Michael A. Innis

Characterization of β-amyloid peptide precursor processing by the yeast Yap3 and Mkc7 proteases

Two proteases, denoted beta- and gamma-secretase, process the beta-amyloid peptide precursor (APP) to yield the Abeta peptides involved in Alzheimers disease. A third protein, alpha-secretase, cleaves APP near the middle of the Abeta sequence and thus prevents Abeta formation. These enzymes have defied identification. Because of its similarity to the systems of mammalian cells the yeast secretory system has provided important clues for finding mammalian processing enzymes. When expressed in Saccharomyces cerevisiae APP is processed by enzymes that possess the specificity of the alpha-secretases of multicellular organisms. APP processing by alpha-secretases occurred in sec1 and sec7 mutants, in which transport to the cell surface or to the vacuole is blocked, but not in sec17 or sec18 mutants, in which transport from the endoplasmic reticulum to the Golgi is blocked. Neutralization of the vacuole by NH4Cl did not block alpha-secretase action. The time course of processing of a pro-alpha-factor leader-APP chimera showed that processing by Kex2 protease, a Golgi protease that removes the leader, preceded processing by alpha-secretase. Deletions of the genes encoding the GPI-linked aspartyl proteases Yap3 and Mkc7 decreased alpha-secretase activity by 56 and 29%, respectively; whereas, the double deletion decreased the activity by 86%. An altered form of APP-695, in which glutamine replaced Lys-612 at the cleavage site, is cleaved by Yap3 at 5% the rate of the wild-type APP. Mkc7 protease cleaved APP (K612Q) at about 20% the rate of wild-type APP. The simplest interpretation of these results is that Yap3 and Mkc7 proteases are alpha-secretases which act on APP in the late Golgi. They suggest that GPI-linked aspartyl proteases should be investigated as candidate secretases in mammalian tissues.

Optimization of PCR: Conversations between Michael and David

Publisher Summary This chapter, written in a dialogue format, discusses the optimization of polymerase chain reaction (PCR). The authors have published the same chapter in PCR Protocols a few years back. Though the basic PCR technique remains the same, numerous and diverse innovations have occurred. For example, availability of multiple thermostable polymerases and proofreading polymerases; multiple hot start strategies; long-range PCR; real-time quantitative PCR; PCR-derived cDNA libraries, PCR strategies for generating normalized libraries, subtractive libraries, and representational difference analysis; buffer optimization, use of cosolvents, and mixtures of enzymes; and optimized primer design tools. Thus, the purpose of this chapter is to provide additional information concerning optimization of PCR to that which was published in PCR Protocols. Recorded as a conversation between the two authors of this book, this chapter explores the long range PCR, magnesium ion concentration, high-fidelity PCR, fluorescent dye labeling, and prime design factors.