Molecular cloning,expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis>) lectin SL-I

Isolation and purification of a α-methyl-mannoside specific lectin (SL-I) of peanut was reported earlier [Singh and Das (1994) Glycoconj J 11:282–285]. Native SL-I is a glycoprotein having ∼31 kDa subunit molecular mass and forms dimer. The gene encoding this lectin is identified from a 6-day old peanut root cDNA library by anti-SL-I antibody and N-terminal amino acid sequence homology to the native lectin. Nucleotide sequence derived amino acid sequence of the re-SL-I shows amino acid sequence homology with the N-terminal and tryptic digests’ amino acid sequence of the native SL-I (nSL-I). Presence of a putative glycosylation (QNPS) site and a hydrophobic adenine-binding (VLVSYDANS) site is also identified in SL-I. Homology modeling of the lectin suggests it to be an archetype of legume lectins. It is expressed as a ~30 kDa apoprotein in E. coli and has the carbohydrate specificity and secondary structure identical to its natural counterpart. The lectin SL-I inhibits cytokinin 6-benzylaminopurine (BA)-induced “delayed leaf senescence” and “cotyledon expansion”. Equilibrium dialysis revealed a single high-affinity binding site for adenine (7.6 × 10⁻⁶ M) and BA (1.09 × 10⁻⁵ M) in the SL-I dimer and thus suggesting that the cytokinin antagonist effect of SL-I is mediated by the direct interaction of SL-I with BA.The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank databases under the Accession No. AJ585523     

The nuclear genome of the phytoseiid <Emphasis Type="Italic">Metaseiulus occidentalis</Emphasis> (Acari: Phytoseiidae) is among the smallest known in arthropods

The genome size of the phytoseiid Metaseiulus (=Typhlodromus or Galendromus) occidentalis (Nesbitt) needs to be estimated before the whole nuclear genome can be sequenced. Two different procedures were used to estimate the genome size of M. occidentalis; (1) flow cytometry (Marescalchi et al. in Genome 33:789–793, 1990) and (2) quantitative real-time PCR (qRT-PCR) (Wilhelm et al. in Nucleic Acids Res 31:e56, 2003). Fluorescence intensity of propidium iodide-stained nuclei of M. occidentalis was measured by flow cytometry using females, males, and eggs. Only the eggs yielded peaks, which ranged in size from 35 to 160 Mb, with a tall peak of 140 Mb in 1-day-old eggs and 65 Mb in 2-day-old eggs, respectively. However, the peaks are broad and do not provide an accurate estimate. The qRT-PCR procedure required single-copy nuclear gene sequences from this phytoseiid. This was accomplished by designing degenerate primers, amplifying the Actin and EF1α sequences from M. occidentalis, and then designing M. occidentalis-specific primers that amplified a unique sequence. The standard qRT-PCR protocol was inefficient and amplification failed frequently, so we developed a high-fidelity qRT-PCR protocol, which utilizes a mix of two DNA polymerases (Taq and a proof-reading Tgo or ACCUZYME) to consistently amplify sequences. This allowed us to estimate the nuclear genome size of M. occidentalis as 88–90 ± 5 Mb. When compared to other arthropod genomes, this appears to be very small.     

Tissue Distribution, Effects of Salinity Acclimation, and Ontogeny of Aquaporin 3 in the Marine Teleost, Silver Sea Bream (Sparus sarba)

