species. In addition, the RT-PCR results indicated that CaM 3 was widely and differentially expressed in guinea pigs. In conclusion, the current study provided valuable information with regard to the cloning and expression of CaM 3 in guinea pig hearts. These findings may be helpful for understanding the function of CaM3 and the possible role of CaM3 in cardiovascular diseases.

Cloning, expression and characterization of the recombinant cold-active type-I pullulanase from Shewanella arctica

An activity-based screening approach led to the identification of a novel glycoside hydrolase family-13 pullulanase gene (pul13A) from a psychrophilic bacterium. The recombinant enzyme exhibited a deduced peptide sequence of 1440 amino acid residues and was produced in a heterologous host in Escherichia coli. Purification from inclusion bodies was achieved by a six-step dialysis protocol enabling mild refolding of the urea-denaturated protein followed by affinity- and size-exclusion chromatography under native conditions. Pul13A is a type-I pullulanase, which is capable of hydrolysing α-1,6-glycosidic bonds in pullulan to produce maltotriose, while maltose and intermediate oligosaccharides are produced from soluble starch and amylopectin. The recombinant enzyme exhibited typical properties of cold-adapted proteins including low thermostability at elevated temperatures. It showed a temperature optimum at 35°C, while at 10°C residual activity (25%) remained. The optimal pH was in the range of 6.0–7.0, with Pul13A being stable at neutral and basic pH, but not in the acidic range. Catalytic activity was increased in the presence of divalent cations calcium and cobalt and both metal ions were also able to restore catalytic activity of EDTA-chelated enzyme samples. Pul13A represents the first type-I pullulanase from a psychrophile that has been produced in recombinant form. Moreover, its favourable enzymatic properties make this enzyme a potential candidate for industrial applications such as starch degradation for ethanol based biofuel production.

Three types of geranylgeranyl diphosphate synthases from the medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

Geranylgeranyl diphosphate synthase (GGPPS) is a key enzyme in the carotenoid biosynthetic pathway, catalyzing the synthesis of its C20 precursor. In the present study, three types of ggpps genes were cloned and analyzed from the Caterpillar Medicinal Fungus Cordyceps militaris, a valued carotenoid-producing species. The sequences were named as ggpps727, ggpps191, and ggpps595. The open reading frame codes for predicted polypeptides of 464, 550, and 431 aa. Three predicted GGPPSs had a high similarity to that from Beauveria bassiana ARSEF 2860 with identity of 73%, 71%, and 56%, respectively. Homology comparison of the deduced peptide sequences of the various GGPPSs revealed highly conserved domains. Both GGPPS727 and GGPPS191 from C. militaris contained all five domains highly conserved among prenyltransferases as well as two aspartate-rich DDXX(XX)D motifs in domains II and V, which have been proven essential for prenyltransferase activity. By constructing the phylogenetic tree of fungal GGPPSs, it was found that fungi-derived GGPPSs could be divided into three clusters, suggesting there were three types of GGPPSs in fungi. Each type may be responsible for a different metabolism. Three types of GGPPSs from C. militaris belonged to the different clusters separately. Expression analysis of three ggpps genes during the fruit body cultivation of C. militaris by real-time polymerase chain reaction (PCR) suggested the ggpps 191 gene may be involved in the synthesis of carotenoids and ggpps 727 may be responsible for primary metabolism. This is the first report of the GGPPS from C. militaris, a valued edible and medicinal fungus.

