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The activity of the M26 meiotic recombination hot spot of Schizosaccharomyces pombe depends on the presence of the heptamer 5-ATGACGT-3. Transplacement of DNA fragments containing the ade6-M26 gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context. In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome. When created by mutagenesis of 1-4 bp in the ade6 and ura4 genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested. Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for M26 hot spot activity.Homologous recombination, the exchange of genetic information between homologous DNA duplexes, plays a vital role in the generation of genetic diversity, in the proper segregation of chromosomes during meiosis, and in the repair of DNA damage in somatic cells. Although homologous recombination occurs throughout the genome, some sites, termed hot spots, exhibit elevated frequencies of exchange. Recombination hot spots have been described in diverse organisms from bacteria to mammals (1, 2). Because hot spots appear to enhance a rate-limiting step of recombination, determination of the molecular mechanism by which hot spots act will increase our understanding of an important stage of homologous recombination.The M26 mutation of the fission yeast Schizosaccharomyces pombe creates a meiotic recombination hot spot in the ade6 gene. M26 is one of 394 mutations in ade6 described by Gutz (3) and results from a G 3 T transversion in the coding region of the ade6 gene (4, 5). Among these 394 mutations, M26 is unique; in heteroallelic crosses, it recombines with other ade6 mutations up to 15-fold more frequently than the adjacent M375 mutation does (3). M375 is an equivalent G 3 T transversion in the preceding codon and thus serves as an ideal genetic control for M26 (4). M26 undergoes gene conversion 10 times more frequently than M375 and demonstrates disparity of conversion, with preferential conversion of M26 to wild type, whereas M375 converts with near parity (3). M26 also increases the frequency of conversion of other ade6 mutations, with which it frequently coconverts; the frequency of coconversion decreases with increasing distance from M26 (6). These properties led Gutz (3) to propose that the M26 mutation creates a site that stimulates recombination near itself and, less frequently, at a distance.M26 is unique among eukaryotic hot spots in that hot spot activity is known to depend on a specific DNA sequence, the heptamer 5Ј-ATGACGT-3Ј, the first T in the heptamer sequence being the site of mut...

The toxicity associated with conventional cancer chemotherapy is primarily due to a lack of specificity for tumour cells. In contrast, intravenously injected clostridial spores exhibit a remarkable specificity for tumours. This is because, following their administration, clostridial spores become exclusively localised to, and germinate in, the hypoxic/necrotic tissue of tumours. This unique property could be exploited to deliver therapeutic agents to tumours. In particular, genetic engineering could be used to endow a suitable clostridial host with the capacity to produce an enzyme within the tumour which can metabolise a systemically introduced, non-toxic prodrug into a toxic metabolite. The feasibility of this strategy (clostridial-directed enzyme prodrug therapy, CDEPT) has been demonstrated by cloning the Escherichia coli B gene encoding nitroreductase (an enzyme which converts the prodrug CB1954 to a highly toxic bifunctional alkylating agent) into a clostridial expression vector and introducing the resultant plasmid into Clostridium beijerinckii (formerly C. acetobutylicum) NCIMB 8052. The gene was efficiently expressed, with recombinant nitroreductase representing 8% of the cell soluble protein. Following the intravenous injection of the recombinant spores into mice, tumour lysates have been shown, by Western blots, to contain the E. coli-derived enzyme.

Cellular uptake and subcellular localisation of the antitumour agent mitoxantrone were studied in a human colon-carcinoma cell line and a mitoxantrone-resistant subline showing features consistent with an atypical multidrug-resistance phenotype involving altered topoisomerase II. Flow cytometry indicated a reduced uptake of mitoxantrone in the resistant line. Confocal microscopy indicated that mitoxantrone-associated fluorescence was primarily found within discrete cytoplasmic inclusions and around the periphery of the nucleus, with low levels being observed within the nucleus. The frequency of cytoplasmic inclusions was reduced in mitoxantrone-resistant cells as compared with parental cells. Fluorescence in cytoplasmic inclusions persisted throughout a 24-h post-treatment period in both cell lines. The results suggest that the persistence of mitoxantrone in cells is a determinant for the continuous induction of DNA damage, perhaps through chronic topoisomerase II trapping, and that modified sequestration may contribute to clinically relevant moderate levels of non-classic multidrug resistance.