The purpose of the present study was to ascertain the tissue-specific expression of the water channel protein, aquaporin 3 (AQP3), during salinity acclimation and larval development of silver sea bream (Sparus sarba). A cDNA fragment encoding aquaporin 3 (aqp3) from silver sea bream gill was cloned and from the deduced amino acid sequence a polyclonal antibody was prepared. AQP3 was found to be present in gill, kidney, liver, brain, heart, and spleen but not in whole blood. The abundance of AQP3 was significantly highest in gills of hypoosmotic (6 ppt) and isoosmotic (12 ppt) acclimated sea bream when compared to seawater (33 ppt) and hypersaline (50 ppt)- acclimated sea bream. Spleen tissue also displayed significantly high levels of AQP3 protein in hypoosmotic and isoosmotic salinities whereas the AQP3 abundance in brain, liver, heart, and kidney remained unchanged across the range of salinities tested. The ontogenetic profile of AQP3 was also investigated from developing sea bream larvae and AQP3 was first detected at 14 days posthatch (dph) and increased steadily up to 28–46 dph. In conclusion, this study has demonstrated that AQP3 expression is modulated in gill and spleen tissue of salinity acclimated sea bream and that it can be detected relatively early during larval development.     

Callus formation,plant regeneration,and transient expression in the halophyte sea aster (<Emphasis Type="Italic">Aster tripolium</Emphasis> L.)

An in vitro regeneration and transient expression systems were developed for the halophyte sea aster (Aster tripolium L.), an important genetic resource for salt tolerance. Adventitious shoots were formed from both leaf explants and suspension-cultured cells in a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) basal salts containing 500 mg l⁻¹ casamino acids, and supplemented with 5.4 μM a-naphthaleneacetic acid (NAA) and 4.7 μM kinetin to the culture medium. Hyperhydricity of shoots was avoided by increasing the ventilation of the culture vessel. Root formation from shoots was promoted in the presence of 26.9 μM NAA. A high yield of protoplasts was isolated using 1% cellulase and 0.25% pectinase from both leaf mesophyll and suspension-cultured cells, and these were used for transient expression. The highest level of transient expression of the green fluorescent protein was obtained with 1 × 10⁵ protoplasts ml⁻¹, 25 μg batch⁻¹ of plasmid vector, and 30% polyethylene glycol 4,000.     

Development of three allele-specific codominant rice <Emphasis Type="Italic">Waxy</Emphasis> gene PCR markers suitable for marker-assisted selection of amylose content and paste viscosity

Four Waxy haplotypes, previously identified as each having a different combination of three single nucleotide polymorphisms (SNPs) in the Waxy gene, were highly correlated with apparent amylose content and pasting properties, which are important grain quality traits for predicting cooked rice (Oryza sativa L.) texture and processing properties (Chen et al. in J Cereal Sci 47:536–545, 2008a; Chen et al. in J Cereal Sci 48:781–788, 2008b). Three allele-specific PCR markers were developed to genotype the three aforementioned functional SNPs in a single PCR amplification. Each marker contained two allele-specific primers and one common primer. For each marker, the two allele-specific primers differed by one base at the 3′-end to provide discrimination of SNP alleles, and were labeled with unique fluorescence probes. An additional mismatched base, the third base from the 3′-end, was inserted in some allele-specific primers to increase selectivity. The amplification step of the PCR thermal cycling program was initially set for 20× touch-down cycles with the annealing temperature of the first cycle approximately 6°C above the thermal melting temperature of all three primers at a touch-down rate of −0.3°C per cycle, and followed by 25× regular thermal cycles with the annealing temperature at their thermal melting temperature. The allelic genotypes for each SNP were distinguished from each other by both their differential primer-allele fluorescences and their amplification product lengths. The simplicity of these assays makes it easy to utilize these markers as part of a marker-assisted selection strategy in rice breeding programs selecting for these important grain quality traits.     

Ebbie: automated analysis and storage of small RNA cloning data using a dynamic web server

Background  

DNA sequencing is used ubiquitously: from deciphering genomes[1] to determining the primary sequence of small RNAs (smRNAs) [2–5]. The cloning of smRNAs is currently the most conventional method to determine the actual sequence of these important regulators of gene expression. Typical smRNA cloning projects involve the sequencing of hundreds to thousands of smRNA clones that are delimited at their 5' and 3' ends by fixed sequence regions. These primers result from the biochemical protocol used to isolate and convert the smRNA into clonable PCR products. Recently we completed a smRNA cloning project involving tobacco plants, where analysis was required for ~700 smRNA sequences[6]. Finding no easily accessible research tool to enter and analyze smRNA sequences we developed Ebbie to assist us with our study.     

