No abstract

Tubercidin, toyocamycin, and the corresponding 5'-o:-D-glucopyranose derivatives of the nucleosides are frequently responsible for muchof the cytotoxicity and antimycotic activity associated with extracts of cultured cyanophytes belonging to the family Scytonemataceae.The 5'-a:-D-glucopyranoses of tubercidin and toyocamycin, for example, are the major cytotoxic and fungicidal nucleosides in Fijian Plectonema radiosum and Hawaiian Tolypothrix tenuis, respectively.The blue green algae provide an excellent source of new bioactive compounds15. Over the past 6 years we have mass cultured over 700 clonal isolates from a variety of terrestrial, freshwater and marine environments and screened hydrophilic and lipophilic extracts of these cyanophytes for cytotoxicity and antifungal activity. About 6% of the extracts show cytotoxicity at <20 jug/ml against the KBcell line (a human epidermoid carcinoma of the nasopharynx) and roughly 9 % of the extracts show antifungal activity at 500 /^g/disc against one or more test organisms, viz. Aspergillus oryzae, Candida albicans, Penicillium notatum, Saccharomyces cerevisiae and Trichophyton mentagrophytes.Several of the active hydrophilic extracts (obtained with 30 % ethanol in water) show both cytotoxicity and antifungal activity and many are cyanophytes belonging to the family Scytonemataceae. Twodistinct classes of compoundsare responsible for the cytotoxicity and fungicidal activity of the Scytonemataceae listed in Table 1 , viz. scytophycin-type macrolides and tubercidin/toyocamycintype nucleosides. Scytophycins account for the cytotoxicity and antifungal activity of Scytonema pseudohofmanni (strain BC-l-2)2>3).Tolytoxin, a scytophycin-related compound that was first isolated from field-collected Tolypothrix conglutinata var. colorata°, is responsible for both the cytotoxicity and antifungal activity of Scytonema mirabile (BY-8-1) and Scytonema ocellatum (DD-8-1) (unpublished results). In an earlier report we showed that tubercidin

This work presents the first example of an anti-mutator role of the sbcC,D genes, and defines a new gene (rmuC) involved in DNA recombination.

Mutator cells that lack the mismatch repair system (MMR ؊ ) occur at rates of 10 ؊5 or less in laboratory populations started from wild-type cells. We show that after selection for recombinants in an interspecies mating between Salmonella enterica serovar Typhimurium and Escherichia coli, the percentage of MMR ؊ cells rises to several percent of the recombinant population, and after a second successive mating and selection, greater than 95% of the recombinants are MMR ؊ . Coupling a single cross and selection with either mutagenesis or selection for spontaneous mutants also results in a dramatic increase in MMR ؊ cells. We discuss how horizontal transfer can result in mutator strains during adaptive evolution.Mutators, cells with higher rates of mutation than normal cells, play a role in human disease and adaptive evolution (see reviews in references 7, 18, and 19). For instance, the human mismatch repair system (MMR), the counterpart of the bacterial and yeast mismatch repair systems (10,21,22,27), is involved in inherited predispositions to colon (HNPCC), endometrial, and ovarian cancer (3,6,12,24). Tumor lines from HNPCC patients are mutators with greatly increased repeattract or microsatellite instability (1,9,12,25,26). How mutator cells arise and proliferate in cell populations as a result of different processes and selective forces have been the object of recent studies (15,20). Reports of these studies previously described how selection for mutants resulting from spontaneous mutations amplifies the mutator subpopulation, to the point where an entire population of cells becomes mutator (15,20). Most of the mutators arising from this process are mismatch repair system deficient (MMR Ϫ ) and make frequent mutations due to their inability to repair replication errors. However, the MMR not only protects against replication errors but also acts as a barrier to recombination between divergent chromosomes. Radman and coworkers showed that MMR Ϫ cells lacking either the MutS or MutL function carry out homeologous recombination resulting from interspecies crosses between Salmonella enterica serovar Typhimurium and Escherichia coli three orders of magnitude more frequently than MMR ϩ cells (28,31). MutS binding to mismatches may limit the heteroduplex region (see reference 28 and references therein; see also references 2 and 34). Since the mutators in a wild-type population have such an elevated frequency of recombination in interspecies crosses, does the selection for recombination in such a mating enhance the mutator fraction among the surviving cells? We describe here how interspecies crosses and homeologous recombination does select for rare MMR Ϫ cells, amplifying them in the population. Two successive homeologous crosses can convert a population with as few as 10 Ϫ5 mutators to greater than 95% mutator. This suggests that horizontal transfer can ultimately be a mutagenic process at the population level. MATERIALS AND METHODSBacterial strains and strain construction. Table 1 lists the strains used in this work...

We have constructed strains that allow a direct selection for mutators of Escherichia coli on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (mutHLS and uvrD). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10−5 in the population. Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second mutator (mutHLS) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.