We estimated the dates of the monocot-dicot split and the origin of core eudicots using a large chloroplast (cp) genomic dataset. Sixty-one protein-coding genes common to the 12 completely sequenced cp genomes of land plants were concatenated and analyzed. Three reliable split events were used as calibration points and for cross references. Both the method based on the assumption of a constant rate and the Li-Tanimura unequal-rate method were used to estimate divergence times. The phylogenetic analyses indicated that nonsynonymous substitution rates of cp genomes are unequal among tracheophyte lineages. For this reason, the constant-rate method gave overestimates of the monocot-dicot divergence and the age of core eudicots, especially when fast-evolving monocots were included in the analysis. In contrast, the Li-Tanimura method gave estimates consistent with the known evolutionary sequence of seed plant lineages and with known fossil records. Combining estimates calibrated by two known fossil nodes and the Li-Tanimura method, we propose that monocots branched off from dicots 140-150 Myr ago (late Jurassic-early Cretaceous), at least 50 Myr younger than previous estimates based on the molecular clock hypothesis, and that the core eudicots diverged 100-115 Myr ago (Albian-Aptian of the Cretaceous). These estimates indicate that both the monocot-dicot divergence and the core eudicot's age are older than their respective fossil records.

This study investigates end‐users' image queries by comparing the features of the queries to those identified in previous studies by Enser and McGregor (1992), Jorgensen (1995), and Fidel (1997). Twenty‐nine college students majoring in art history were recruited. They were required to finish a term paper including at least 20 images. The participants' image queries were collected by pre‐ and postsearch questionnaires, and three human reviewers mapped these queries into the previously identified features. Enser and McGregor's categories of Unique and Nonunique, and Jorgensen's classes of Location, Literal Object, Art Historical Information, People, and People‐Related Attributes received high degrees of matching by three reviewers. This finding can be applied to add more details to Enser and McGregor's four categories (Unique, Nonunique, Unique with refiners, and Nonunique with refiners) and to regroup Jorgensen's 12 classes of image attributes.

BackgroundTo achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome.ResultsA technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO), that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei), a beta-glucosidase (from a cow rumen fungus), a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol.ConclusionsThis study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools.

Whether turns play an active or passive role in protein folding remains a controversial issue at this juncture. Here we use a photolabile cage strategy in combination with laser-flash photolysis and photoacoustic calorimetry to study the effects of different turns on the kinetics of ␤-hairpin refolding on a nanosecond time scale. This strategy opens up a temporal window to allow the observation of early kinetic events in the protein refolding process at ambient temperature and pH without interference from any denaturants. Our results provide direct evidence demonstrating that even a one-residue difference in the turn region can change the refolding kinetics of a peptide. This observation suggests an active role for turn formation in directing protein folding. R everse turns, with the ability of significantly restricting the conformational space available to the folding polypeptide chain and bringing distant parts of the chain into proximity, have long been suggested to play an important role in the initiation of protein folding (1, 2). In proteins, turns can play an important role in determining the structural stability as well as the details of the folding pathway (3-7). Although changes in the sequence of the turn or loop region can alter thermal stability and folding kinetics, there are mutations that remain tolerant of the change, depending on the role of the turn or the loop formation in the overall folding process. Several peptide models with a basic hairpin structure have been used to examine the relationship between turn sequence and turn conformation. They are either designed peptides (8-13) or short peptide segments adopted from protein sequences like ubiquitin (14-17) and the protein G B1 domain (18,19). From these studies, there is little doubt that turn residues determine the conformation and stability of ␤-hairpins. However, although it has been proven that turns can indeed affect the equilibrium states of peptides, there have been only a few studies directed toward the effect of turns on kinetic properties. The paucity of data in this matter is perhaps due to the limitation of techniques in the detection of very fast events involving turns. The formation of hairpins is a very rapid process. It occurs much faster than the dead time of standard stoppedflow mixing devices (1 Ϸ 2 ms) or that of the continuous-flow method (Ͼ45 s) (20-22), and therefore folding is lost in the burst phase (missing amplitude) of the kinetic traces recorded by using these methods. On the other hand, time-resolved infrared spectroscopy and fluorescence spectroscopy in response to laserinduced temperature jump, which can quickly perturb the temperature of the system and hence the equilibrium between folded and unfolded states, have been successfully applied to the study of the rapid refolding of ␤-hairpins (23-25). Direct observation of the folding of peptides with different turns, however, remains elusive.Here, we present a new method to initiate and interrogate rapid peptide refolding in real time. The strategy is based on...