Loss of <Emphasis Type="Italic">Tc-arrow</Emphasis> and canonical Wnt signaling alters posterior morphology and pair-rule gene expression in the short-germ insect, <Emphasis Type="Italic">Tribolium castaneum</Emphasis>

Wnt signaling has been implicated in posterior patterning in short-germ insects, including the red flour beetle Tribolium castaneum (Bolognesi et al. Curr Biol 18:1624–1629, 2008b; Angelini and Kaufman Dev Biol 283:409–423, 2005; Miyawaki et al. Mech Dev 121:119–130, 2004). Specifically, depletion of Wnt ligands Tc-Wnt1 and Tc-WntD/8 produces Tribolium embryos lacking abdominal segments. Similar phenotypes are produced by depletion of Tc-porcupine (Tc-porc) or Tc-pangolin (Tc-pan), indicating that the signal is transmitted through the canonical Wnt pathway (Bolognesi et al. Curr Biol 18:1624–1629, 2008b). Here we show that RNAi for the receptor Tc-arrow produced similar truncated phenotypes, providing additional evidence supporting canonical signal transduction. Furthermore, since in Tribolium segments are defined sequentially by a pair-rule gene circuit that, when interrupted, produces truncated phenotypes (Choe et al. Proc Natl Acad Sci U S A 103:6560–6564, 2006), we investigated the relationship between loss of Wnt signaling and this pair-rule gene circuit. After depletion of the receptor Tc-arrow, expression of Tc-Wnt1 was noticeably absent from the growth zone, while Tc-WntD/8 was restricted to a single spot of expression in what remained of the posterior growth zone. The primary pair-rule genes Tc-runt (Tc-run) and Tc-even-skipped (Tc-eve) were expressed normally in the anterior segments, but were reduced to a single spot in the remnants of the posterior growth zone. Thus, expression of pair-rule genes and Tc-WntD/8 are similarly affected by depletion of Wnt signal and disruption of the posterior growth zone.     

<Emphasis Type="Italic">Anisakis nascettii</Emphasis> n. sp. (Nematoda: Anisakidae) from beaked whales of the southern hemisphere: morphological description,genetic relationships between congeners and ecological data

A new anisakid nematode, Anisakis nascettii n. sp., is described from beaked whales Mesoplodon spp. off the coast of New Zealand and South Africa. Morphological and molecular (allozymes and mtDNA cox2 sequence) data were used for diagnostic and identification purposes. Among the 19 allozymes studied, 10 were found to be unique and characteristic for A. nascettii n. sp. Analysis of allozymes demonstrated reproductive isolation from A. ziphidarum Paggi, Nascetti, Webb, Mattiucci, Cianchi & Bullini, 1998 and mtDNA cox2 sequences depict this Anisakis species as a distinct and unique entity. Key morphological diagnostic traits for A. nascettii with respect to the genetically closely related species A. ziphidarum include: spicule length, the spicule/body length ratio, the arrangement of the caudal papillae and the shape of the plectanes of the adult males. Genetic data confirmed that Anisakis sp. A of Pontes et al. (2005), which was partly described by Iglesias et al. (2008), and Anisakis sp. of Valentini et al. (2006) are conspecific with A. nascettii. Both molecular and morphological data indicate that the new species belongs to the ‘ziphidarum-group’; however, it is genetically very distinct from A. ziphidarum (D _Nei  = 0.69, K2P = 0.09), as well as from all of the previously genetically characterised Anisakis spp. All tree topologies inferred by different methods (MP, NJ and Bayesian) support the finding that A. nascettii n. sp. and A. ziphidarum are sister-species. It is also confirmed that A. nascettii n. sp. is, at the adult stage, a parasite of beaked whales of the genus Mesoplodon, whereas, as a larva, it has been identified from the squid Moroteuthis ingens Smith. Furthermore, Mesoplodon bowdoini Andrews represents a new host record for A. ziphidarum. The parallelism between the clade formed by these two anisakine taxa, i.e. A. ziphidarum and A. nascettii, and that formed by their definitive hosts further supports the hypothesis of host–parasite co-evolutionary relationships, as previously suggested for Anisakis spp. and their cetacean hosts.     

