The 1H, 13C and 15N NMR assignments of the backbone and side-chain resonances of rat S100 beta were made at pH 6.5 and 37 degrees C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE correlations, together with amide exchange rate and 1H alpha, 13C alpha and 13C beta chemical shift data, provided extensive secondary structural information. Thus, the secondary structure of S100 beta was determined to comprise four helices (Leu3-Ser18, helix I; Lys29-Leu40, helix II; Gln50-Glu62, helix III; and Phe70-Ala83, helix IV), four loops (Gly19-His25, loop I; Ser41-Glu49, loop II; Asp63-Gly66, loop III; and Cys84-Glu91, loop IV) and two beta-strands (Lys26-Lys28, beta-strand I and Glu67-Asp69, beta-strand II). The beta-strands were found to align in an antiparallel manner to form a very small beta-sheet. This secondary structure is consistent with predictions that S100 beta contains two 'helix-loop-helix' Ca(2+)-binding motifs known as EF-hands. The alignment of the beta-sheet, which brings the two EF-hand domains of S100 beta into close proximity, is similar to that of several other Ca(2+)-ion-binding proteins.

Understanding of vertebrate neuromuscular junction (NMJ) development has been advanced by experimentation with cultures of dissociated embryonic nerve and skeletal muscle cells, particularly those derived from Xenopus and chick embryos. We previously developed a rodent (rat) nerve‐muscle coculture system that is characterized by extensive induction of acetylcholine receptor (AChR) aggregation at sites of axonal contact with myotubes (Dutton et al., 1995). In this article, we report modifications of this culture system and examples of its application to the study of NMJ development: (1) We describe improved methods for the enrichment of myoblasts to give higher yields of myotubes with equal or greater purity. (2) We demonstrate lipophilic dye labeling of axons in cocultures by injection of dye into neuron aggregates and show the feasibility of studying the growth of living axons on myotubes during synapse formation. (3) We describe the preparation of a better‐defined coculture system containing myotubes with purified rat motoneurons and characterize the system with respect to axon‐induced AChR aggregation. (4) We demonstrate dependence of the pattern of axon‐induced AChR aggregation on muscle cell species, by the use of chick‐rat chimeric co‐cultures. (5) We provide evidence for the role of alternatively‐spliced agrin isoforms in synapse formation by using single cell RT‐PCR with neurons collected from co‐cultures after observation of axon‐induced AChR aggregation. Microsc. Res. Tech. 49:26–37, 2000. Published 2000 Wiley‐Liss, Inc.

Escherichia coli Acya-283 is a 75-bp in-frame deletion overlapping the 5' end of Acya-854; Acya-201 is a 41-bp frameshift deletion overlapping the 3' end of Acya-854. Sequence repeats were found at the boundaries of Acya-283 and Acya-201, suggesting a mechanism for deletion formation. Recombinant DNA procedures were used to construct a strain in which the total cya structural gene in the chromosome was replaced by the kanamycin resistance gene.Numerous studies on the physiology of Escherichia coli have taken advantage of strains that are incapable of accumulating cyclic AMP (cAMP). Brickman et al. (3) described three spontaneous deletions in the adenylate cyclase gene (cya). One of these mutations (Acya-854) has become the most widely used Acya background. Recently, Glaser et al. (7) have characterized the Acya-854 mutation as a 200-bp frameshift deletion. This study was designed to characterize the remaining two cya deletions described by Brickman et al. and to design a cya total deletion strain.(A preliminary report of some of these findings has been presented as an abstract [18].)Characterization of adenylate cyclase deletions. Adenylate cyclase activity in toluene-treated cells (11) of the parent strain CA8000 (3) was compared with those of cells of the Acya-201 (strain CA8300) and Acya-283 (strain CA8303) deletion strains. Although there was easily detectable activity in the wild-type cells, there was no detectable activity in either of the deletion strains. The lack of adenylate cyclase activity in the deletion strains might be due either to the absence of the protein, resulting from a premature translation termination or instability of the protein product, or, alternatively, to the accumulation of an inactive protein. The analyses described below were performed to evaluate these possibilities.Polymerase chain reaction (PCR) analysis of Acya-283 compared with that of the wild-type DNA indicates clearly that the deletion is located somewhere between bases 901 and 1320 (Fig. 1A, lanes 4 and 5) and that the size of the deletion is approximately 80 bp. Similarly, PCR analysis of Acya-201 compared with that of the wild-type DNA shows that the deletion is located in the same fragment (Fig. 1B, lanes 4 and 5) and that the deletion size is approximately 50 bp.A precise analysis of the locations of Acya-283 and Acya-201 was made by sequencing asymmetric PCR products (Fig. 2). Acya-283 was found to span the region from bases 1002 to 1076 (numbering as described in reference 1). This deletion is * Corresponding author. t Present address: Department of Neurology, School of Medicine, University of Maryland at Baltimore, Baltimore, MD 21201. therefore a 75-bp in-frame deletion and might be expected to produce a truncated gene product. Acya-201 was found to cover the region from bases 1219 to 1259; this corresponds to a 41-bp deletion. Since this deletion is out of frame, it is likely that premature translation termination would result. Inspection of the cya sequence (1) indicates that there is a stop codon (TGA) in the...

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