We describe a dominant behavioral marker, rol‐6(su‐1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double‐strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single‐stranded oligonucleotide was co‐injected with the double‐stranded DNA.

The molecular analysis of gene structure, function, and regulation depends upon the ability to correlate physiological, genetic, and structural data relating to a specific gene or set of genes. Many mutants of the yeast Saccharomyces cerevisiae have been isolated, and techniques for the manipulation and mapping of the associated genetic characteristics are routinely performed (1). Genetically defined yeast DNA sequences have been isolated in the form of viable molecular hybrids with bacteriophage X or Escherichia coli plasmids (2-5). Derivatives of the cloned his3 gene that delete DNA sequences near or in the structural gene have been isolated and physically defined (unpublished data). The demonstration by Hinnen et al. (5) that recombinant DNA containing cloned yeast genes can be used to transform yeast cells clearly expands the potential of molecular analysis considerably. These workers showed that yeast transformation occurred at low frequency and that it was usually accompanied by homologous recombination between the transforming DNA and the host chromosomal DNA.The present paper reports two additional modes of yeast transformation. In both, the transformation event occurs at high frequency and is associated with autonomous replication of the transforming DNA. Yeast vectors useful for a wide variety of genetic manipulations have been constructed by combining the three mechanistically different modes of transformation with structural information of the endogenous yeast plasmid (6) MATERIALS AND METHODS Organisms, DNAs, and Enzymes. The following strains were used: yeast-A3617C (a his3-532 gal2) (3) and D13-IA (a his3-532 trpl gal2); E. coli-trpC 9830 (7), hisB 463, SF8, C600 (rK -mK+) (2), and MB1000 (rK-mK+lac-trp-pyrF-) (D. Botstein, personal communication); phage-Xgt-Sc2601 (2), X590 (8), and Xgt-Sc4104; plasmid DNAs-pMB9-Sc2601 (3), Scpl (6), pBR322 (9), pGT2-Sc2605, pBR322-Sc2676 (unpublished data), and pMB1068 (D. Botstein, personal communication). Propagation of strains and preparation of DNAs have been described (2, 3, 6).EcoRI, E. coli DNA ligase, and deoxynucleotidyl terminal transferase were the gifts of Marj Thomas, Robert Alazard, and Tom St. John, respectively. Other restriction endonucleases were purchased from New England BioLabs and Bethesda Research Laboratories (Rockville, MD) and used as directed. Cloning procedures have been described (2, 10).Where appropriate p2,EK1 conditions, as described by the National Institutes of Health Guidelines fo Recombinant DNA Research, were used.Rapid Yeast DNA Preparations. Total yeast DNA was prepared from 5-ml cultures of cells grown to the stationary phase. Yeast cells were harvested and resuspended in 0.4 ml of 0.9 M sorbitol/50 mM potassium phosphate, pH 7.5/14 mM 2-mercaptoethanol. Lyticase (25 units) (a gift from R. Schekman) was added and spheroplast formation was allowed to proceed for 30 min at 300C. At this stage, the procedure for rapid phage DNA preparations (6)

A yeast DNA sequence that behaves as a chromosomal replicator, ars1 (autonomously replicating sequence), has been isolated. On transformation, ars1 allows autonomous replication of all co-linear DNA. The replicator can integrate into other replication units and can function in multimeric form. The 850-base pair ars1 element has no detectable homology to other yeast sequences. Such replicator-containing plasmids can be used for the isolation of DNA sequences in yeast cells as well as for the study of chromosomal DNA replication.

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DNA was introduced into the germ line of the nematode Caenorhabditis elegans by microinjection.Approximately 10% of the injected worms gave rise to transformed progeny. Upon injection, supercoiled molecules formed a high-molecular-weight array predominantly composed of tandem repeats of the injected sequence. Injected linear molecules formed both tandem and inverted repeats as if they had ligated to each other. No worm DNA sequences were required in the injected plasmid for the formation of these highmolecular-weight arrays. Surprisingly, these high-molecular-weight arrays were Similarly, techniques for DNA transformation of multicellular organisms facilitate the molecular analysis of developmental processes. DNA transformation of multicellular organisms has been achieved by microinjection of fertilized eggs of mice (6,10,18,53), frogs (14), sea urchins (16), and Drosophila melanogaster (39). In D. melanogaster (17,42,45) and in several cases in mice (7,19,50) the reintroduced genes appear to be properly regulated during development. Thus, DNA transformation permits studies of the expression of isolated sequences in specific tissues at specific times. DNA transformation may also be used to identify and isolate other sequences that encode functions vital for determination and development.The nematode Caenorhabditis elegans has been used widely for studies of developmental genetics. Its simple anatomy and life cycle have permitted the characterization of mutants defective in particular tissues, behaviors, and developmental pathways (5). The cell lineage of the entire worm is known (25,48,49) and developmental physiology of the nematode. Here we describe the introduction of foreign DNA into the C. elegans genome by microinjection into the' gonad. The exogenous DNA formed a high-molecular-weight concatamer which was extrachromosomal and heritable. These experiments, demonstrating DNA transformation of C. elegans, provide a foundation for studying transformation and expression of developmentally interesting genes. MATERIALS AND METHODSStrains and media. C. elegans var. Bristol N2 (5) or a strain lacking the major DNase of the worm [nuc-J(e1392)X] (47) were used for the injections. DSB40 (alias Escherichia coli HB101) was used for bacterial transformation experiments. C. elegans w,as grown and manipulated as described by Brenner (5). 'To genetically manipulate the transformants, males were isolated from a population 'of 'transformed hermaphrodites that had been incubated at 30°C for 5 h. Individual males were mated with MT465 [dpy-5(e61)I bli-2(e768)1I; unc-32(e189)III] and MT464 [unc-5(e53)IV; dpy-11(e224)V; lon-2(e678)XJ provided by the C. elegans Genetic Stock Center. Bacteria were grown and manipulated as described by Davis et al. (11). J3acterial strains harboring plasmids used for injection were generously provided by P. Southern (pSV2neo) and R. Jefferson (pCEV70.3-neo). YRp17 has been described previously (46). YRp17-unc was constructed by inserting the 3.8-kilobase-pair (kpb) BglII fragment of the unc-54 ge...