Coinjection of Xenopus Oocytes With cDNA-Produced and Native mRNAs: A Molecular Biological Approach to the Tissue-Specific Processing of Human Cholinesterases

This chapter discusses a combination of molecular–biological, classical–biochemical, and immunocytohistochemical techniques for monitoring the biogenesis of synthetic butyrylcholinesterase (BuChE) in Xenopus oocytes. Synthetic BuChE mRNA encodes the complete amino-acid sequence of human serum BuChE—a putative signal peptide containing a yet-unaccounted-for upstream open-reading frame, a 3' untranslated sequence, and a poly-A tail. In the experiment described in the chapter, synthetic RNA was injected alone and in conjunction with tissue-extracted poly-A(+) RNA from fetal human brain, muscle, and liver. The following observations were made in this chapter: (1) Synthetic BuChE mRNA alone is sufficient to direct the synthesis in oocytes of an enzymatic activity characteristic of normal human serum BuChE and clearly distinct from acetylcholinesterase (AChE), (2) synthetic BuChE undergoes limited subunit assembly in the oocytes to generate functional dimeric molecules, (3) synthetic BuChE associates with the oocyte membrane where it can be seen to interact primarily with the oocyte's outermost extracellular matrix and not with the plasmalemma itself, (4) BuChE aggregates are preferentially but not exclusively deposited at the animal pole of the oocyte, and (5) co-injection with brain or muscle RNA induces the appearance of complex oligomeric forms of BuChE in a tissue-specific manner and with the enhanced aggregation of synthetic BuChE in a manner consistent with ChE organization in the native tissues. The structural heterogeneity characteristic of AChE is defined by sequence variations at the COOH terminal of the molecule. Thus, distinct cDNA clones representing both globular and asymmetric forms have been isolated, diverging only in the 3' terminal region of the coding sequence.