Purification and characterization of a lectin,BPL-3, from the marine green alga <Emphasis Type="Italic">Bryopsis plumosa</Emphasis>

A novel lectin was isolated and characterized from Bryopsis plumosa (Hudson) Agardh and named BPL-3. This lectin showed specificity to N-acetyl-d-galactosamine as well as N-acetyl-d-glucosamine and agglutinated human erythrocytes of all blood types, showing slight preference to the type A. SDS-PAGE and MALDI-TOF MS data showed that BPL-3 was a monomeric protein with molecular weight of 11.5 kDa. BPL-3 was a non-glycoprotein with pI value of ∼7.0. It was stable in high temperatures up to 70°C and exhibited optimum activity in pH 5.5–10. The N-terminal and internal amino acid sequences of the lectin were determined by Edman degradation and enzymatic digestion, which showed no sequence homology to any other reported proteins. The full sequence of the cDNA encoding this lectin was obtained from PCR using cDNA library, and the degenerate primers were designed from the N-terminal amino acid sequence. The size of the cDNA was 622 bp containing single ORF encoding the lectin precursor. This lectin showed the same sugar specificity to previously reported lectin, Bryohealin, involved in protoplast regeneration of B. plumosa. However, the amino acid sequences of the two lectins were completely different. The homology analysis of the full cDNA sequence of BPL-3 showed that it might belong to H lectin group, which was originally isolated from Roman snails.     

Comparison of adherence, cytotoxicity, and Gal/GalNAc lectin gene structure in Entamoeba histolytica and Entamoeba dispar

The recent development of axenic culture for E. dispar allowed us to examine this ameba's ability to bind and lyse target cells and compare it to E. histolytica which has been axenically cultured for years. We found that under axenic conditions, E. dispar's adherence to target cells, ligand binding, and cytotoxicity were less than that of E. histolytica. These events were Gal/GalNAc-inhibitable supporting the idea that E. dispar expresses a lectin similar to E. histolytica. Genetic analysis showed that E. dispar had at least two members of the lectin heavy subunit family and four members of the lectin light subunit family that hybridized to ehhgl and ehlgl gene probes. A library screen produced clones which were isolated and sequenced. Derived amino acid sequences showed that the E. dispar heavy and light subunit clones were 86% and 79% identical, respectively, to their E. histolytica counterparts. In particular, the region which contains the epitopes for two adherence-enhancing monoclonal antibodies and a complement-sensitizing monoclonal antibody (amino acids 882–959 of the lectin heavy subunit) were conserved between the species.     

Fine mapping and DNA fiber FISH analysis locates the tobamovirus resistance gene <Emphasis Type="Italic">L</Emphasis><Superscript>3</Superscript> of <Emphasis Type="Italic">Capsicum chinense</Emphasis> in a 400-kb region of R-like genes cluster embedded in highly repetitive sequences

The tobamovirus resistance gene L ³ of Capsicum chinense was mapped using an intra-specific F2 population (2,016 individuals) of Capsicum annuum cultivars, into one of which had been introduced the C. chinense L ³ gene, and an inter-specific F2 population (3,391 individuals) between C. chinense and Capsicum frutescence. Analysis of a BAC library with an AFLP marker closely linked to L ³-resistance revealed the presence of homologs of the tomato disease resistance gene I2. Partial or full-length coding sequences were cloned by degenerate PCR from 35 different pepper I2 homologs and 17 genetic markers were generated in the inter-specific combination. The L ³ gene was mapped between I2 homolog marker IH1-04 and BAC-end marker 189D23M, and located within a region encompassing two different BAC contigs consisting of four and one clones, respectively. DNA fiber FISH analysis revealed that these two contigs are separated from each other by about 30 kb. DNA fiber FISH results and Southern blotting of the BAC clones suggested that the L ³ locus-containing region is rich in highly repetitive sequences. Southern blot analysis indicated that the two BAC contigs contain more than ten copies of the I2 homologs. In contrast to the inter-specific F2 population, no recombinant progeny were identified to have a crossover point within two BAC contigs consisting of seven and two clones in the intra-specific F2 population. Moreover, distribution of the crossover points differed between the two populations, suggesting linkage disequilibrium in the region containing the L locus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. R. Tomita and J. Murai contributed equally to this work.     

