The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound oa-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (-1,100 residues), that for glucan binding (-240 residues), and that of unknown function (-260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus.Mutans streptococci produce several extracellular glucosyltransferases (GTFs) which synthesize water-soluble glucans and water-insoluble glucans (ISGs) containing co-1,6-and a-1,3-glucosyl linkages [ISG(1,6) and ISG(1,3), respectively]. The ISGs adhere to smooth tooth surfaces and facilitate aggregation of oral bacteria, so that GTFs are believed to play a key role in the formation of dental plaque (12,20).In view of this etiological importance, a number of studies of the properties of GTFs to understand the mechanism of ISG synthesis have been conducted over the past two decades (2,5,8,9,11,23,24,30,33). From the culture fluids of a strain of Streptococcus sobrinus (previously named Streptococcus mutans 6715), we purified an enzyme responsible for ISG synthesis (GTF-I) from sucrose in the presence of a-1,6 soluble glucan such as dextran T10 as a primer (9). In this reaction, sucrose is split into fructose and an enzymebound glucosyl moiety (26); the latter is transferred to the C-3 position of the glucose residue of the glucan, resulting in the formation of a-1,3-glucosyl polymer (GTF-I activity) (9). In the absence of the primer glucans, however, the glucosyl moiety is transferred to water (hydrolysis of sucrose or sucrase activity). Further studies have not been made because of a limited supply of purified GTF-I.In the meantime, the structure and function of GTFs have been studied by biochemical and recombinant DNA techniques. Glucan-binding fragments were isolated by tryptic digestion of GTF proteins from S. sobrinus, but none of them showed glucan-synthesizing activity (18,22). Of several molecular cloning studies of GTF genes from mutans streptococci (1,7,10,13,14,25...

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Gene duplication is a major genetic event that can produce multiple protein isoforms. Comparative sequence and functional analysis of related gene products can provide insights into protein family evolution. To characterize the Caenorhabditis elegans troponin I family, we analyzed gene structures, tissue expression patterns and RNAi phenotypes of four troponin I isoforms. Tissue expression patterns were determined using lacZ/gfp/rfp reporter gene assays. The tni-1 , tni-2 / unc-27 and tni-3 genes, each encoding a troponin I isoform, are uniquely expressed in body wall, vulval and anal muscles but at different levels; tni-4 was expressed solely in the pharynx. Expressing tni-1 and -2 gene RNAi caused motility defects similar to unc-27 ( e155 ) mutant, a tni-2 null allele. The tni-3 RNAi expression produced egg laying defects while the tni-4 RNAi caused arrest at gastrulation. Overlay analyses were used to assay interactions between the troponin I and two troponin C isoforms. The three body wall troponin I isoforms interacted with body wall and pharyngeal troponin C isoforms; TNI-4 interacted only with pharyngeal troponin C. Our results suggest the body wall genes have evolved following duplication of the pharynx gene and provide important data about gene duplication and functional differentiation of nematode troponin I isoforms.

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