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The nucleotide sequence and transcript initiation site of the Synechococcus sp. strain PCC 7002 recA gene have been determined. The deduced amino acid sequence of the RecA protein of this cyanobacterium is 56% identical and 73% similar to the Escherichia coli RecA protein. Northern (RNA) blot analysis indicates that the Synechococcus strain PCC 7002 recA gene is transcribed as a monocistronic transcript 1,200 bases in length. The 5' endpoint of the recA mRNA was mapped by primer extension by using synthetic oligonucleotides of 17 and 27 nucleotides as primers. The nucleotide sequence 5' to the mapped endpoint contained sequence motifs bearing a striking resemblance to the heat shock (sigma 32-specific) promoters of E. coli but did not contain sequences similar to the E. coli SOS operator recognized by the LexA repressor. An insertion mutation introduced into the recA locus of Synechococcus strain PCC 7002 via homologous recombination resulted in the formation of diploids carrying both mutant and wild-type recA alleles. A variety of growth regimens and transformation procedures failed to produce a recA Synechococcus strain PCC 7002 mutant. However, introduction into these diploid cells of the E. coli recA gene in trans on a biphasic shuttle vector resulted in segregation of the cyanobacterial recA alleles, indicating that the E. coli recA gene was able to provide a function required for growth of recA Synechococcus strain PCC 7002 cells. This interpretation is supported by the observation that the E. coli recA gene is maintained in these cells when antibiotic selection for the shuttle vector is removed.

DNA-based computing uses the tendency of nucleotide bases to bind (hybridize) in preferred combinations to do computation. Depending on reaction conditions, oligonucleotides can bind despite noncomplementary base pairs. These mismatched hybridizations are a source of false positives and negatives, which limit the efficiency and scalability of DNA-based computing. The ability of specific base sequences to support error-tolerant Adleman-style computation is analyzed, and criteria are proposed to increase reliability and efficiency. A method is given to calculate reaction conditions from estimates of DNA melting. [S0031-9007 (97)04987-9] PACS numbers: 89.70. + c, 87.15.By, 89.80. + h Adleman [1] introduced a way to do computations with DNA, and applied the technique to the solution of an NPcomplete problem, the Hamiltonian path problem (HPP) [2]. In general, a DNA-based computation involves three steps. First, the problem instance is encoded in a collection of DNA oligonucleotides. Second, template matching reactions, or hybridizations, between oligonucleotides produce double-stranded molecules, which ligase forms into longer molecules. These long molecules potentially represent the result of the computation. Third, the results are extracted with techniques, such as polymerase chain reaction (PCR) and gel electrophoresis. The basic processing power of a DNA-based computation, as suggested by Adleman [1], is in the massive number of string comparisons that occur during the template matching reactions between DNA oligonucleotides. Thus, a fundamental step in a DNA computation is the hybridization between oligonucleotides. Other proposals for DNA computation [3][4][5] continue to rely on the mechanism of the template-matching hybridization reaction. Most assume that the hybridizations between oligonucleotides occur error free. Nevertheless, errors, i.e., double strands which are not fully Watson-Crick complementary, are a consequence of the cooperative and uncertain nature of the chemistry on which the technique is based, and cannot be eliminated entirely.To make DNA-based computing a reliable technique, the first step is to ensure that false positives and negatives occur with negligible probabilities. If many incorrect or mismatched hybridizations are possible, then false positives (i.e., DNA strands which appear to be valid solutions, but actually are not) can occur. Likewise, if DNA oligonucleotides are used up in unproductive mismatches, there will be fewer available for formation of the result, and a false negative, or the failure to detect a correct answer when one is present, is possible. The probability of a less than perfect hybridization depends on the reaction conditions of the hybridization, with temperature being the most significant [6,7]. In this paper, the Hamming distance between oligonucleotides is explored as a criterion for reliable DNA solution of HPP. As a first estimate for a reliable encoding, the required distance can be estimated from the melting temperature, which is the temperature at which ...

A mosquitocidal cyanobacterium has been developed by introducing the mosquito-toxic crylVD gene from Bacillus thuringiensis subsp. israelensis into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002). The cryIVD gene was introduced into the cyanobacterium on a derivative of the PR-6 expression vector pAQE19ASal in which the cryIVD gene was translationally fused to the initial coding sequence of the highly expressed PR-6 cpcB gene. Coomassie blue staining and immunoblot analysis of gel-fractionated cell extract polypeptides indicate that the cpcB-cryIVD gene fusion is expressed at high levels in the cyanobacterial cells, with little or no apparent degradation of the cryIVD gene product. Larvicidal assays revealed that freshly hatched Culexpipiens mosquito larvae readily ingested the transformed cyanobacteria and that the cells proved to be toxic to the larvae.

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