Molecular cloning and the cDNA-derived amino acid sequence of Narcissus tazetta isolectins

Recently several complete cDNAs encoding the Narcissus tazetta lectins (NTL) were cloned. The sequence analyses of the cloned DNAs reveal that there are at least three unidentical positive clones for NTLs. The primary structure of the three NTL clones contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension beyond the C-terminal amino acids Thr-Gly. There are two fixed-position cysteines within the protein domain (amino acids 29 and 52), which are probably involved in the disulfide-bond linkage within the molecules to confer the secondary structure of the mature lectin. One third of the deduced amino acid composition consisted of glycine, leucine, and asparagine. From the cDNA-derived amino acid sequences the three NTL clones are not identical and are suggested to be isolectins present in N. tazetta var. chinensis. This study further confirms the previous isolation of mannose-specific isolectins from Chinese daffodil leaves [Ooi et al. (2000), J. Protein Chem. 19, 163-168].     

Purification,characterization and molecular cloning of a monocot mannose-binding lectin from <Emphasis Type="Italic">Remusatia vivipara</Emphasis> with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80°C and under wide range of pH (2.0–9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 μg/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.     

Expressed sequence tags of young floral buds and characterization of a bud- preferential lectin- like cDNA fromPharbitis nil

To identify the genes involved in flower development, we analyzed 207 expressed sequence tags (ESTs) from a young floral bud cDNA library ofPharbitis nil. Of these, 87 clones (42%) showed significant homology to known protein sequences in the NCBI database. Four of these had not been reported previously in the plant kingdom, indicating that 1.9% of the ESTs were newly identified in plants. Functional categorization revealed that the genes involved in metabolic pathways, such as glycolysis and photosynthesis, were most abundant Reverse-northern and northern analyses showed that one clone,PnFP161, was expressed preferentially in floral buds. DNA sequence analysis indicated that this clone contained 147 bp of 5′-UTR, 264 bp of -UTR, and an open reading frame of 233 amino acids, thereby sharing 33% identity with a lectin fromCalystegia sepium. The C-terminal regionof PnFPI61 had well-conserved residue with that of the lectins. Southern blot analysis demonstrated thatPnFPI61 exists as a multigene family.     

Morphology and phylogenetics of two holoparasitic plants, <Emphasis Type="Italic">Balanophora japonica</Emphasis> and <Emphasis Type="Italic">Balanophora yakushimensis</Emphasis> (Balanophoraceae), and their hosts in Taiwan and Japan

Balanophora japonica and B. yakushimensis are two putatively agamospermic taxa previously reported from southern Japan. Their inflorescences superficially represent those of B. laxiflora and B. fungosa. In this study we confirmed their presence in Taiwan by morphological and phylogenetic analysis using nuclear 18S rDNA and nrITS sequences with related taxa. B. japonica, B. yakushimensis, and B. laxiflora formed a well-supported clade that is distinct from other Balanophora. All three taxa also show considerable differences on morphological and nucleotide sequence differences, therefore the name of B. yakushimensis is retained. The results provide new insights on the intrageneric classification of Balanophora and suggest the positioning of female flowers should be down-weighted. We also successfully identify the hosts of B. japonica and B. yakushimensis by amplifying chloroplast matK sequences from the connected root tissues. The results showed that B. japonica parasitizes on Symplocos species, and that B. yakushimensis parasitizes on Distylium racemosum in Japan and Schima superba in Taiwan’s population.     

An intersubspecific genetic map of<Emphasis Type="Italic"> Lens</Emphasis>

A Lens map was developed based on the segregational analysis of five kinds of molecular and morphological genetic markers in 113 F₂ plants obtained from a single hybrid of Lens culinaris ssp. culinaris × L. c. ssp. orientalis. A total of 200 markers were used on the F₂ population, including 71 RAPDs, 39 ISSRs, 83 AFLPs, two SSRs and five morphological loci. The AFLP technique generated more polymorphic markers than any of the others, although AFLP markers also showed the highest proportion (29.1%) of distorted segregation. At a LOD score of 3.0, 161 markers were grouped into ten linkage groups covering 2,172.4 cM, with an average distance between markers of 15.87 cM. There were six large groups with 12 or more markers each, and four small groups with two or three markers each. Thirty-nine markers were unlinked. A tendency for markers to cluster in the central regions of large linkage groups was observed. Likewise, clusters of AFLP, ISSR or RAPD markers were also observed in some linkage groups, although RAPD markers were more evenly spaced along the linkage groups. In addition, two SSR, three RAPD and one ISSR markers segregated as codominant. ISSR markers are valuable tools for Lens genetic mapping and they have a high potential in the generation of saturated Lens maps.Communicated by H.C. Becker     

