Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells.

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.     

Recombination of homologous DNA fragments transfected into mammalian cells occurs predominantly by terminal pairing.

The mechanism by which double-strand cleavages stimulate the joining of plasmid DNA fragments introduced into cultured mammalian cells was investigated by cotransfecting pairs of plasmids encoding deletion mutations in a dominant selectable gene into LMtk- cells. Plasmid recombination substrates were produced by creating deletions of different sizes within the neo coding region of the pSV2neo plasmid. Complementing pairs of deleted plasmid DNAs were linearized at specific unique sites before cotransfection into mouse LMtk- cells by the calcium phosphate precipitation method. Cleaving one donor plasmid produced a 4- to 10-fold stimulation in the production of colonies able to survive in medium containing G-418. The linearization of the second plasmid further increased the efficiency by another factor of 6 to 15 when the cut was made on the opposite side of the homology, approximately equidistant from the center of the overlap. Fifty-seven individual G-418-resistant colonies representing the products of individual crosses were isolated, and the genomic DNAs containing the presumably integrated, functional recombinant neo genes were analyzed on Southern blots. A band consistent with the exchange of markers flanking the neo gene was present in 90% of the DNAs examined. In only one case was the pattern indicative of either a double crossover or a gene conversion event. These results support the idea that homologous extrachromosomal DNA fragments are joined through annealing of overlapping single-stranded ends. This DNA-joining phenomenon may represent the activity of cellular DNA repair enzymes; its relationship to genetic recombination occurring at the chromosomal level remains to be determined.     

New, small circular DNA in transfected mammalian cells.

Circular DNA isolated by the Hirt procedure from transfected mammalian cells was examined by electron microscopy. Typically, the number of small (1- to 5-kilobase) DNA circles increased about fivefold even though DNA of larger size classes (5 to 15 kilobases) has been transferred. In one case, where extensive rearrangement of the transferred DNA was observed, the rearrangement products were cloned and analyzed. In most cases, however, no rearrangement could be detected, but the amount of small circular DNA was still increased. This effect was seen with two transfection procedures (erythrocyte ghost fusion and calcium phosphate precipitation) and with various combinations of transfecting DNA and recipient cell type. The origin of the new small circular DNA is discussed.     

Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative.

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.     

DNA homologous recombination repair in mammalian cells

DNA double-strand breaks (DSBs) are the most serious DNA damage. Due to a great variety of factors causing DSBs, the efficacy of their repair is crucial for the cell's functioning and prevents DNA fragmentation, chromosomal translocation and deletion. In mammalian cells DSBs can be repaired by non-homologous end joining (NHEJ), homologous recombination (HRR) and single strand annealing (SSA). HRR can be divided into the first and second phase. The first phase is initiated by sensor proteins belonging to the MRN complex, that activate the ATM protein which target HRR proteins to obtain the second response phase--repair. HRR is precise because it utilizes a non-damaged homologous DNA fragment as a template. The key players of HRR in mammalian cells are MRN, RPA, Rad51 and its paralogs, Rad52 and Rad54.     

Effects of methotrexate on transfected DNA stability in mammalian cells.

The chromosomal locations, amounts, and level of expression of transfected, amplified c-myc and dihydrofolate reductase sequences were measured in cells cultured in the presence and absence of methotrexate. These studies show that the location and amount of transfected sequences, as well as the level of expression, were more variable when the cells were cultured in methotrexate.     

Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells.

The nucleoprotein structures formed on various plasmid expression vectors transfected into mammalian cells by both the calcium phosphate and DEAE-dextran methods have been studied. We demonstrate by a variety of means that mammalian cells are capable of rapidly assembling non-integrated circular plasmids (both replicating and non-replicating) into typical "minichromosomes" containing nucleosomes with a 190 bp repetitive spacing. Treatment of recipient cells with sodium butyrate for a short period of time (12-16 h) immediately following transfection markedly increased the DNase I digestion sensitivity of the newly assembled plasmid chromatin. Furthermore, minichromosomes isolated from such butyrate-treated cells are depleted in histone H1 and contain highly acetylated forms of histone H4. These findings are entirely consistent with our earlier speculation (Gorman et al., Nucleic Acids Res. 11, 1044; 1983) that appropriate butyrate treatment might stimulate transient expression of newly transfected genes by facilitating their assembly into an "active" type of chromatin structure.     

Specificity of mutations induced in transfected DNA by mammalian cells

DNA transfected into mammalian cells is subject to the high mutation frequency of approximately 1% per gene. We present data bearing on the derivation of the two main classes of mutations detected, base substitutions and deletions. The DNA sequence change is reported for nearly 100 independent base substitution mutations that occurred in shuttle vectors as a result of passage in simian cells. All of the mutations occur at G:C base pairs and involve either transition to A:T or transversion to T:A. To identify possible mutational intermediates, various topological forms of the vector DNA were introduced separately. Supercoiled and relaxed DNA are mutated at equal frequencies. However, linearized DNA leads to a greatly elevated frequency of deletions. Nicked and gapped templates stimulate both deletions and base substitutions. We discuss a model involving intracellular degradation of the transfected DNA which explains these observations.     

Characterization of homologous DNA recombination activity in normal and immortal mammalian cells.

