(1/119) A method for estimating nucleotide diversity from AFLP data.
A method for estimating the nucleotide diversity from AFLP data is developed by using the relationship between the number of nucleotide changes and the proportion of shared bands. The estimation equation is based on the assumption that GC-content is 0.5. Computer simulations, however, show that this method gives a reasonably accurate estimate even when GC-content deviates from 0.5, as long as the number of nucleotide changes per site (nucleotide diversity) is small. As an example, the nucleotide diversity of the wild yam, Dioscorea tokoro, was estimated. The estimated nucleotide diversity is 0.0055, which is larger than estimations from nucleotide sequence data for Adh and Pgi. (+info)
(2/119) Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha.
A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha. (+info)
(3/119) Pulsatile influxes of H+, K+ and Ca2+ lag growth pulses of Lilium longiflorum pollen tubes.
Fluxes of H+, K+ and Ca2+ were measured with self-referencing ion-selective probes, near the plasma membrane of growing Lilium longiflorum pollen tubes. Measurements from three regions around short, steady-growing tubes showed small, steady influx of H+ over the distal 40 microm and a region of the tube within 50-100 microm of the grain with larger magnitude efflux from the grain. K+ fluxes were immeasurable in short tubes. Measurements of longer tubes that were growing in a pulsatile manner revealed a pulsatile influx of both H+ and K+ at the growing tip. The average fluxes at the cell surface during the peaks of the H+ and K+ pulses were 489+/-81 and 688+/-144 pmol cm-2 second-1, respectively. Growth was measured by tracking the pollen tips with a computer vision system that achieved a spatial resolution of approximately 1/10 pixel. The high spatial resolution enabled the detection of growth, and thus the changes in growth rates, with a temporal sampling rate of 1 frame/second. These data show that the H+ and K+ pulses have a phase lag of 103+/-9 and 100+/-11 degrees, respectively, with respect to the growth pulses. Calcium fluxes were also measured in growing tubes. During steady growth, the calcium influx was relatively steady. When pulsatile growth began, the basal Ca2+ influx decreased and a pulsatile component appeared, superimposed on the reduced basal Ca2+ flux. The peaks of the Ca2+ pulses at the cell surface averaged 38.4+/-2.5 pmol cm-2 second-1. Longer tubes had large pulsatile Ca2+ fluxes with smaller baseline fluxes. The Ca2+ influx pulses had a phase lag of 123+/-9 degrees with respect to the growth pulses. (+info)
(4/119) Nucleotide sequence analysis of the 3'-terminal region of two Korean isolates of lily symptomless Carlavirus and expression of the coat protein in E. coli.
The 3'-terminal regions of the genomic RNAs of two Korean isolates of the lily symptomless Carlavirus (LSV), LSV-Ko and LSV-KII, were cloned and their nucleotide sequences were determined. The nucleotide sequence analysis and protein analysis by the Western blot revealed that E. coli expressed a 32-kDa protein that is the viral coat protein (CP) for the LSV. The two Korean strains share 98.4% and 98.3% sequence identities at the nucleotide and amino acid levels, respectively. The CP gene of LSV-Ko showed 99.1% and 87.0% nucleotide sequence identities, and 99.0% and 96.6% amino acid sequence identities with those of the Netherlands and the Japanese LSV strains, respectively. A pairwise amino acid sequence comparison revealed a sequence similarity of 29.6% to 69.8% between LSV-Ko and other species of the carlavirus. The 16 kDa protein of LSV-Ko shares 17.6% to 42.7% amino acid similarity with those of 8 other the carlaviruses, and they are variable in the N-terminal region. The Cys repeated zinc finger nucleic acid binding domain was found in the 16 kDa protein for all of the LSV strains. Sequence comparisons of the 7 kDa protein of LSV in the strain level showed significant identities from 100.0% to 98.4%. LSV-Ko shares 21.9% to 42.2% amino acid similarity with those of 8 other carlaviruses, 4 members of the potexviruses, and a closterovirus. LSV is closely related to blueberry scorch virus (BISV) based upon the phylogenetic tree analyses of the three proteins, indicating LSV to be a quite distinct member of the genus Carlavirus. (+info)
(5/119) Effect of Yucca schidigera on ruminal fermentation and nutrient digestion in heifers.
