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Tracts of the alternating dinucleotide polydeoxythymidylic-guanylic [d(TG)] polydeoxyadenylic-cytidylic acid [d(AC)], present throughout the human genome, are capable of readily forming left-handed Z-DNA in vitro. We have analyzed the effects of the Z-DNA motif d(TG)30 upon homologous recombination between two nonreplicating plasmid substrates cotransfected into human cells in culture. In this study, the sequence d(TG)30 is shown to stimulate homologous recombination up to 20-fold. Enhancement is specific to the Z-DNA motif; a control DNA fragment of similar size does not alter the recombination frequency. The stimulation of recombination is observed at a distance (237 to 1,269 base pairs away from the Z-DNA motif) and involves both gene conversion and reciprocal exchange events. Maximum stimulation is observed when the sequence is present in both substrates, but it is capable of stimulating when present in only one substrate. Analysis of recombination products indicates that the Z-DNA motif increases the frequency and alters the distribution of multiple, unselected recombination events. Specifically designed crosses indicate that the substrate containing the Z-DNA motif preferentially acts as the recipient of genetic information during gene conversion events. Models describing how left-handed Z-DNA sequences might promote the initiation of homologous recombination are presented.Homologous recombination is the process of exchange of genetic information between stretches of DNA containing sequence similarity. Although the degree of recombination is generally proportional to the length of homology, there are regions of DNA in which the rate of exchange is nonlinear with respect to distance, resulting in disparity between genetic and physical maps. The sequences responsible for this disparity, called "recombination hotspots," increase the rate of genetic exchange between homologous DNA molecules. The 8-base-pair (bp) Chi sequence in Escherichia coli is the most fully characterized recombination hotspot, shown to increase recombination in bacteriophage lambda and E. coli DNA via the RecA-RecBCD pathway (reviewed in reference 34). Recombination hotspots have also been described in fungi (12,25) and in mammalian cells (37; W. P. Wahls, L. J. Wallace, and P. D. Moore, Cell, in press).A short fragment of mammalian DNA from the human ,Bglobin gene cluster, MG-1 (29), promotes unusual recombination events during yeast meiosis (41). Molecular evidence indicates that the DNA sequence responsible is an 80-bp stretch of the simple repeating dinucleotide polydeoxythymidylic-guanylic polydeoxyadenylic-cytidylic acid, abbreviated hereafter as d(TG)n.The repeating dinucleotide d(TG)" is not detectable in the genomes of eubacteria, archaebacteria, or mitochondria (11) but is ubiquitously present in eucaryotic chromosomes (13,29,40). It is estimated that there are on the order of 105 copies of this dinucleotide repeat, each approximately 10 to 50 bp in length, scattered throughout the mammalian genome. A striking feature of these r...

We have examined the ability of singlestranded DNA to participate in homologous recombination reactions in mammalian cells and nuclear extracts derived from them. We have inserted a fragment of the neo gene into the single-stranded DNA phage vector M13 mphl. The neo fragment was derived from a deletion derivative of the prokaryotic-eukaryotic shuttle vector pSV2neo. The resulting singlestranded DNA was mixed with a double-stranded deletion derivative of pSV2neo and tested for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells. We were able to obtain recombinant molecules containing wild-type neo genes in all three systems. Examination of the products of recombination indicated that they resulted from correction of the deletion in the double-stranded DNA substrate. We were unable to detect any extensive conversion of single-stranded DNA into its double-stranded counterpart before it participated in the recombination reaction. We have also tested the ability of single-stranded DNA to yield transfectants. When a single-stranded DNA derivative of the herpes simplex virus thymidine kinase (TK) gene was introduced into mouse L-M(TK-) cells, we were able to obtain TKV colonies.From these results, we conclude that single-stranded DNA can participate in transfection as well as homologous recombination reactions in mammalian cells.Integration of exogenous DNA into mammalian chromosomes by homologous recombination would be the preferred strategy for gene replacement therapy and in vivo gene modification. Mammalian somatic cells have been shown to contain all of the enzymes required to mediate recombination between homologous viral or plasmid sequences cotransfected into cells or between chromosomal sequences and their homologous counterparts (1-6). The exact nature of the proteins involved and the mechanism by which homologous recombination occurs in mammalian cells is not understood.We have developed a set of three systems to study the genetic and biochemical aspects of homologous recombination in mammalian cells (7-9). All of these involve the use of two derivatives of the prokaryotic-eukaryotic shuttle vector pSV2neo, a plasmid containing the bacterial gene for aminoglycoside 3'-phosphotransferase, which can be expressed in bacterial as well as in mammalian cells, resulting in neomycin or kanamycin resistance in bacteria and G418 resistance in mammalian cells. It also has an ampicillinresistance (AmpR) gene. Each of the two recombination substrates is a nonoverlapping, nonreverting deletion derivative of pSV2neo. The two deletion derivatives are referred to as pSV2neo DL and pSV2neo DR (7). In the first system, homologous recombination is studied by cotransfecting the two deletion plasmids into mammalian cells and selecting the cells for the expression of the normal neo gene, which can be generated by homologous recombination (7). The second method, somewhat analogous to studying bacteriophage recombination, involves introducing these substrates into monkey COS cells (10) ...

In Escherichia coli a single copy of Tn10 confers high-level resistance to tetracycline. Resistance itself results from expression of three distinct mechanisms which normally act together (Shales et al., 1980). In cells containing two copies of Tn10, the level of resistance to tetracycline was reduced. This was not due to overproduction of the repressor which controls the resistance genes, because strains diploid for an operator-constitutive allele of Tn10 also exhibited reduced expression of resistance. The negative gene dosage effect resulted from decreased expression of two mechanisms (1 and 2) consequent on enhanced expression of the third mechanism. The net result of increasing the copy number was a decrease in resistance because mechanism 3 was less efficient than mechanisms 1 and 2 in protecting the cell against tetracycline. The DNA sequence responsible for the reduced expression of resistance was contained in the internal BglII fragment of Tn10. This sequence, which is probably unique to Tn10, may encode the protein which mediates mechanism 3. Elevated levels of this protein probably cause decreased expression of mechanisms 1 and 2.

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