A new species of <Emphasis Type="Italic">Litomosoides</Emphasis> Chandler, 1931 (Nematoda: Filarioidea) from the long-nosed hocicudo <Emphasis Type="Italic">Oxymycterus nasutus</Emphasis> Waterhouse (Rodentia: Cricetidae) in Uruguay

A new species of Litomosoides Chandler, 1931 was collected from the abdominal cavity of Oxymycterus nasutus Waterhouse (Rodentia: Cricetidae) in the grassland of the Departamento Rocha, Uruguay. Litomosoides nasuti n. sp. belongs to the ‘sigmodontis group’, and is characterised by: salient amphids; two ventral and one dorsal labial papillae, but no cephalic papillae; a buccal capsule with a transparent anterior segment and an annular asymmetrical thickening; a muscular oesophagus; a bottle-shaped buccal cavity; the male with symmetrically situated cloacal papillae (one pair ad-cloacal and three pairs post-cloacal); phasmids displaced laterally to the longitudinal axis; and microfilariae without terminal nuclei in the tail tip. It resembles five known species; three of which have been recovered from Oxymycterus spp. in neighbouring countries. However, the new species can be differentiated from L. sigmodontis Chandler, 1931 by the shape and size of the buccal capsule; from L. navonae Notarnicola, 2005 by the muscular oesophagus; from L. legerae Bain, Petit & Berteaux, 1980 by the length of the oesophagus and the cephalic papillae; from L. anguyai Notarnicola, Bain & Navone, 2002 by the absence of lappets in the female tail; and from L. oxymycteri Notarnicola, Bain & Navone, 2000 by absence of pre-cloacal papillae. L. legerae from O. quaestor and L. sigmodontis from Sigmodon hispidus in North America are closely related species, as indicated by Brant & Gardner’s phylogenetic tree based on morphological characters. However, a new analysis is needed to include the recently described Argentinean species for a better understanding of the diversification of this genus.     

Class 1 KNOX Gene Expression Supports the <Emphasis Type="Italic">Selaginella</Emphasis> Rhizophore Concept

The spikemoss is marked by the unique root-producing pleurogeous rhizophore as well as the lycophytic microphyll. Imaichi and Kato (Bot Mag Tokyo 102:369–380, 1989; Am J Bot 78:1694–1703, 1991) revealed that the exogenous developmental process in the rhizophore is clearly distinguishable from the developmental process in the endogenous root, argued that the axial organ could be coordinate with other fundamental organs including the root and stem, and demonstrated the “rhizophore concept.” In this paper, we report on the expression pattern of the spikemoss Selaginella class 1 KNOX gene, SuKNOX1, in the rhizophore. We show that the SuKNOX1 mRNA is specifically accumulated at the tip of the rhizophore as well as the shoot apical apex, but not in the root tip. This result supports the “rhizophore concept” at the molecular level.     