In a replicated 3 x 3 Latin square experiment, six heifers (443 +/- 6.1 kg) fed a 61% barley grain:39% alfalfa silage diet (DM basis) were given intraruminal doses of powdered Yucca schidigera (YS). Doses of 0 (control), 20, or 60 g/d were given at 0800 daily. Ruminal content was sampled 0, 2, 4, and 6 h after dosing. Acidity, concentrations of reducing sugars, free amino acids, and peptides in the rumen were not affected (P > .05) by YS. Relative to control, ruminal ammonia concentration was reduced (P < .05) 2 h after YS dosing. Ruminal propionate concentration was increased (P < .05) by YS. Protozoal numbers in the rumen were lower (P < .05) with YS than without. Yucca did not affect (P > .05) rate or extent of in situ DM degradability. Fibrolytic, amylolytic, and proteolytic activities in ruminal contents were similar among treatments (P > .05). Dry matter intake, apparent digestibilities of DM, NDF, and CP, nitrogen balance, and microbial protein synthesis in the rumen were not affected (P > .05) by treatment. The effect of YS on ruminal ammonia concentration likely resulted from a decreased concentration of protozoa and, presumably, from ammonia binding by YS. The effect on ruminal propionate was probably a result of a selective inhibitory effect of YS on rumen microbial species. (+info)
(6/119) A lipid transfer-like protein is necessary for lily pollen tube adhesion to an in vitro stylar matrix.
Flowering plants possess specialized extracellular matrices in the female organs of the flower that support pollen tube growth and sperm cell transfer along the transmitting tract of the gynoecium. Transport of the pollen tube cell and the sperm cells involves a cell adhesion and migration event in species such as lily that possess a transmitting tract epidermis in the stigma, style, and ovary. A bioassay for adhesion was used to isolate from the lily stigma/stylar exudate the components that are responsible for in vivo pollen tube adhesion. At least two stylar components are necessary for adhesion: a large molecule and a small (9 kD) protein. In combination, the two molecules induced adhesion of pollen tubes to an artificial stylar matrix in vitro. The 9-kD protein was purified, and its corresponding cDNA was cloned. This molecule shares some similarity with plant lipid transfer proteins. Immunolocalization data support its role in facilitating adhesion of pollen tubes to the stylar transmitting tract epidermis. (+info)
(7/119) High genetic diversity, distant phylogenetic relationships and intraspecies recombination events among natural populations of Yam mosaic virus: a contribution to understanding potyvirus evolution.
To evaluate the genetic diversity and understand the evolution of Yam mosaic virus (YMV), a highly destructive pathogen of yam (Dioscorea sp.), sequencing was carried out of the C-terminal part of the replicase (NIb), the coat protein (CP) and the 3'-untranslated region (3'-UTR) of 27 YMV isolates collected from the three main cultivated species (Dioscorea alata, the complex Dioscorea cayenensis-Dioscorea rotundata and Dioscorea trifida). YMV showed the most variable CP relative to eight other potyviruses. This high variability was structured into nine distant molecular groups, as revealed by phylogenetic analyses and validated by assessment of the molecular evolutionary noise. No correlation was observed between the CP and 3'-UTR diversities and phylogenies. The most diversified and divergent groups included isolates from Africa. The remaining groups clustered in a single clade and a geographical distinction between isolates from the Caribbean, South America and Africa was observed. The role of the host in the selection of particular isolates was illustrated by the case of a divergent cultivar from Burkina Faso. Phylogenetic topological incongruence and complementary statistical tests highlighted the fact that recombination events, with single and multiple crossover sites, largely contributed to the evolution of YMV. We hypothesise an African origin of YMV from the yam complex D. cayenensis-D. rotundata, followed by independent transfers to D. alata and D. trifida during virus evolution. (+info)
(8/119) Structural characterisation of the native fetuin-binding protein Scilla campanulata agglutinin: a novel two-domain lectin.
The three-dimensional structure of a 244-residue, multivalent, fetuin-binding lectin, SCAfet, isolated from bluebell (Scilla campanulata) bulbs, has been solved at 3.3 A resolution by molecular replacement using the coordinates of the 119-residue, mannose-binding lectin, SCAman, also from bluebell bulbs. Unlike most monocot mannose-binding lectins, such as Galanthus nivalis agglutinin from snowdrop bulbs, which fold into a single domain, SCAfet contains two domains with approximately 55% sequence identity, joined by a linker peptide. Both domains are made up of a 12-stranded beta-prism II fold, with three putative carbohydrate-binding sites, one on each subdomain. SCAfet binds to the complex saccharides of various animal glycoproteins but not to simple sugars. (+info)