Approaches to library screening

Fuelled by the drive to complete the Human Genome Project, many laboratories have developed new methods of screening clone libraries. From PCR-based strategies to pooling schemes and increased automation, the tedious task of library screening has become less labour-intensive and more cost-efficient. Currently, two main screening methods dominate: hybridization and polymerase chain reaction (PCR). In the following article, we present a brief overview of hybridization and PCR-based screening of yeast and bacterial libraries. Multi-faceted approaches combining different techniques, as well as less frequently employed methods such as fingerprinting are also described.     

Bcl-x(L) complements Saccharomyces cerevisiae genes that facilitate the switch from glycolytic to oxidative metabolism

All eukaryotic organisms have mechanisms to adapt to changing metabolic conditions. The mammalian cell survival gene Bcl-x(L) enables cells to adapt to changes in cellular metabolism. To identify genes whose function can be substituted by Bcl-x(L) in a unicellular eukaryote, a genetic screen was performed using the yeast Saccharomyces cerevisiae. S. cerevisiae grows by anaerobic glycolysis when glucose is available, switching to oxidative phosphorylation when carbohydrate in the media becomes limiting (diauxic shift). Given that Bcl-x(L) appears to facilitate the switch from glycolytic to oxidative metabolism in mammalian cells, a library of yeast mutants was tested for the ability to efficiently undergo diauxic shift in the presence and absence of Bcl-x(L). Several mutants were identified that have a defect in growth when switched from a fermentable to a nonfermentable carbon source that is corrected by the expression of Bcl-x(L). These genes include the mitochondrial chaperonin TCM62, as well as previously uncharacterized genes. One of these uncharacterized genes, SVF1, promotes cell survival in mammalian cells in response to multiple apoptotic stimuli. The finding that TCM62 and the analogous human prohibitin gene also inhibit mammalian cell death following growth factor withdrawal implicates mitochondrial chaperones as regulators of apoptosis. Further characterization of the genes identified in this screen may enhance our understanding of Bcl-x(L) function in mammalian cells, and of cell survival pathways in general.     

A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.     

Chlorobium Tepidum: Insights into the Structure,Physiology, and Metabolism of a Green Sulfur Bacterium Derived from the Complete Genome Sequence

Green sulfur bacteria are obligate, anaerobic photolithoautotrophs that synthesize unique bacteriochlorophylls (BChls) and a unique light-harvesting antenna structure, the chlorosome. One organism, Chlorobium tepidum, has emerged as a model for this group of bacteria primarily due to its relative ease of cultivation and natural transformability. This review focuses on insights into the physiology and biochemistry of the green sulfur bacteria that have been derived from the recently completed analysis of the 2.15-Mb genome of Chl. tepidum. About 40 mutants of Chl. tepidum have been generated within the last 3 years, most of which have been made based on analyses of the genome. This has allowed a nearly complete elucidation of the biosynthetic pathways for the carotenoids and BChls in Chl. tepidum, which include several novel enzymes specific for BChl c biosynthesis. Facilitating these analyses, both BChl c and carotenoid biosynthesis can be completely eliminated in Chl. tepidum. Based particularly on analyses of mutants lacking chlorosome proteins and BChl c, progress has also been made in understanding the structure and biogenesis of chlorosomes. In silico analyses of the presence and absence of genes encoding components involved in electron transfer reactions and carbon assimilation have additionally revealed some of the potential physiological capabilities, limitations, and peculiarities of Chl. tepidum. Surprisingly, some structural components and biosynthetic pathways associated with photosynthesis and energy metabolism in Chl. tepidum are more similar to those in cyanobacteria and plants than to those in other groups of photosynthetic bacteria.     

Alkaline environmental pH has no effect on ammonia excretion in the mudskipper Periophthalmodon schlosseri but inhibits ammonia excretion in the related species Boleophthalmus boddaerti