cDNA Cloning and Functional Expression of the α-<Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin Frutalin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

cDNA clones encoding frutalin, the α-d-galactose-binding lectin expressed in breadfruit seeds (Artocarpus incisa), were isolated and sequenced. The deduced amino acid sequences indicated that frutalin may be encoded by a family of genes. The NCBI database searches revealed that the frutalin sequence is highly homologous with jacalin and mornigaG sequences. Frutalin cDNA was re-amplified and cloned into the commercial expression vector pET-25b(+) for frutalin production in Escherichia coli. An experimental factorial design was employed to maximise the soluble expression of the recombinant lectin. The results indicated that temperature, time of induction, concentration of IPTG and the interaction between the concentration of IPTG and the time of induction had the most significant effects on the soluble expression level of recombinant frutalin. The optimal culture conditions were as follows: induction with 1 mM IPTG at 22°C for 20 h, yielding 16 mg/l of soluble recombinant frutalin. SDS-PAGE and Western blot analysis revealed that recombinant frutalin was successfully expressed by bacteria with the expected molecular weight (17 kDa). These analyses also showed that recombinant frutalin was mainly produced as insoluble protein. Recombinant frutalin produced by bacteria revealed agglutination properties and carbohydrate-binding specificity similar to the native breadfruit lectin.     

Molecular cloning of mannose-binding lectins from Clivia miniata

Screening of a cDNA library constructed from total RNA isolated from young developing ovaries of Clivia miniata Regel with the amaryllis lectin cDNA clone resulted in the isolation of four different isolectin clones which clearly differ from each other in their nucleotide sequences and hence also in their deduced amino acid sequences. Apparently the lectin is translated from an mRNA of ca. 800 nucleotides encoding a precursor polypeptide of 163 amino acids. Northern blot analysis of total RNA isolated from different tissues of Clivia miniata has shown that the lectin is expressed in most plant tissues with very high lectin mRNA concentrations in the ovary and the seed endosperm.     

Purification and functional characterization of lectin with phenoloxidase activity from the hemolymph of cockroach,Periplaneta americana

Lectins also identified as hemagglutinins are multivalent proteins and on account of their fine sugar‐binding specificity play an important role in immune system of invertebrates. The present study was carried out on the hemolymph lectin of cockroach, Periplaneta americana with appropriate screening and purification to understand its molecular as well as functional nature. The lectin from the hemolymph was purified using ion‐exchange chromatography. The approximate molecular weight of purified lectin was 340 kDa as determined by FPLC analysis. Rabbit erythrocytes were highly agglutinated with purified lectin from the hemolymph of P. americana. The hemagglutination activity (HA) of lectin was specifically inhibited by fucose. Glycoproteins also inhibited the HA activity of lectin. The amino acid sequences of the purified lectin revealed homology with amino acid sequences of allergen proteins from P. americana. Purified lectin showed the highest phenoloxidase activity against dopamine. The activators such as exogenous proteases and LPS from Escherichia coli and Salmonella minnesota significantly enhanced the PO activity of the purified lectin. Besides, the presence of copper and hemocyanin conserved domain in the purified lectin provided a new facet that insects belonging to the ancient clade such as cockroaches retained some traces of evolutionary resemblance in possessing lectin of ancient origin.     

New insights into DNA barcoding of seagrasses

Taxonomists find some plant genera challenging because of the few morphological differences or unclear characters among closely related species, which leads to the misidentification of taxa. DNA barcoding is an approach to identify species by using short orthologous DNA sequences, known as ‘DNA barcodes’. Concatenated rbcL and matK sequences are considered DNA barcodes for seagrasses. However, these markers are not applicable to all members of seagrasses at the species level, especially within the genus Halophila. Our previous studies indicated that the internal transcribed spacer (ITS) showed higher species resolution than the concatenated rbcL and matK sequences in the case of Halophila ovalis and closely related species. In this study, 26 ITS, two rbcL and two matK consensus sequences from 18 seagrass taxa belonging to four families collected in India, Vietnam, Germany, Croatia and Egypt were processed. Molecular ITS analysis resolved five clades. The results also indicate that the Cymodoceaceae family might be a non-monophyletic group. In conclusion, ITS could be applied as a DNA barcode for seagrasses instead of the rbcL/matK system previously proposed.     

