White and green screening with circular polymerase extension cloning for easy and reliable cloning

Cloning is an essential prerequisite to test protein design and engineering ideas. However, it is often time consuming, unreliable, and therefore frustrating. Here, we present a streamlined cloning strategy that incorporates a powerful white and green screening protocol to identify colonies with inserts. We use circular polymerase extension cloning, which is both ligation and sequence independent. Furthermore, our entire procedure requires only three quick steps and one enzyme making it easy to use, inexpensive, and tractable. We anticipate that this method will be particularly useful for protein engineers who frequently subclone or make focused deletion, insertion, or substitution libraries.     

Ca2+ channels in rat central and peripheral neurons: high-threshold current resistant to dihydropyridine blockers and omega-conotoxin.

Block of Ca2+ channel current by dihydropyridines and by omega-conotoxin (omega-CgTx) was studied in a variety of freshly dissociated rat neurons. In most neurons, including those from dorsal root ganglia, sympathetic ganglia, spinal cord, cerebral cortex, and hippocampus, nitrendipine and omega-CgTx each blocked a fraction of the high-threshold current, but a substantial fraction of current remained even when the two blockers were applied together at saturating concentrations. An extreme case was cerebellar Purkinje neurons, in which very little current was blocked by either nitrendipine or omega-CgTx. These results demonstrate the existence in mammalian neurons of high-threshold channels that are resistant to both omega-CgTx and dihydropyridine blockers. Such channels might underlie instances of synaptic transmission and other processes that depend on Ca2+ entry but are not sensitive to these blockers.     

Rarity,geography, and plant exposure to global change in the California Floristic Province

Aim

Rarity and geographic aspects of species distributions mediate their vulnerability to global change. We explore the relationships between species rarity and geography and their exposure to climate and land use change in a biodiversity hotspot.

Location

California, USA.

Taxa

One hundred and six terrestrial plants.

Methods

We estimated four rarity traits: range size, niche breadth, number of habitat patches, and patch isolation; and three geographic traits: mean elevation, topographic heterogeneity, and distance to coast. We used species distribution models to measure species exposure—predicted change in continuous habitat suitability within currently occupied habitat—under climate and land use change scenarios. Using regression models, decision-tree models and variance partitioning, we assessed the relationships between species rarity, geography, and exposure to climate and land use change.

Results

Rarity, geography and greenhouse gas emissions scenario explained >35% of variance in climate change exposure and >61% for land use change exposure. While rarity traits (range size and number of habitat patches) were most important for explaining species exposure to climate change, geographic traits (elevation and topographic heterogeneity) were more strongly associated with species' exposure to land use change.

Main conclusions

Species with restricted range sizes and low topographic heterogeneity across their distributions were predicted to be the most exposed to climate change, while species at low elevations were the most exposed to habitat loss via land use change. However, even some broadly distributed species were projected to lose >70% of their currently suitable habitat due to climate and land use change if they are in geographically vulnerable areas, emphasizing the need to consider both species rarity traits and geography in vulnerability assessments.