Abstract. The vacuolar protease B of Saccharomyces cerevisiae is a subtilisin-like protease encoded by the PRB1 gene. Antibodies raised against a synthetic peptide and an Escherichia coli-derived PRB1 open reading frame (ORF) protein cross-react with authentic protease B from yeast. By using these antibodies, the posttranslational biosynthetic pathway of protease B has been elucidated. Preproprotease B is a 76-kD unglycosylated precursor that enters the endoplasmic reticulum (ER), where it receives one asparaginelinked (Asn-linked) and an undetermined number of non-Asn-linked carbohydrate side chains. The large glycosylated intermediate is proteolytically processed to a 39-kD form before exiting the ER. In the Golgi complex, the 39-kD form becomes 40 kD, due to elaboration of the Asn-linked side chain. The carboxyterminal end of the 40-kD proprotease B undergoes protease A-mediated processing to a 37-kD intermediate, which in turn is quickly processed to 31-kD mature protease B. The ultimate processing step removes a peptide containing the Asn-linked chain; mature PrB has only non-Asn-linked carbohydrate.T nF. lysosome-like vacuole of the yeast Saccharomyces cerevisiae contains several hydrolytic activities, including protease A (PrA),~ protease B (PrB), carboxypeptidase Y (CpY), a 600-kD aminopeptidase, a repressible alkaline phosphatase, and at least one RNase (39,65). In all cases studied to date, these hydrolases are synthesized as inactive glycoprotein precursors (reviewed in reference 28) that require the PEP4 gene product, protease A, for activation (2, 28, 66). Vacuolar hydrolases may be thought of as internally secreted proteins, as they traverse the ER and Golgi apparatus en route to the vacuole. Mutations that block transport from the ER or Golgi apparatus affect internally and externally secreted proteins equally (31, 57).Some preliminary work on the structure of PrB and its precursors has been reported. In kinetic experiments using a 20-min pulse of [35S]methionine, Mechler et al. (43) identiffed a 42-kD precursor of PrB that is subsequently processed to the mature form. The relative molecular mass of the precursor was 3 kD smaller in tunicamycin-treated cells, but the relative molecular mass of the mature protease was unaffected (43). This kinetic precursor had the same relative molecular mass as the zymogen form of protease B observed to accumulate in a pep4 mutant (44). Mature protease B has been reported to be a glycoprotein that contains "no more than 0.5%" carbohydrate (61). A different group reported C. M. Moehle's present address is Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, MD 20892. PrB to be a 33-kD glycoprotein with 8-9% neutral sugars and 1.5 % amino sugars (32). The neutral/amino sugar ratio was suggestive of an Asn-linked carbohydrate moiety. Abbreviations used in this paperWe recently reported the cloning and sequencing of PRB1 and also the purification and amino-terminal sequence of PrB (46,47). Unexpectedly, analysis of the...

pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.

The cheilanthoid ferns have long resisted efforts to circumscribe well‐defined, phylogenetically natural generic and infrageneric groups, presumably because of homoplastic morphologies associated with their xeric habitats. This cladistic analysis of phylogenetically informative chloroplast DNA restriction site data from 14 enzymes and seven taxa in the cheilanthoid genus Pellaea provides new insights into the phylogenetic relationships of the P. glabella complex. It also assesses the congruence of results based on restriction site data at inter‐ and intraspecific levels in these sexually and apogamously reproducing ferns with those of earlier morphological and isozyme analyses of the same group. Wagner parsimony yielded a single most parsimonious tree of 187 steps and 11% homoplasy, based on a data matrix of 166 restriction sites of which 66 were phylogenetically informative. Phylogenetic analysis based on user‐defined stepmatrix character‐state weighting of site gains over losses produced an identical single most parsimonious tree. Dollo parsimony yielded two most parsimonious trees, one of which was topologically identical to the Wagner tree. Specific and infraspecific relationships in the P. glabella complex determined by the completely independent restriction site and isozyme data sets are identical. This lends confidence to the ongoing use of restriction site data in a broader study of Pellaea and other cheilanthoid taxa and to the present conclusions that P. atropurpurea is sister to the P. glabella complex, whereas P. breweri, previously considered the closest relative of this complex, is actually more distantly related to it than are the other taxa in this study.

PurposeThe purpose of this paper is to identify best practices from high‐technology industries that face many of the same challenges around uncertainty, complexity, and risk that are faced by the pharmaceutical industry.Design/methodology/approachThis research has conducted an extensive review of risk management literature and research conducted in high‐technology industries to collect some of the key best practices for high‐risk research projects.FindingsA literature review of recent risk management publications from three high‐technology industries yielded 13 best practices in project risk management that could potentially be applied to pharmaceutical R&D projects to improve managing risks and uncertainties of managing projects. By reviewing these lessons learned from industries that share many of the challenges of the pharmaceutical R&D projects, it is suggested that the implementation of risk management in the context of drug development projects will require adaptation to the specific needs and challenges of those projects.Research limitations/implicationsImplementing a risk management process is very challenging for pharmaceutical R&D projects, as there are high degrees of complexity, uncertainty, and large amounts of resources at stake. Many of the techniques could be applied to all stages of drug development, but some are clearly most applicable to particular stages. Some will work for small, medium and large pharmaceuticals, though the way they are implemented should be modified to meet the needs and resources of the particular company.Practical implicationsThis study will serve as a basis for exploration and discussion that will result in controlled application and experimentation with these approaches, and this in turn could lead to real improvements in the use of risk management in pharmaceutical companies.Originality/valueAn exploration of the methods they have employed to address risk in R&D projects, as well as the outcomes of the application of those methods, should reveal tools, techniques, processes, training, and approaches that can be effectively applied to pharmaceutical development projects, and support the value of spending resources to employ risk management practices.

The cheilanthoid ferns have long resisted efforts to circumscribe well-defined, phylogenetically natural generic and infrageneric groups, presumably because of homoplastic morphologies associated with their xeric habitats. This cladistic analysis of phylogenetically informative chloroplast DNA restriction site data from 14 enzymes and seven taxa in the cheilanthoid genus Pellaea provides new insights into the phylogenetic relationships of the P. glabella complex. It also assesses the congruence of results based on restriction site data at inter-and intraspecific levels in these sexually and apogamously reproducing ferns with those of earlier morphological and isozyme analyses of the same group. Wagner parsimony yielded a single most parsimonious tree of 187 steps and II % homoplasy, based on a data matrix of 166 restriction sites of which 66 were phylogenetically informative. Phylogenetic analysis based on user-defined stepmatrix character-state weighting of site gains over losses produced an identical single most parsimonious tree. Dollo parsimony yielded two most parsimonious trees, one of which was topologically identical to the Wagner tree. Specific and infraspecific relationships in the P. glabella complex determined by the completely independent restriction site and isozyme data sets are identical. This lends confidence to the ongoing use of restriction site data in a broader study of Pellaea and other cheilanthoid taxa and to the present conclusions that P. atropurpurea is sister to the P. glabella complex, whereas P. breweri, previously considered the closest relative of this complex, is actually more distantly related to it than are the other taxa in this study.