The Role of Organic Acids in the Domestication of <Emphasis Type="Italic">Oxalis tuberosa</Emphasis>: A New Model for Studying Domestication Resulting in Opposing Crop Phenotypes<Superscript>1</Superscript>

The Role of Organic Acids in the Domestication of Oxalis tuberosa: A New Model for Studying Domestication Resulting in Opposing Crop Phenotypes. Though few crops display directly opposing domesticated phenotypes, these crops may be the key to understanding domestication processes that address conflicting selective pressures in the agricultural ecosystem. Two relatively well-known examples are cassava (Manihot esculenta Crantz), which has high-cyanide and low-cyanide varieties, and potato (Solanum section Petota). Among the potatoes are several species, including the common potato (Solanum tuberosum L.), that have low levels of glycoalkaloids and there are other species of “bitter potato” with elevated levels of glycoalkaloids. We propose that Oxalis tuberosa Molina, “oca,” may represent a third example of such a crop system, with opposing high organic acid and low organic acid cultivars. Each cultivar set has different cultural food preparation practices (“use-categories”), similar to the “use-categories” that have been described for potatoes in the Andes (Brush et al. Economic Botany 35;70–88, 1981; Zimmerer Journal of Biogeography 18;165–178, 1991). Our initial analyses suggest that organic acids in tubers may be an important biochemical difference between use-categories, based on both oxalic acid and pH data. Here, we review our understanding of organic acids in oca tubers, while highlighting areas that merit further investigation.     

On Primordial Sense–Antisense Coding

The genetic code is implemented by aminoacyl-tRNA synthetases (aaRS). These 20 enzymes are divided into two classes that, despite performing same functions, have nothing common in structure. The mystery of this striking partition of aaRSs might have been concealed in their sterically complementary modes of tRNA recognition that, as we have found recently, protect the tRNAs with complementary anticodons from confusion in translation. This finding implies that, in the beginning, life increased its coding repertoire by the pairs of complementary codons (rather than one-by-one) and used both complementary strands of genes as templates for translation. The class I and class II aaRSs may represent one of the most important examples of such primordial sense–antisense (SAS) coding (Rodin and Ohno, Orig Life Evol Biosph 25:565–589, 1995). In this report, we address the issue of SAS coding in a wider scope. We suggest a variety of advantages that such coding would have had in exploring a wider sequence space before translation became highly specific. In particular, we confirm that in Achlya klebsiana a single gene might have originally coded for an HSP70 chaperonin (class II aaRS homolog) and an NAD-specific GDH-like enzyme (class I aaRS homolog) via its sense and antisense strands. Thus, in contrast to the conclusions in Williams et al. (Mol Biol Evol 26:445–450, 2009), this could indeed be a “Rosetta stone” gene (Carter and Duax, Mol Cell 10:705–708, 2002) (eroded somewhat, though) for the SAS origin of the two aaRS classes.     

Hadamard Phylogenetic Methods and the <Emphasis Type="Italic">n</Emphasis>-taxon Process

The Hadamard transform (Hendy and Penny, Syst. Zool. 38(4):297–309, 1989; Hendy, Syst. Zool. 38(4):310–321, 1989) provides a way to work with stochastic models for sequence evolution without having to deal with the complications of tree space and the graphical structure of trees. Here we demonstrate that the transform can be expressed in terms of the familiar P[τ]=e ^Q[τ] formula for Markov chains. The key idea is to study the evolution of vectors of states, one vector entry for each taxa; we call this the n-taxon process. We derive transition probabilities for the process. Significantly, the findings show that tree-based models are indeed in the family of (multi-variate) exponential distributions.     

Phylogenetic Position of <Emphasis Type="Italic">Corchoropsis</Emphasis> Siebold &; Zucc. (Malvaceae s.l.) Inferred from Plastid DNA Sequences

