Jac A. Nickoloff

Site-directed mutagenesis of virtually any plasmid by eliminating a unique site

We describe an efficient site-specific mutagenesis procedure that is effective with virtually any plasmid, requiring only that the target plasmid carry a unique, nonessential restriction site. The procedure employs two mutagenic oligonucleotide primers. One primer contains the desired mutation and the second contains a mutation in any unique, nonessential restriction site. The two primers are annealed to circular single-stranded DNA (produced by heating circular double-stranded DNA) and direct synthesis of a new second strand containing both primers. The resulting DNA is transformed into a mismatch repair defective (mut S) Escherichia coli strain, which increases the probability that the two mutations will cosegregate during the first round of DNA replication. Transformants are selected en masse in liquid medium containing an appropriate antibiotic and plasmid DNA is prepared, treated with the enzyme that recognizes the unique, nonessential restriction site, and retransformed into an appropriate host. Linearized parental molecules transform bacteria inefficiently. Plasmids with mutations in the unique restriction site are resistant to digestion, remain circular, and transform bacteria efficiently. By linking a selectable mutation in a unique restriction site to a nonselectable mutation, the latter can be recovered at frequencies of about 80%. Since most plasmids share common vector sequences, few primers, targeted to shared restriction sites, are needed for mutagenizing virtually any plasmid. The procedure employs simple procedures, common materials, and it can be performed in as little as 2 days.

Chromosomal Double-Strand Breaks Induce Gene Conversion at High Frequency in Mammalian Cells

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

Transcription enhances intrachromosomal homologous recombination in mammalian cells.

The influence of transcription on homologous intrachromosomal recombination between direct and inverted repeats has been examined by using Chinese hamster ovary cells. Recombination was monitored between two integrated neomycin (neo) genes, including one silent allele and a second allele regulated by the inducible mouse mammary tumor virus promoter. Transcription of mouse mammary tumor virus neo alleles was regulated with the glucocorticoid hormone dexamethasone. Alleles transcribed at high levels recombined about two- to sevenfold more frequently than identical alleles transcribed at low levels. Direct repeats recombined primarily by a gene conversion mechanism; inverted repeats produced a variety of rearranged products. These results are discussed in relation to recombinational processes that regulate gene expression, influence gene family structures, and mediate genomic instability associated with cellular transformation and tumorigenesis.

Electroporation protocols for microorganisms

Electroporation Theory: Concepts and Mechanisms. Instrumentation. Direct Plasmid Transfer Between Bacterial Species and Electrocuring. Transfer of Episomal and Integrated Plasmids from Saccharomyces cerevisiae to Escherichia coli by Electroporation. Production of cDNA Libraries by Electroporation. Electroporation of RNA into Saccharomyces cerevisiae. Electrofusion of Yeast Protoplasts. Escherichia coli Electrotransformation. Electrotransformation in Salmonella. Electrotransformation of Pseudomonas. Electroporation of Xanthomonas. Transformation of Species with Suicide and Broad Host-Range Plasmids. Electroporation of Francisella tularensis. A Simple and Rapid Method for Transformation of Vibrio Species by Electroporation. Genetic Transformation of Bacteroides spp. Using Electroporation. Electrotransformation of Agrobacterium. Electroporation of Helicobacter pylori. Electrotransformation of Streptococci. Transformation of Lactococcus by Electroporation. Transformation of Lactobacillus by Electroporation. Electrotransformation of Staphylococci. Electroporation and Efficient Transformation of Enterococcus faecalis Grown in High Concentrations of Glycine. Introduction of Recombinant DNA into Clostridium spp. Electroporation of Mycobacteria. Electrotransformation of the Spirochete Borrelia burgdorferi. Yeast Transformation and the Preparation of Frozen Spheroplasts for Electroporation. Ten-Minute Electrotransformation of Saccharomyces cerevisiae. Electroporation of Schizosaccharomyces pombe. Gene Transfer by Electroporation of Filamentous Fungi. Transformation of Candida maltosa by Electroporation. Electroporation of Physarum polycephalum. Electroporation of Dictyostelium discoideum. Gene Transfer by Electroporation of Tetrahymena. Transfection of the African and American Trypanosomes. Electroporation in Giardia lamblia. Index.

Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

WIL2-NS and TK6 are two distinct human lymphoblast cell lines derived from a single male donor. WIL2-NS cells are significantly more resistant to the cytotoxic effects of X-irradiation but considerably more sensitive to induced mutation. In an effort to determine the mechanistic basis for these differences, we analyzed the physical structures of thymidine kinase (tk)-deficient mutants isolated after X-ray treatment of tk heterozygotes derived from TK6 and the more mutable WIL2-NS. Southern analysis showed that while 84% of TK6-derived mutants had arisen by loss of heterozygosity (LOH), all 106 mutants from WIL2-NS derivatives arose with LOH at tk and all but one showed LOH at other linked loci on chromosome 17. We adapted a fluorescence in situ hybridization technique to distinguish between LOH due to deletion, which results in retention of only one tk allele, and LOH due to a mechanism involving the homologous chromosome (e.g., recombination), which results in the retention of two alleles. Among the LOH mutants derived that were analyzed in this way, 9 of 26 from WIL2-NS and 11 of 17 from TK6 cell lines arose by deletion. The remaining mutants retained two copies of the tk gene and thus arose by a mechanism involving the homologous allele. Since many of these mutants arising by a homologous mechanism retained partial heterozygosity of chromosome 17, they must have arisen by recombination or gene conversion, and not chromosome loss and reduplication. Finally, the recombinational capacities of WIL2-NS and TK6 were compared in transfection assays with plasmid recombination substrates. Intermolecular recombination frequencies were greater in WIL2-NS than in TK6. These data are consistent with a model suggesting that a recombinational repair system is functioning at a higher level in WIL2-NS than in TK6; the greater mutability of the tk locus in WIL2-NS results from more frequent inter- and intramolecular recombination events.

Fine-resolution mapping of spontaneous and double-strand break-induced gene conversion tracts in Saccharomyces cerevisiae reveals reversible mitotic conversion polarity.

Spontaneous and double-strand break (DSB)-induced gene conversion was examined in alleles of the Saccharomyces cerevisiae ura3 gene containing nine phenotypically silent markers and an HO nuclease recognition site. Conversions of these alleles, carried on ARS1/CEN4 plasmids, involved interactions with heteroalleles on chromosome V and were stimulated by DSBs created at HO sites. Crossovers that integrate plasmids into chromosomes were not detected since the resultant dicentric chromosomes would be lethal. Converted alleles in shuttle plasmids were easily transferred to Escherichia coli and analyzed for marker conversion, facilitating the characterization of more than 400 independent products from five crosses. This analysis revealed several new features of gene conversions. The average length of DSB-induced conversion tracts was 200 to 300 bp, although about 20% were very short (less than 53 bp). About 20% of spontaneous tracts also were also less than 53 bp, but spontaneous tracts were on average about 40% longer than DSB-induced tracts. Most tracts were continuous, but 3% had discontinuous conversion patterns, indicating that extensive heteroduplex DNA is formed during at least this fraction of events. Mismatches in heteroduplex DNA were repaired in both directions, and repair tracts as short as 44 bp were observed. Surprisingly, most DSB-induced gene conversion tracts were unidirectional and exhibited a reversible polarity that depended on the locations of DSBs and frameshift mutations in recipient and donor alleles.

Plant cell electroporation and electrofusion protocols

Part I. Theory And Instrumentation. Electroporation Theory: Concepts and Mechanisms. Effects of Pulse Length and Strength on Electroporation Efficiency. Instrumentation. Part II. Electroporation Protocols. Electroporation of Agrobacterium tumefaciens. Electroporation of DNA into the Unicellular Green Alga Chlamydomonas reinhardtii. Pollen Electrotransformation in Tobacco. Electroporation of Tobacco Leaf Protoplasts Using Plasmid DNA or Total Genomic DNA. Electroporation of Brassica. Transformation of Maize by Electroporation of Embryos. Transient Gene Expression Analysis in Electroporated Maize Protoplasts. Reporter Genes and Transient Assays for Plants. Part III. Electrofusion Protocols. Electrofusion of Plant Protoplasts: Selection and Screening for Somatic Hybrids of Nicotiana. Protoplast Electrofusion and Regeneration in Potato. Polymer- Supported Electrofusion of Protoplasts: A Novel Method and a Synergistic Effect. Index.

