Despite the absence of 2' hydroxyls, single-stranded DNA can adopt structures that promote divalent-metal-dependent self-cleavage via an oxidative mechanism. These results suggest that an efficient DNA enzyme might be made to cleave DNA in a biological context.

The sweet melon fruit is characterized by a metabolic transition during its development that leads to extensive accumulation of the disaccharide sucrose in the mature fruit. While the biochemistry of the sugar metabolism pathway of the cucurbits has been well studied, a comprehensive analysis of the pathway at the transcriptional level allows for a global genomic view of sugar metabolism during fruit sink development. We identified 42 genes encoding the enzymatic reactions of the sugar metabolism pathway in melon. The expression pattern of the 42 genes during fruit development of the sweet melon cv Dulce was determined from a deep sequencing analysis performed by 454 pyrosequencing technology, comprising over 350,000 transcripts from four stages of developing melon fruit flesh, allowing for digital expression of the complete metabolic pathway. The results shed light on the transcriptional control of sugar metabolism in the developing sweet melon fruit, particularly the metabolic transition to sucrose accumulation, and point to a concerted metabolic transition that occurs during fruit development.

A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ''deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a k obs of 0.2 min ؊1 , whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ''restriction enzymes'' for the site-specific cleavage of single-stranded DNA.DNA in biological systems exists primarily in duplex form where it serves almost exclusively as a storage system for genetic information. Outside the confines of cells, DNA in its single-stranded form can be made to perform both molecular recognition and catalysis-biochemical operations that were until recently thought to be possible only with macromolecules made of protein or RNA. For example, a number of DNA ''aptamers'' (1) can be made that function as ligands for proteins or as highly specific receptors for small organic molecules (2-4). In addition, certain single-stranded DNAs act as artificial enzymes (4-6), catalyzing such chemical reactions as phosphoester transfer (7-12), phosphoester formation (13), porphyrin metalation (14), phosphoramidate cleavage (15), and DNA cleavage (16). Most likely, DNA can be made to perform a much broader repertoire of catalytic activities (6).These capabilities of DNA conceivably can be exploited to create a variety of structured DNAs that perform useful tasks, either in vitro or in vivo, involving molecular recognition and catalysis. Such functional DNAs offer several advantages, including ease of synthesis and chemical stability, that might make attractive properties for polymers that serve as artificial receptors or as biocatalysts for various applications. Characteristics such as thermostability and solvent͞solute preferences could be conferred upon deoxyribozymes by using both rational and combinatorial methods of molecular design. Catalytic DNAs may offer distinct advantages over natural protein enzymes for operation under nonbiological conditions.In a previous study (16), we reported the isolation of two distinct types of deoxyribozymes (classes I and II) that undergo oxidative self-cleavage in the presence of copper ions. By using in vitro selection (17), class II self-cleaving DNAs have been further optimized for catalytic function, and the most active structure obtained from this process has been engineered to act as a ''restriction endonuclease'' for single-stranded DNA substrates. MATERIALS AND METHODSOligonucleotides. Synthetic DNAs were prepared by automated chemical synthesis (Keck Biotechnology Resource Laboratory, Yale University) and were purified by denaturing (8 M urea) PAGE prior to use. Double-stran...

SummaryRaf®nose and stachyose are ubiquitous galactosyl-sucrose oligosaccharides in the plant kingdom which play major roles, second only to sucrose, in photoassimilate translocation and seed carbohydrate storage. These sugars are initially metabolised by a-galactosidases (a-gal). We report the cloning and functional expression of the ®rst genes, CmAGA1 and CmAGA2, encoding for plant a-gals with alkaline pH optima from melon fruit (Cucumis melo L.), a raf®nose and stachyose translocating species. The alkaline a-gal genes show very high sequence homology with a family of unde®ned`seed imbibition proteins' (SIPs) which are present in a wide range of plant families. In order to con®rm the function of SIP proteins, a representative SIP gene, from tomato, was expressed and shown to have alkaline a-gal activity. Phylogenetic analysis based on amino acid sequences shows that the family of alkaline a-gals shares little homology with the known prokaryotic and eukaryotic a-gals of glycosyl hydrolase families 27 and 36, with the exception of two cross-family conserved sequences containing aspartates which probably function in the catalytic step. This previously uncharacterised, plant-speci®c a-gal family of glycosyl hydrolases, with optimal activity at neutral-alkaline pH likely functions in key processes of galactosyl-oligosaccharide metabolism, such as during seed germination and translocation of RFO photosynthate.

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