The seed lectins of black locust (robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes

Two lectins were isolated from Robinia pseudoacacia (black locust) seeds using affinity chromatography on fetuin-agarose, and ion exchange chromatography on a Neobar CS column. The first lectin, R. pseudoacacia seed agglutinin I, referred to as RPsAI, is a homotetramer of four 34 kDa subunits whereas the second lectin, referred to as RPsAII, is composed of four 29 kDa polypeptides. cDNA clones encoding the polypeptides of RPsAI and RPsAII were isolated and their sequences were determined. Both polypeptides are translated from mRNAs of ca. 1.2 kb encoding a precursor carrying a signal peptide. Alignment of the deduced amino acid sequences of the different clones indicates that the 34 and 29 kDa seed lectin polypeptides show 95% sequence identity. In spite of this striking homology, the 29 kDa polypeptide has only one putative glycosylation site whereas the 34 kDa subunit has four of these sites. Carbohydrate analysis revealed that the 34 kDa possesses three carbohydrate chains whereas the 29 kDa polypeptide is only partially glycosylated at one site. A comparison of the deduced amino acid sequences of the two seed and three bark lectin polypeptides demonstrated unambiguously that they are encoded by different genes. This implies that five different genes are involved in the control of the expression of the lectins in black locust.Abbreviations LECRPAs cDNA clone encoding Robinia pseudoacacia seed lectin - LoLI Lathyrus ochrus isolectin I - PsA Pisum sativum agglutinin - RPbAI Robinia pseudoacacia bark agglutinin I - RPbAII Robinia pseudoacacia bark agglutinin II - RPsAI Robinia pseudoacacia seed agglutinin I - RPsAII Robinia pseudoacacia seed agglutinin II     

Genetic Diversity in Lens Species Revealed by EST and Genomic Simple Sequence Repeat Analysis

Low productivity of pilosae type lentils grown in South Asia is attributed to narrow genetic base of the released cultivars which results in susceptibility to biotic and abiotic stresses. For enhancement of productivity and production, broadening of genetic base is essentially required. The genetic base of released cultivars can be broadened by using diverse types including bold seeded and early maturing lentils from Mediterranean region and related wild species. Genetic diversity in eighty six accessions of three species of genus Lens was assessed based on twelve genomic and thirty one EST-SSR markers. The evaluated set of genotypes included diverse lentil varieties and advanced breeding lines from Indian programme, two early maturing ICARDA lines and five related wild subspecies/species endemic to the Mediterranean region. Genomic SSRs exhibited higher polymorphism in comparison to EST SSRs. GLLC 598 produced 5 alleles with highest gene diversity value of 0.80. Among the studied subspecies/species 43 SSRs detected maximum number of alleles in L. orientalis. Based on Nei’s genetic distance cultivated lentil L. culinaris subsp. culinaris was found to be close to its wild progenitor L. culinaris subsp. orientalis. The Prichard’s structure of 86 genotypes distinguished different subspecies/species. Higher variability was recorded among individuals within population than among populations.     

Polymorphism of lectin genes in <Emphasis Type="Italic">Lathyrus</Emphasis> plants

The carbohydrate-binding sequences of the lectin genes from spring vetchling Lathyrus vernus (L.) Bernh., marsh vetchling L. palustris (L.), and Gmelin’s vetchling L. gmelinii (Fitsch) (Fabaceae) were determined. Computer-aided analysis revealed substantial differences between nucleotide and predicted amino acid sequences of the lectin gene regions examined in each of the three vetchling species tested. In the phylogenetic trees based on sequence similarity of carbohydrate-biding regions of legume lectins, the sequences examined formed a compact cluster with the lectin genes of the plants belonging to the tribe Fabeae. In each plant, L. vernus, L. palustris, and L. gmelinii, three different lectin-encoding genes were detected. Most of the substitutions were identified within the gene sequence responsible for coding the carbohydrate-binding protein regions. This finding may explain different affinity of these lectins to different carbohydrates, and as a consequence, can affect the plant host specificity upon development of symbiosis with rhizobium bacteria.     

Structure and organization of the rDNA intergenic spacer region in <Emphasis Type="Italic">Pythium ultimum</Emphasis>

Nucleotide sequences of the rDNA intergenic spacer (IGS) region in Pythium ultimum were determined in 16 clones obtained from three isolates differing in production of sexual organs. Several sequences with different lengths were detected in each isolate, showing heterogeneity in the IGS region. In addition, several tandem repeat regions were detected in all the clones. The sequences, length, and number of each copy largely varied among repeat regions. Length heterogeneity arose from the complex combination of the number of copy within the repeat regions. Furthermore, the nucleotide sequence of each copy and the number of repetition varied not only between isolates but also between clones from an isolate. Based on the sequence similarity and the number of copies in repeat regions, specific patterns different between homothallic P. ultimum and the Pythium group HS (hyphal swellings) were recognized in a few regions. These results suggest that these two groups have slight genetic differences in the IGS region, although the differences in most of the repeat regions were not enough to identify each group.     