A role for p53 in DNA end rejoining by human cell extracts

The tumor suppressor p53 is a major regulator in the response of human cells to DNA damage. In this study we assessed the role of p53 in the repair of DNA double-strand breaks in plasmid DNA using cell extracts from three human lymphoblastoid cell lines derived from the same donor. TK6, WI-L2-NS and TK6-E6-5e cells express wild-type, mutated and essentially no p53 protein, respectively. Total cellular extracts from TK6, WI-L2-NS and TK6-E6-5e cells were incubated with EcoRI linearized pUC19 DNA. Southern blot analysis of end-rejoined DNA indicated that the major products formed were linear multimers. There was approximately 2-fold greater end rejoining in WI-L2-NS and TK6-E6-5e extracts compared with TK6 extracts. Total DNA from end-rejoining reactions was purified and used to transform bacteria. Using the lacZ reporter gene as a measure of repair fidelity we found that misrepair, as indicated by white colonies, occurred at 4.1% to 6.5% of transformants, with no significant difference between the three cell lines. Gel analysis revealed that misrepair involved only deletions. Sequence analysis of 11 misrepaired products from each cell line showed 12 different deletions from 4 to 48 bp in length, but each cell line yielded similar product types. These results indicate that total cellular extracts from human lymphoblastoid cells lacking p53 or expressing mutated p53 have increased end-rejoining activity as compared with extracts from cells expressing wild-type p53. However, the p53 status does not influence the ratio of misrepair:correct repair, or the type of misrepair events.

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.

Animal cell electroporation and electrofusion protocols

Part I. Theory and Instrumentation. Electroporation Theory: Concepts and Mechanisms. Effects of Pulse Length and Strength on Electroporation Efficiency. Instrumentation. Part II. Electroporation Protocols. The Introduction of Proteins into Mammalian Cells by Electroporation. Electroporation of Antigen-Presenting Cells for T-Cell Recognition and Cytotoxic T-Lymphocyte Priming. Electroporation of Antibodies into Mammalian Cells. Electroporation of Adherent Cells In Situ for the Introduction of Nonpermeant Molecules. Electrotransformation of Chinese Hamster Ovary Cells. Electroporation of Rat Pituitary Cells. Electroporation of Plasmid DNA into Normal Human Fibroblasts. Electroporation-Mediated Gene Transfer into Hepatocytes. Electroporation of Human Lymphoblastoid Cells. The Use of Electroporated Bovine Spermatozoa to Transfer Foreign DNA into Oocytes. Electroporation of Embryonic Stem Cells for Generating Transgenic Mice and Studying In Vitro Differentiation. Electrotransfection with Intracellular Buffer. Effect of Cis-Located Human Satellite DNA on Electroporation Efficiency. Quantitation of Transient Gene Expression. Stable Integration of Vectors at High Copy Number for High-Level Expression in Animal Cells. Electroporation of Drosophila Embryos. Transformation of Fish Cells and Embryos. Electroporation of Cardiac Cells. Electroporation for Gene Therapy. Part III. Electrofusion Protocols. Electrofusion of Mammalian Cells. Stabilizing Antibody Secretion of Human Epstein Barr Virus-Activated B-Lymphocytes with Hybridoma Formation by Electrofusion. Electrofusion of Mammalian Oocytes and Embryonic Cells. Nuclear Transfer in Bovine Embryos. Electrofusion of Mouse Embryos to Produce Tetraploids. Spectrofluorometric Assay for Cell-Tissue Electrofusion. Cytometric Detection and Quantitation of Cell-Cell Electrofusion Products. Index.