Cloning of Physarum actin sequences in an exonuclease-deficient bacterial host.

Abstract

A genomic library of Physarum was constructed in the replacement vector EMBL3. Efficient propagation of the recombinant phages occurred only on the recBCsbcB-host Escherichia coli CES200, which is deficient in the exonucleases I and V. Thirteen different recombinants with actin-related sequences were detected and 10 were purified from 90,000 plaques (the equivalent of6 Physarum genomes) on strain CES200. Comparison of the plating efficiencies of the library and the actin-related isolates suggests that palindromic DNA sequences are responsible for the instability of Physarum DNA in E. coli. In one of these isolates, XPpA10, and in a 2. (7,8). The stability of palindromic structures, artificially constructed in plasmid (9) and X DNA (10), has also been studied. It has been found that propagation of these sequences is permitted only in bacterial hosts lacking both exonucleases I and V, the products of the sbcB and recBC genes, respectively (for reviews, see refs. 11 and 12). It has been shown by electron microscopy (13, 14) that in singlestranded Physarum DNA, "foldback" structures can be observed after intramolecular reassociation; these structures are regularly spaced throughout the genome. Here we present evidence that palindromic structures in Physarum DNA may cause low plating efficiencies of a genomic Physarum library in the X replacement vector EMBL3 on Rec' E. coli hosts and that these structures also may cause instability of actinrelated sequences obtained from this library that are cloned either in phage X or the plasmid pBR322. MATERIALS AND METHODSConstruction of the Genomic Library. DNA was isolated from nuclei of axenically grown Physarum amoebae, strain Cld-Axe (15). The nuclei were lysed in 50 mM Tris Cl, pH 8/0.1 M EDTA/proteinase K (100 gg/ml)/0.5% N-lauroylsarcosine for 1 hr at room temperature. High molecular weight DNA was obtained by phenol extraction, RNase treatment, and extensive dialysis against 10 rnM Tris Cl, pH 8/1 mM EDTA (16). Electrophoresis in a 0.4% agarose gel revealed a major DNA band at -100 kilobases (kb) and a minor band at 60 kb, which probably represents the intact linear, extrachromosomal ribosomal DNA of Physarum. The DNA was partially digested with restriction endonuclease Mbo I, and 15-to 20-kb fragments were isolated from a sucrose gradient (1). DNA from bacteriophage EMBL3 was digested with BamHI and EcoRI, and the small linker fragment was removed by isopropanol-precipitation (17). The Physarum DNA fragments were then ligated to the BamHI sites of the phage arms in a 1:4 molar ratio. The ligated DNA was packaged in vitro according to Mullins et al. (18) or with an in vitro packaging kit (Amersham).Isolation and Characterization of Bacteriophage and Plasmid DNA. Bacteriophages were prepared from cultures of infected E. coli strains LE392 and CES200 in L-broth, and DNA was isolated as described (19,20 mapping by multiple digestion were done as described (23,24). For DNA blotting and hybridization we followed the procedure of Southern (25) with modifications (3...

Characterization of N-myc amplification units in human neuroblastoma cells.

“…Therefore, subsequent walking experiments employed an EMBL3 phage library of NGP DNA sequences which allowed us to overcome these blocks to cloning. CES200 or DB1316 cells were used to facilitate bacteriophage cloning of sequences which are difficult to clone in standard bacteria such as KH802 recA, the strain used as the host for the cosmid library (22). Fragments were prepared from digests of phage or cosmid clones and used for hybridization to Southern transfers of cloned and genomic NGP DNA.…”

RecA-independent high-frequency deletion of recombinant cosmid DNA in Escherichia coli

“…In both ;1 and plasmid cloning, a recB sbcB host and a recB recC sbcB host have been reported to prevent the deletions of some kinds of recombinant DNAs that were not stably maintained in usual rec+ or recA hosts (Collins et al, 1982;Leach & Stahl, 1983;Wyman et al, 1985;Nader et al, 1985). Therefore, we examined the effects of such E. coli hosts on the cosmid deletion events.…”

Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster

“…were plated on the Escherichia coli strain CES200 [22] and transferred to nitrocellulose. The probe used to screen for the Drosophila EH gene was the portion of the Munduca EH gene encoding the 62-amino-acid peptide (Manduca coding region probe).…”