Homologous DNA recombination levels were measured in normal and spontaneously immortalized murine and human fibroblasts, and in a number of primate and murine established fibroblast cell lines. Immortal cell lines and tumor-derived clones homologously recombined extrachromosomal plasmid substrates at frequencies approximately 100-fold higher than did normal cells. To further explore the mechanism responsible for this phenotype, homologous recombination frequency was measured using nuclear extracts derived from normal and immortalized murine and human fibroblasts. Extracts prepared from immortal cells catalyzed high levels of homologous recombination, whereas very little recombination activity was detected in extracts prepared from normal fibroblasts. Similarly, only extracts derived from immortal cells contained strand-transferase activity as measured by the recently described pairing-on-membrane assay. Mixing experiments indicated that a recombination enhancing factor or factors present in immortal cells, rather than a recombination inhibitor in normal cells, was responsible for the enhanced homologous recombination activity observed using extracts derived from the former.     

Efficient transfer of chromosome-based DNA constructs into mammalian cells

Artificial chromosomes, engineered minichromosomes and other chromosome-based DNA constructs are promising new vectors for use in gene therapy, protein production and transgenics. However, a major drawback in the application of chromosome-based DNA is the lack of a suitable and convenient procedure for large-scale cellular introduction, which is particularly frustrated by their size (1 by 2 microm). Here we present a method to transfer Artificial Chromosome Expression systems (ACEs) into mammalian cells, which relies on a combined approach of using cationic amphiphiles and high frequency ultrasound. Thus, when cells were preincubated with liposomes consisting of the cationic lipid SAINT-2 and the phospholipid dioleoylphosphatidylethanolamine (molar ratio 1:1), followed by ultrasound, ACEs could be introduced into mammalian cells, which resulted in the expression of ACEs-harbored reporter genes, such as Green Fluorescent Protein. Depending on cell type, transfection efficiencies ranged from 12% to 53%. Interestingly, no detectable delivery occurred when cells were treated alone with either ultrasound or liposomes. Evidence is provided, based on cellular entry of differently sized beads and trypan-blue permeation, which supports a mechanism in which integration of the lipids creates unstable membrane domains, which are particularly prone to ultrasound-induced pore formation. Time- and temperature-dependent experiments indicate that these pores display a transient stability. Hence, following ultrasound, the pores disappear as a function of time as suggested by a time-window for ACEs entry, and trypan blue exclusion, 80% of the cells becoming stained immediately following ultrasound, dropping to approximately 20% after 30 min. Co-expression of different genes in conjunction with fluorescence in situ hybridization (FISH) analysis indicates that the current procedure provides a means to introduce functionally active artificial chromosomes into eukaryotic cells.     

Characterization of nonconservative homologous junctions in mammalian cells.

Homologous recombination in mammalian cells between extrachromosomal molecules, as well as between episomes and chromosomes, can be mediated by a nonconservative mechanism. It has been proposed that the key steps in this process are the generation (by double-strand cleavage) of overlapping homologous ends, the creation of complementary single-strand ends (either by strand-specific exonuclease degradation or by unwinding of the DNA helix), and finally the creation of heteroduplex DNA by the annealing of the single-strand ends. We have analyzed in detail the structure of nonconservative homologous junctions and determined the contribution of each end to the formation of the junction. We have also analyzed multiple descendants from single recombination events. Two types of junctions were found. The majority (90%) of the junctions were characterized by a single crossover site. These crossover sites were distributed randomly throughout the junction. The remaining 10% of the junctions had mosaic patterns of parental markers. Furthermore, in 9 of 10 cases, multiple descendants from a single recombination event were identical. Thus, it appears that in most cases few parental markers were involved in junction formation. This finding suggests that nonconservative homologous junctions are mediated mainly by short heteroduplexes of a few hundred base pairs or less. These results are discussed in terms of the current models of nonconservative homologous recombination.     

Efficient transient and stable expression in mammalian cells of transfected genes using erythrocyte ghost fusion

We have modified the erythrocyte ghost fusion transfection method (F.C. Wiberg, P. Sunnerhagen, K. Kaltoft, J. Zeuthen, and G. Bjursell (1983) Nucleic Acids Res. 11, 7287) by changing the resealing buffer to a Ca2+-containing ghost shrinkage solution, which considerably enhances DNA loading and transfection efficiency. In addition, we have demonstrated the generality of the transfection method by successfully transfecting different cell types, including lymphoid cells, obtaining both transient and long-term expression.     

Efficient expression of an Epstein-Barr nuclear antigen in Drosophila cells transfected with Epstein-Barr virus DNA.

In a search for exogenous promoters which function in cultured Drosophila cells, we have co-transfected a D. melanogaster cell line with an Epstein-Barr virus (EBV) cosmid clone which encodes the Epstein-Barr nuclear antigen (EBNA-1). Here we report that Drosophila cells containing stably integrated copies of EBNA-1 encoding DNA synthesise a polypeptide of mol. wt. identical to that of authentic EBNA-1, which is detectable with EBNA-positive but not EBNA-negative human serum. As in EBV-transformed lymphoblastoid cells, this neo-antigen is associated with the nucleus of transfected cells suggesting that cellular localisation signals which operate in mammalian cells are also recognised in insect cells.