Mol Cell Biol 5(12): 3484-3496

Extrachromosomal DNA transformation of Caenorhabditis elegans.

Abstract

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 high-molecular-weight arrays. Surprisingly, these high-molecular-weight arrays were extrachromosomal and heritable. On average 50% of the progeny of a transformed hermaphrodite still carried the exogenous sequences. In situ hybridization experiments demonstrated that approximately half of the transformed animals carried foreign DNA in all of their cells; the remainder were mosaic animals in which some cells contained the exogenous sequences while others carried no detectable foreign DNA. The presence of mosaic and nonmosaic nematodes in transformed populations may permit detailed analysis of the expression and function of C. elegans genes.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.4M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Image
on p.3489

Image
on p.3491

Image
on p.3493

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

Albertson DG. Localization of the ribosomal genes in Caenorhabditis elegans chromosomes by in situ hybridization using biotin-labeled probes. EMBO J. 1984 Jun;3(6):1227–1234.[PMC free article] [PubMed] [Google Scholar]
Albertson DG, Thomson JN. The kinetochores of Caenorhabditis elegans. Chromosoma. 1982;86(3):409–428. [PubMed] [Google Scholar]
Bishop JM. Cellular oncogenes and retroviruses. Annu Rev Biochem. 1983;52:301–354. [PubMed] [Google Scholar]
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974 May;77(1):71–94.[PMC free article] [PubMed] [Google Scholar]
Brinster RL, Chen HY, Trumbauer M, Senear AW, Warren R, Palmiter RD. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell. 1981 Nov;27(1 Pt 2):223–231.[PMC free article] [PubMed] [Google Scholar]
Brinster RL, Ritchie KA, Hammer RE, O'Brien RL, Arp B, Storb U. Expression of a microinjected immunoglobulin gene in the spleen of transgenic mice. Nature. 1983 Nov 24;306(5941):332–336.[PMC free article] [PubMed] [Google Scholar]
Capecchi MR. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980 Nov;22(2 Pt 2):479–488. [PubMed] [Google Scholar]
Clarke L, Carbon J. Genomic substitutions of centromeres in Saccharomyces cerevisiae. Nature. 1983 Sep 1;305(5929):23–28. [PubMed] [Google Scholar]
Costantini F, Lacy E. Introduction of a rabbit beta-globin gene into the mouse germ line. Nature. 1981 Nov 5;294(5836):92–94. [PubMed] [Google Scholar]
Emmons SW, Klass MR, Hirsh D. Analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1333–1337.[PMC free article] [PubMed] [Google Scholar]
Emmons SW, Yesner L, Ruan KS, Katzenberg D. Evidence for a transposon in Caenorhabditis elegans. Cell. 1983 Jan;32(1):55–65. [PubMed] [Google Scholar]
Etkin LD, Roberts M. Transmission of integrated sea urchin histone genes by nuclear transplantation in Xenopus laevis. Science. 1983 Jul 1;221(4605):67–69. [PubMed] [Google Scholar]
Files JG, Carr S, Hirsh D. Actin gene family of Caenorhabditis elegans. J Mol Biol. 1983 Mar 5;164(3):355–375. [PubMed] [Google Scholar]
Goldberg DA, Posakony JW, Maniatis T. Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line. Cell. 1983 Aug;34(1):59–73. [PubMed] [Google Scholar]
Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7380–7384.[PMC free article] [PubMed] [Google Scholar]
Grosschedl R, Weaver D, Baltimore D, Costantini F. Introduction of a mu immunoglobulin gene into the mouse germ line: specific expression in lymphoid cells and synthesis of functional antibody. Cell. 1984 Oct;38(3):647–658. [PubMed] [Google Scholar]
Herman RK. Analysis of genetic mosaics of the nematode Caneorhabditis elegans. Genetics. 1984 Sep;108(1):165–180.[PMC free article] [PubMed] [Google Scholar]
