Methods in Enzymology Vol.326 Applications of Chimeric Genes and Hybrid Proteins Part A

This chapter provides an introduction to gene fusion. The development of gene fusion approaches came from studies on the lac operon of E. coli. The first fusions of lac were obtained unwittingly as revertants of strong polar mutations in the lacZ gene. Selection for restoration of the activity of the downstream lacY gene yielded many deletions that removed the polar mutation site, the promoter of lac, and fused the lacY gene to an upstream promoter of an unknown neighboring gene. Another important step in the evolution of uses of gene fusions came with the concept of signal sequence traps. The first development of this concept came out of the recognition that the bacterial enzyme alkaline phosphatase is active when it is exported to the periplasm but inactive when it is retained in the cytoplasm. Thus, alkaline phosphatase without its signal sequence provides an assay for export signals via gene fusion approaches—that is, alkaline phosphatase will only be active if one attaches a region of DNA that encodes a signal sequence, thus reallowing its export. Extending beyond the differentiation of exported vs cytosolic proteins, gene fusion techniques can be evolved to determine the subcellular localization of proteins more generally. The use of green fluorescent protein (GFP) fusions enhances this ability. In addition, reporter proteins that sense the specific features of organelle environment may provide a tool for detecting location and genetically manipulating signals for the localization process.

• Series: Methods in Enzymology (Book 326)
• Hardcover: 617 pages
• Publisher: Academic Press;
  
• Language: English
• ISBN-10: 0121822273
• ISBN-13: 978-0121822279
• Product Dimensions: 9.3 x 6.3 x 1.3 inches

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