Phylogeny and taxonomic reassessment of pikas Ochotona pallasii and O. argentata (Mammalia,Lagomorpha)

We examined a cranial morphometric data set consisting of 186 specimens from the entire distribution range of Ochotona pallasii sensu lato and O. argentata, as well as 67 complete sequences of the COI gene and 28 sequences of the MGF and PRKCI introns from these and closely allied species. Our results show that the two allopatric morphologically similar taxa composing O. pallasii sensu lato – from Mongolia and adjacent territories and Kazakhstan – are paraphyletic relative to O. argentata. Genetic distances between these three taxa are larger than the intraspecific variation known for the subgenus Pika, in which the species under consideration belong; these distances are even larger than the interspecific differences among closely related species such as O. hyperborea, O. mantchurica and O. hoffmanni. Thus, the three focal taxa are recognized here as distinct species. Inspection of the type specimen of O. pallasii indicated that this specimen was not collected in Kazakhstan, has previously been theorized. The most probable place of the holotype's origin is Russian south‐eastern Altai (Chuyskaya Steppe); whatever its exact origin, it definitively originates from the ‘Mongolian’ taxon. Based on this evidence, the senior synonym for the Kazakh pika is O. opaca Argyropulo, 1930. Thus, we propose to recognize three separate species in the O. pallasii species group: O. pallasii (Mongolia and adjacent territories), O. opaca (eastern Kazakhstan) and O. argentata (Helan Shan Range, China).     

Classification and Characterization of Species within the Genus Lens Using Genotyping-by-Sequencing (GBS)

Lentil (Lens culinaris ssp. culinaris) is a nutritious and affordable pulse with an ancient crop domestication history. The genus Lens consists of seven taxa, however, there are many discrepancies in the taxon and gene pool classification of lentil and its wild relatives. Due to the narrow genetic basis of cultivated lentil, there is a need towards better understanding of the relationships amongst wild germplasm to assist introgression of favourable genes into lentil breeding programs. Genotyping-by-sequencing (GBS) is an easy and affordable method that allows multiplexing of up to 384 samples or more per library to generate genome-wide single nucleotide Polymorphism (SNP) markers. In this study, we aimed to characterize our lentil germplasm collection using a two-enzyme GBS approach. We constructed two 96-plex GBS libraries with a total of 60 accessions where some accessions had several samples and each sample was sequenced in two technical replicates. We developed an automated GBS pipeline and detected a total of 266,356 genome-wide SNPs. After filtering low quality and redundant SNPs based on haplotype information, we constructed a maximum-likelihood tree using 5,389 SNPs. The phylogenetic tree grouped the germplasm collection into their respective taxa with strong support. Based on phylogenetic tree and STRUCTURE analysis, we identified four gene pools, namely L. culinaris/L. orientalis/L. tomentosus, L. lamottei/L. odemensis, L. ervoides and L. nigricans which form primary, secondary, tertiary and quaternary gene pools, respectively. We discovered sequencing bias problems likely due to DNA quality and observed severe run-to-run variation in the wild lentils. We examined the authenticity of the germplasm collection and identified 17% misclassified samples. Our study demonstrated that GBS is a promising and affordable tool for screening by plant breeders interested in crop wild relatives.     

Structural and functional differences of Litopenaeus vannamei crustins

Penaeid crustins were described in Litopenaeus vannamei and L. setiferus as proteins belonging to an antibacterial peptide family with similar sequences but different sizes. Six crustin-coding clones were isolated from a cDNA library from L. vannamei hemocytes, sequenced and compared. Two different isoforms (named I and P) were found, based on two nucleotide differences that produce one change in amino acid sequence (Ile/Pro). Other single differences in nucleotide sequences were also noted, but they did not change the translated product. The mRNA steady state levels of crustin I, but not of crustin P, were down regulated by Vibrio alginolyticus inoculation. Thus, the differences among penaeid crustins seem to be associated with one amino acid substitution, which affects their expression after bacterial inoculation. By structural similarity, shrimp crustins seem to belong to an antibacterial WAP-domain containing protein family.