The growth of clones of human genomic DNA fragments in a bacteriophage X vector has been examined in a number of different Escherichia coli hosts. A large proportion (8.9%) of the phages carrying different fragments of the human genome fail to grow on standard rec' hosts but will grow on hosts carrying mutations in the recB, recC, and sbcB genes. Heteroduplex analysis in the electron microscope of DNA from four of these phages revealed substantial secondary structure, including snap-back regions 200-500 base pairs in length. Such structures were not found in phages from the same DNA library that grow in rec' hosts. Recombinant DNA libraries in bacteriophage X vectors are generally found to contain most of the sequences of the genomes from which they are derived (1, 2). Nevertheless, anecdotal reports of sequences that cannot be found in libraries of eukaryotic genomes are fairly common. One striking example is the hypervariable region 3' to the a-globin locus, which has resisted strenuous efforts at cloning (S. Goodbourn and S. Orkin, personal communications). Another example is the junction fragment from a translocation between chromosomes 6 and 10 in a murine plasmacytoma (3). A related phenomenon is the observation of discrepancies between cloned and genomic sequences that are the result of deletions occurring during cloning of mammalian DNA (3)(4)(5). Only some of these deletions can be prevented by growth on a recA host.For the past few years we have been studying a highly polymorphic locus in humans, now known as D14S1, which was identified by hybridization of gel transfers of genomic DNA using an adjacent single-copy fragment as probe (6-8).Efforts to obtain clones of the sequences surrounding and including the polymorphic site itself from the Maniatis human genomic library (2) and from libraries made in our laboratory, using conventional cloning conditions as well as hosts carrying mutations in the recA gene, were all unsuccessful. Given the rarity in general of highly polymorphic DNA sequences in the human genome and the striking difficulty in recovering the variable segments in two of the most studied ones (a globin and D14S1), we became concerned that there might be a systematic difficulty in cloning certain human DNA sequences. Further, we speculated that the difficulty might sometimes be associated with a high degree of polymorphism.Recently, Leach and Stahl (9) reported that inverted repetitions cannot be cloned into phage X vectors unless mutant hosts are used. We report here that such a host, which contains mutant recB, recC, and sbcB genes, will propagate many phage X clones containing random human DNA fragments, including D14S1, that cannot be grown on the Escherichia coli hosts commonly used in the selection and propagation of recombinant DNA genome libraries. MATERIALS AND METHODSBacterial Strains. With the exception of strains BNN45 and SF8, all strains used are derivatives of the E. coli K-12 strain AB1157; they have the following markers in common: leu6 aral4 his4 thrl thil lac Y1...
The potential for exposure to mycotoxins in indoor environments is of increasing concern. In order to evaluate the potential for mycotoxin production by toxigenic fungi growing on waterdamaged building materials, two aflatoxin producing strains of Aspergillus f1avus (American Type Culture Collection 16875 and 15547) were inoculated onto culture media, plain wallboard, and vinyl wallpapered wallboard (cellulose-based and wheatbased wallpaper paste) and incubated at high relative humidity and room temperature for up to 16 weeks. Each sample was extracted with 60% methanol and aflatoxins in the crude extract were collected by immunoaffinity chromatography and quantified by fluorometry. Analysis by high performance liquid chromatography was performed for confirmation. Varying degrees of fungal growth were evident on all tested substrate types. Up to 4800 ppb of aflatoxin was detected when strain ATCC 16875 was grown on potato dextrose agar. However, when inoculation was standardized to minimize initial aflatoxin concentration in the inoculum, aflatoxin production was not detected on any wallboard sample under any of the incubation conditions provided. The presence of a toxigenic fungal strain on an indoor substrate does not necessarily indicate that the fungus is producing mycotoxins and our data provide evidence that wet wallboard is unlikely to provide appropriate conditions for aflatoxin production.
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