Neuropeptide Y mRNA and peptide in the night-migratory redheaded bunting brain

This study investigated the distribution of neuropeptide Y (NPY) in the brain of the night-migratory redheaded bunting (Emberiza bruniceps). We first cloned the 275-bp NPY gene in buntings, with ≥95% homology with known sequences from other birds. The deduced peptide sequence contained all conserved 36 amino acids chain of the mature NPY peptide, but lacked 6 amino acids that form the NPY signal peptide. Using digosigenin-labeled riboprobe prepared from the cloned sequence, the brain cells that synthesize NPY were identified by in-situ hybridization. The NPY peptide containing cell bodies and terminals (fibers) were localized by immunocytochemistry. NPY mRNA and peptide were widespread throughout the bunting brain. This included predominant pallial and sub-pallial areas (cortex piriformis, cortex prepiriformis, hyperpallium apicale, hippocampus, globus pallidus) and thalamic and hypothalamic nuclei (organum vasculosum laminae terminalis, nucleus (n.) dorsolateralis anterior thalami, n. rotundus, n. infundibularis) including the median eminence and hind brain (n. pretectalis, n. opticus basalis, n. reticularis pontis caudalis pars gigantocellularis). The important structures with only NPY-immunoreactive fibers included the olfactory bulb, medial and lateral septal areas, medial preoptic nucleus, medial suprachiasmatic nucleus, paraventricular nucleus, ventromedial hypothalamic nucleus, optic tectum, and ventro-lateral geniculate nucleus. These results demonstrate that NPY is possibly involved in the regulation of several physiological functions (e.g. daily timing feeding, and reproduction) in the migratory bunting.

Isolation and characterization of a candidate gene for resistance to cereal cyst nematode from Aegilops variabilis in China

Cereal cyst nematode (CCN) ( Heterodera avenae Woll.) is one of the most economically damaging endoparasite pests of wheat worldwide. We isolated and characterized a novel cereal CCN resistance candidate gene, CreV8 , from Aegilops variabilis (2n = 28, UUSvSv). The gene was 3,568 bp long and showed high nucleotide similarity with the known nematode resistance genes Cre3, CreZ, and go35 . CreV8 consisted of two exons, one intron and a complete ORF of 2,763 bp. The deduced peptide sequence consisted of 920 amino acid residues and shared 97.83% amino acid identity with Cre3. The sequence harbored a signal peptide domain, a nucleotide-binding ARC (NB-ARC) domain, and a leucine-rich repeat (LRR) domain, all of which are typical characteristics of resistance genes. We proposed the resistance mechanism of CreV8 based on functional analysis and predictions from its conserved domains and tertiary structure. Real-time polymerase chain reaction (PCR) showed that CreV8 was expressed in CCN-inoculated roots of resistant plants, and its expression levels were 2.80 and 27.45 times higher than that in the untreated control at 3 and 11 days post-inoculation, respectively. These results indicate that CreV8 belongs to the nucleotide binding site (NBS)-LRR resistance gene family and that it is a candidate CCN-resistance gene. Key words : Aegilops variabilis , CreV8, cereal cyst nematode (CCN), Heterodera avenae, tertiary structure.

Functional analysis of Rig, a novel Ras family small GTPase protein, in neurons

Starting with mRNA from field bean (Vicia faba) cotyledons, PCR fragments were obtained by using degenerate primers from conserved domains of ras-related GTP-binding proteins. These were used to isolate a number of clones from a field bean cotyledon cDNA-library. Homologs of rab1 (ypt1), rab2, rab11 (ypt3) and rab5 (ypt5) were identified. DNA sequencing yielded complete sequences of five clones named Vfa-ypt1, Vfa-ypt3a, Vfa-ypt3b, Vfa-ypt3x, and Vfa-ypt5. The deduced peptide sequences showed clear homology to the family of small GTP-binding proteins, including the effector loop region, the four conserved domains involved in GTP-binding, and the C-terminal cysteine-motif. Vfa-ypt3a and -3b are 99% and 100% identical to pea Pra6 and Pra5, respectively. Vfa-ypt3x is 60% identical to Ara1 and Ara4 from A. thaliana, Vfa-ypt1 is 93 % identical to Ara5 and 100 % identical to Pra9c from pea, and Vfa-ypt5 is 90% identical to Rhn1 from tobacco. RNA blotting showed a relatively low expression level of these clones, the highest level was observed with clone Vfa-ypt3 b. The five clones were expressed in bacteria and the products were shown to bind GTP. Antibodies were raised against synthetic peptides from variable regions and used to detect the protein expressed in bacteria as well as proteins in membrane preparations from different organs of field bean.