Sugar‐free or reduced‐sugar foods and beverages are very popular in the United States and other countries, and the sweeteners that make them possible are among the most conspicuous ingredients in the food supply. Extensive scientific research has demonstrated the safety of the 5 low‐calorie sweeteners currently approved for use in foods in the United States–acesulfame K, aspartame, neotame, saccharin, and sucralose. A controversial animal cancer study of aspartame conducted using unusual methodology is currently being reviewed by regulatory authorities in several countries. No other issues about the safety of these 5 sweeteners remain unresolved at the present time. Three other low‐calorie sweeteners currently used in some other countries–alitame, cyclamate, and steviol glycosides–are not approved as food ingredients in the United States. Steviol glycosides may be sold as a dietary supplement, but marketing this product as a food ingredient in the United States is illegal. A variety of polyols (sugar alcohols) and other bulk sweeteners are also accepted for use in the United States. The only significant health issue pertaining to polyols, most of which are incompletely digested, is the potential for gastrointestinal discomfort with excessive use. The availability of a variety of safe sweeteners is of benefit to consumers because it enables food manufacturers to formulate a variety of good‐tasting sweet foods and beverages that are safe for the teeth and lower in calorie content than sugar‐sweetened foods.

To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication, we isolated and sequenced the transfer origins (ori7) of the promiscuous IncP plasmids RP4 and R751. The central initiating event at the transfer origin of a conjugative plasmid is the cleavage at a unique site (nic) of the strand to be transferred to a recipient cell. This process can be triggered after the assembly of "relaxosomes" (plasmid DNA-protein relaxation complexes), requiring plasmid-encoded gene products. We analyzed the nicking reaction for plasmid RP4 and demonstrated that one of the plasmid strands is specifically cleaved within onT. The fully functional oriT of RP4 represents an intergenic DNA region of -350 base pairs. Dissection of oriT revealed that a portion carrying nic and symmetric sequence repeats determines oriT specificity. This part of oriT is contiguous to a region that is essential for efficient mobilization of oriT plasmids. In addition, oriT contains potential promoter sites allowing divergent transcription of two operons flanking oriT. We overproduced gene products and, from analyzing the products of dermed deletion mutants, deduced the gene arrangements. Formation of RP4 relaxosomes is likely to depend on the presence of at least two plasmid-encoded components, which act in trans. Corresponding genes map on one side of onT. Purification of the traJ product revealed it to be an 11-kDa polypeptide that binds to oriT DNA in vitro. The protein recognizes the part of oriT that is responsible for oriT specificity. While most of the transfer functions, including the matingpair formation system, can be utilized by both plasmids, the interaction at the transfer origin of tra gene products is plasmid specific (6). We used this observation to map the genes encoding oriT-specific functions within the regions immediately flanking oriT. Furthermore, an electrophoretic assay was developed to analyze rapidly the nicking reaction; the assay allowed us to locate genes required for relaxosome nicking within the region encoding oriT-specific functions. Expression-vector cloning of fragments carrying oriTspecific functions facilitated the analysis ofgene organization and the overproduction of gene products. One of these proteins, the traJ gene product, specifically binds to the oriT region in vitro, thus suggesting an important role of the protein in triggering the initial events of transfer DNA replication.Conjugation is the process that allows efficient gene transfer from one bacterial cell to another through plasmid-encoded functions. Conjugative plasmids of the IncP group are of particular interest because they are capable of mediating efficient DNA transfer between virtually any Gram-negative species (1). This promiscuity is important because of its role in the spread of antibiotic resistance and its application to gene manipulation in widely different bacteria.Conjugative transfer requires both a cis-acting site, the origin of transfer (oriT), and a number of trans-acting functions t...

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The nucleotide sequence of an almost complete cDNA copy of chalcone synthase mRNA from cultured parsley cells (Petroselinum hortense) has been determined. The cDNA copy comprised the complete coding sequence for chalcone synthase, a short A‐rich stretch of the 5′ non‐coding region and the complete 3′ non‐coding region including a poly(A) tail. The amino acid sequence deduced from the nucleotide sequence of the cDNA is consistent with a partial N‐terminal sequence analysis, the total amino acid composition, the cyanogen bromide cleavage pattern, and the apparent mol. wt. of the subunit of the purified enzyme.

The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred. The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons. This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.