A procedure is described which allows the rapid permeabilization of yeast cells, Schizosaccharomyces pombe and Saccharomyces cerevisiae, for quantitative in situ assays of beta-galactosidase activity. Yeast cells are permeabilized by incubation in buffer containing 0.2% of the detergent sodium lauroyl sarcosinate without any need for washing or vortexing. This procedure is equally applicable to fresh and frozen samples. It is compared to earlier reported methods and found to be superior by being more accurate and less time-consuming.

A procedure is described which allows the rapid permeabilization of yeast cells, Schizosaccharomyces pombe and Saccharomyces cerevisiae, for quantitative in situ assays of beta-galactosidase activity. Yeast cells are permeabilized by incubation in buffer containing 0.2% of the detergent sodium lauroyl sarcosinate without any need for washing or vortexing. This procedure is equally applicable to fresh and frozen samples. It is compared to earlier reported methods and found to be superior by being more accurate and less time-consuming.

BackgroundHEAT and ARM repeats occur in a large number of eukaryotic proteins. As these repeats are often highly diverged, the prediction of HEAT or ARM domains can be challenging. Except for the most clear-cut cases, identification at the individual repeat level is indispensable, in particular for determining domain boundaries. However, methods using single sequence queries do not have the sensitivity required to deal with more divergent repeats and, when applied to proteins with known structures, in some cases failed to detect a single repeat.Methodology and Principal FindingsTesting algorithms which use multiple sequence alignments as queries, we found two of them, HHpred and COACH, to detect HEAT and ARM repeats with greatly enhanced sensitivity. Calibration against experimentally determined structures suggests the use of three score classes with increasing confidence in the prediction, and prediction thresholds for each method. When we applied a new protocol using both HHpred and COACH to these structures, it detected 82% of HEAT repeats and 90% of ARM repeats, with the minimum for a given protein of 57% for HEAT repeats and 60% for ARM repeats. Application to bona fide HEAT and ARM proteins or domains indicated that similar numbers can be expected for the full complement of HEAT/ARM proteins. A systematic screen of the Protein Data Bank for false positive hits revealed their number to be low, in particular for ARM repeats. Double false positive hits for a given protein were rare for HEAT and not at all observed for ARM repeats. In combination with fold prediction and consistency checking (multiple sequence alignments, secondary structure prediction, and position analysis), repeat prediction with the new HHpred/COACH protocol dramatically improves prediction in the twilight zone of fold prediction methods, as well as the delineation of HEAT/ARM domain boundaries.SignificanceA protocol is presented for the identification of individual HEAT or ARM repeats which is straightforward to implement. It provides high sensitivity at a low false positive rate and will therefore greatly enhance the accuracy of predictions of HEAT and ARM domains.

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Elementary differences in the regulation of cellular physiology separate prokaryotic and eukaryotic organisms. As will be emphasized throughout this paper, fundamental disparities are already apparent at the unicellular level of eukaryotic organization. Regarding their universality in eukaryotes almost inevitably leads to the suggestion that some of these early acquisitions of the developing eukaryotic cell were of prominent evolutionary significance for the later emergence of eukaryotic complexity and diversity. The superior influence of interactions between cellular compartments on cellular evolution has been pointed out previously.' A more pervasive statement is the principal notion of this paper that those interactions culminating in mechanisms for the integration of today's semiautonomous organelles were even indispensable for the evolution of the eukaryotic cell out of an endocytobiotic organization. Hence, the endosymbiotic theory may not only account for the origin of mitochondria and plastids but equally for a substantial part of the developmental potency arising in conjunction with the first eukaryotic organisms. Two essential features of cell regulation ubiquitous in eukaryotes are suggested to have evolved as a consequence of mutual interactions between partners in a cellular symbiotic consortium developing towards the eukaryotic cell: endogenous rhythms and the calcium system of intracellular signaling. These two characters unique to eukaryotic cells may be intertwined, the calcium system being an integral part of the cellular clock mechanism.The presence of a nucleus as the defining characteristic of eukaryotic cells is at the same time the most easy means to distinguish them from their prokaryotic counterparts. Equally eyecatching are their usually much larger size and the complex structure of the cytoplasm harboring various organelles. Differences in cellular morphology like these have been dealt with extensively; likewise those in essential metabolic pathways and the biochemistry of subcellular components. More recently, differences in molecular biology between the two basic subdivisions of life have constituted an 476