Experiments were performed to evaluate the effects of alkaline environmental pH on urea and ammonia excretion rates and on tissue urea, ammonia, and free amino acid concentrations in two mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti. Periophthalomodon schlosseri is known to be capable of actively excreting ammonia. The rate of ammonia excretion in B. boddaerti exposed to 50% seawater (brackish water, BW) at pH 9 decreased significantly during the first 2 d of exposure when compared with that of specimens exposed to pH 7 or 8. This suggested that B. boddaerti was dependent on NH(3) diffusion for ammonia excretion, as in most fishes. It was incapable of detoxifying the accumulating endogenous ammonia to urea but could store and tolerate high concentrations of ammonia in the muscle, liver, and plasma. It did not undergo reductions in proteolysis and/or amino acid catabolism in alkaline water, probably because the buildup of endogenous ammonia was essential for the recovery of the normal rate of ammonia excretion by the third day of exposure to a pH 9 medium. Unlike B. boddaerti, P. schlosseri did not accumulate ammonia in the body at an alkaline pH (i.e., pH 9) because it was capable of actively excreting ammonia. Periophthalmodon schlosseri did not undergo partial amino acid catabolism (no accumulation of alanine) either, although there might be a slight reduction in amino acid catabolism in general. The significant decrease in blood pCO(2) in B. boddaerti at pH 9 might lead to respiratory alkalosis in the blood. In contrast, P. schlosseri was able to maintain its blood pH in BW at pH 9 despite a decrease in pCO(2) in the blood. With 8 mM NH(4)Cl in BW at pH 7, both mudskippers could actively excrete ammonia, although not to the same extent. Only P. schlosseri could sustain ammonia excretion against 8 mM NH(4)Cl in BW at pH 8. In BW containing 8 mM NH(4)Cl at pH 9, both mudskippers died within a short period of time. Boleophthalmus boddaerti consistently died faster than did P. schlosseri. This indicates that the body surfaces of these mudskippers were permeable to NH(3), but the skin of P. schlosseri might be less permeable to NH(3) than that of B. boddaerti. Both mudskippers excreted acid (H(+)) to alter the pH of the alkaline external medium. Such a capability, together with modifications in gill morphology and morphometry as in P. schlosseri, might be essential to the development of an effective mechanism for the active excretion of NH+4.     

CCK-58 is the only detectable endocrine form of cholecystokinin in rat

CCK-58 differs from CCK-8 in patterns of expression of pancreatic secretion of fluid and amylase and gallbladder contraction. These differences have physiological relevance only if CCK-58 release is stimulated by nutrients entering the intestine and if CCK-58 circulates in sizeable amounts. In this study, we report that when radiolabeled CCK-58 is added to rat blood and plasma is formed, there is extensive loss and degradation of the radioactive peptide. Therefore, a new method was developed to minimize loss and degradation of this label. This method recovered >85% of the label with no detectable degradation. Furthermore, the optimized method recovered all unlabeled exogenous cholecystokinin molecular forms in >80% yields. Blood from fasted rats and rats in which cholecystokinin release was stimulated by the trypsin inhibitor camostat contained only CCK-58 (3.5 +/- 0.5 and 17 +/- 1.5 fmol/ml, respectively). Because CCK-58 predominates in the blood, this molecular form should be used in studies on the physiology and pathophysiology of cholecystokinin.     

The sleeper Bostrichthys sinensis (family Eleotridae) stores glutamine and reduces ammonia production during aerial exposure

Bostrichthys sinensis inhabits brackish water, living in the crevices of the river mouths of Shang Xi and Guangdong, China. In its natural habitat, it may encounter aerial exposure frequently during low tides, and it usually remains quiescent in the absence of water. Upon aerial exposure in the laboratory, the ammonia excretion rate decreased to one-fourth that of the submerged control. Although all the enzymes of the ornithine-urea cycle were detected in the liver of this fish, the activity of hepatic carbamoyl phosphate synthetase was too low for the cycle to be functioning. Indeed, ammonia accumulated in the tissues and was not converted to urea. Results indicate that ammonia produced through amino acid catabolism was detoxified to glutamine during the first 24 h of aerial exposure. The excess amount of glutamine stored in the muscle during this period couldaccount approximately for the reduction in ammonia equivalent excreted. There was indeed a significant increase in the activity of glutamine synthetase from the liver of specimens exposed to terrestrial conditions. In contrast to the production of alanine, formation of glutamine is energetically expensive. Since B. sinensis remained relatively inactive on land, the reduction in energy demand for muscular activity might provide it with the opportunity to exploit glutamine formation as a means to detoxify ammonia. After 72 h of aerial exposure, B. sinensis reduced internal ammonia production, possibly through reductions in proteolysis and amino acid catabolism, to avoid excessive accumulation of ammonia.