Recent phylogenetic research suggests that Malvaceae s.l. comprises formerly Tiliaceae, Byttneriaceae, Bombacaceae, and Sterculiaceae. Corchoropsis is traditionally included in Tiliaceae or Sterculiaceae and is distributed in China, Korea, and Japan. One to three species have been recognized for this genus. Phylogenetic relationships among the Malvacean taxa have been intensively studied with molecular data, and the evolution of their morphological characteristics has been re-interpreted accordingly. However, no Corchoropsis species have been included for their phylogenetic position. Here, three chloroplast coding regions—rbcL, atpB, and ndhF, from Corchoropsis psilocarpa and Corchoropsis crenata—were amplified and sequenced, then compared with other Malvacean taxa. This analysis of the three plastid gene sequences now places Corchoropsis species in Dombeyoideae, as previously proposed by Takeda (Bull Misc Inform Kew 365, 1912), Tang (Cathaya 4:131–150, 1992), and Bayer and Kubitzki (2003). Within Dombeyoideae, Corchoropsis forms a strongly supported sister relationship with the Dombeya–Ruizia clade.     

Molecular Relationships of New Guinean Three-Striped Dasyures, (Myoictis, Marsupialia: Dasyuridae)

Complete nucleotide sequences of the cytochrome b and 12S rRNA genes and partial sequences of the mitochondrial 16S rRNA gene and the nuclear ɛ-globin gene were obtained from multiple exemplars of the New Guinean dasyurid, Myoictis. Allozyme data were also obtained from most of the same animals. The molecular data show that the genus comprises a number of genetically distinct lineages which correspond with groups proposed by Woolley (2005) on the basis of a number of morphological traits, including the form of the tail i.e. Myoictis leucura (sp. nov.), M. melas, M. wallacei and M. wavicus (new status). Divergence dates estimated from the weighted-average distances for the combined cytochrome b and 12S rRNA data, calibrated with a dasyurid-thylacine divergence 25 million years ago, suggest that the early cladogenic events separating Myoictis took place in the late Miocene. Subsequent separation of M. wavicus and M. leucura from a common ancestor as well as some genetic differentiation within M. melas, took place in the medial Pliocene.     

Four new species of <Emphasis Type="Italic">Aloe</Emphasis> (Aloaceae) from Ethiopia,with notes on the ethics of describing new taxa from foreign countries

Subsequent to the treatment of the Aloaceae, with 38 species of Aloe, in the Flora of Ethiopia (Sebsebe Demissew & Gilbert 1997), four more species, Aloe bertemariae Sebsebe & Dioli (2000), A. friisii Sebsebe & M. G. Gilbert (2000), A. clarkei L. E. Newton (2002) and A. elkerriana Dioli & T. A. McCoy (2007) have been described from that country. Here four additional new species are described: Aloe benishangulana Sebsebe & Tesfaye from near Assosa, Benishangul-Gumuz in Welega floristic region; A. ghibensis Sebsebe & Friis from the Ghibe Gorge, Kefa floristic region; A. weloensis Sebsebe from near Dessie in Welo floristic region and A. welmelensis Sebsebe & Nordal along the Welmel River in Bale floristic region. The phytogeographical positions of the new species are assessed by comparison with the previously known species. Complications with the deposition of type material of A. clarkei and A. elkerriana is used to raise various issues regarding the ethics of describing new taxa from foreign countries.     

Recombinant DNA-methyltransferase M1.Bst19I from <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis> 19: Purification,properties, and amino acid sequence analysis

The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5′-GCATC-3′) in Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW that carries a tandem of thermo inducible promoters P _R /P _L from phage λ. Highly purified enzyme has been isolated by chromatography on various resins from Escherichia coli cells where it is accumulated in a soluble form. The study of M1.Bst19I properties has revealed that the enzyme has a temperature optimum at 50°C and demonstrates maximal activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5′-GCATC-3′. Kinetic parameters of M1.Bst19I DNA methylation reaction have been determined as follows: Km for λ DNA is 0.68 ± 0.07 μM, Km for S-adenosyl-L-methionine is 2.02 ± 0.31 μM. Catalytical constant (k _cat) is 1.8 ± 0.05 min⁻¹. Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and other α-N6-DNA-methyltransferases has allowed us to suggest the presence of two types of the enzymes containing ATG or ATC triplets in the recognition sequence.