The nucleotide sequence of Bacillus phage phi 29 genes 14 (g14) and 15 (g15) have been determined and shown to encode proteins with molecular weights of 15,014 and 28,022, respectively. The g14 open reading frame (ORF) was confirmed by sequencing a sus14(1241) mutant. Gene product 15 (gp15) has considerable homology with Salmonella phage P22 lysozyme and lesser homology with Escherichia coli phage T4 lysozyme. Putative translation signals are identified. In addition, the role of a previously described promoter, B2, is discussed.

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The mam immunogemc region (MIR) of the acetylcholine receptor (AChR) is the target for the majority of high-affinity autoantibodtes produced in myasthenia gravis patients. Some monoclonal antibodies (mAbs) to the MIR bmd specifically, but with low affinity, to synthetic AChR a subunit peptides with the sequence ~67-76. Studies of synthetic peptides suggest that ammo acids a68 and a71 may be especially important to the antigenic structure of the MIR. We have studted the contribution of amino acids a68 and a71 to the anttgemcity of the MIR on intact AChR by replacing a68 (N) and a71 (D) of Torpedo AChR IX with a68 (D) and x71 (K) by site-directed mutagenests, expressmg the mutated transcripts in Xenopus oocytes along with wild-type Torpedo 8, 7 and b subumts. and analyzing the expressed AChR for the binding of mAbs to the MIR. These mutations of the MIR greatly dtmimshed bmding of mAbs to the MIR. Thus, both ~68 and a71 are crtttcal to the antigemcity of the MIR in intact AChRs.

Abstract. To study pathways by which acetylcholine receptor (AChR) subunits might assemble, Torpedo a subunits were expressed in Xenopus oocytes alone or in combination with ~, 3', or t5 subunits. The maturation of the conformation of the main immunogenic region (MIR) on a subunits was measured by binding of mAbs and the maturation of the conformation of the AChR binding site on a subunits was measured by binding of a-bungarotoxin (aBgt) and cholinergic ligands. The size of subunits and subunit complexes was assayed by sedimentation on sucrose gradients. It is generally accepted that native AChRs have the subunit composition a2/~3'6. Torpedo a subunits expressed alone resulted in an amorphous range of complexes with little affinity for aBgt or mAbs to the MIR, rather than in a unique 5S monomeric assembly intermediate species. A previously recognized temperature-dependent failure in a subunit maturation may cause instability of the monomeric assembly intermediate and accumulation of aggregated denatured a subunits. Coexpression of a with/~ subunits also resulted in an amorphous range of complexes. However, coexpression of a subunits with 3' or t5 subunits resulted in the efficient formation of 6.5S oe3' or a~ complexes with high affinity for mAbs to the MIR, aBgt, and small cholinergic ligands. These a3' and at5 subunit pairs may represent normal assembly intermediates in which Torpedo a is stabilized and matured in conformation. Coexpression of a, 3', and t5 efficiently formed 8.8S complexes, whereas complexes containing a/~ and 3' or a/3 and t5 subunits are formed less efficiently. Assembly of/3 subunits with complexes containing a3' and 5 subunits may normally be a ratelimiting step in assembly of AChRs.