Herman RK, Madl JE, Kari CK. Duplications in Caenorhabditis elegans. Genetics. 1979 Jun;92(2):419–435.[PMC free article] [PubMed] [Google Scholar]
Hirsh D, Oppenheim D, Klass M. Development of the reproductive system of Caenorhabditis elegans. Dev Biol. 1976 Mar;49(1):200–219. [PubMed] [Google Scholar]
Karn J, Brenner S, Barnett L. Protein structural domains in the Caenorhabditis elegans unc-54 myosin heavy chain gene are not separated by introns. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4253–4257.[PMC free article] [PubMed] [Google Scholar]
Kimble J, Hirsh D. The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. Dev Biol. 1979 Jun;70(2):396–417. [PubMed] [Google Scholar]
Kimble J, Hodgkin J, Smith T, Smith J. Suppression of an amber mutation by microinjection of suppressor tRNA in C. elegans. Nature. 1982 Sep 30;299(5882):456–458. [PubMed] [Google Scholar]
Liao LW, Rosenzweig B, Hirsh D. Analysis of a transposable element in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3585–3589.[PMC free article] [PubMed] [Google Scholar]
MacLeod AR, Karn J, Brenner S. Molecular analysis of the unc-54 myosin heavy-chain gene of Caenorhabditis elegans. Nature. 1981 Jun 4;291(5814):386–390. [PubMed] [Google Scholar]
Mandel M, Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. [PubMed] [Google Scholar]
McDonell MW, Simon MN, Studier FW. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. [PubMed] [Google Scholar]
McKnight GS, Hammer RE, Kuenzel EA, Brinster RL. Expression of the chicken transferrin gene in transgenic mice. Cell. 1983 Sep;34(2):335–341. [PubMed] [Google Scholar]
Nasmyth KA. Molecular genetics of yeast mating type. Annu Rev Genet. 1982;16:439–500. [PubMed] [Google Scholar]
NIGON V, BRUN J. L'évolution des structures nucléaires dans l'ovogenèse de Caenorhabditis elegans Maupas 1900. Chromosoma. 1955;7(2-3):129–169. [PubMed] [Google Scholar]
Orr-Weaver TL, Szostak JW, Rothstein RJ. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358.[PMC free article] [PubMed] [Google Scholar]
Pardue ML, Gall JG. Nucleic acid hybridization to the DNA of cytological preparations. Methods Cell Biol. 1975;10:1–16. [PubMed] [Google Scholar]
Rigby PW, Dieckmann M, Rhodes C, Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. [PubMed] [Google Scholar]
Rubin GM, Spradling AC. Genetic transformation of Drosophila with transposable element vectors. Science. 1982 Oct 22;218(4570):348–353. [PubMed] [Google Scholar]
Sakaguchi J, Yamamoto M. Cloned ural locus of Schizosaccharomyces pombe propagates autonomously in this yeast assuming a polymeric form. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7819–7823.[PMC free article] [PubMed] [Google Scholar]
Scangos G, Ruddle FH. Mechanisms and applications of DNA-mediated gene transfer in mammalian cells - a review. Gene. 1981 Jun-Jul;14(1-2):1–10. [PubMed] [Google Scholar]
Scholnick SB, Morgan BA, Hirsh J. The cloned dopa decarboxylase gene is developmentally regulated when reintegrated into the Drosophila genome. Cell. 1983 Aug;34(1):37–45. [PubMed] [Google Scholar]
Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed] [Google Scholar]
Southern PJ, Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
Spradling AC, Rubin GM. The effect of chromosomal position on the expression of the Drosophila xanthine dehydrogenase gene. Cell. 1983 Aug;34(1):47–57. [PubMed] [Google Scholar]
Stinchcomb DT, Mann C, Davis RW. Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol. 1982 Jun 25;158(2):157–190. [PubMed] [Google Scholar]
Sulston JE. Post-embryonic development in the ventral cord of Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci. 1976 Aug 10;275(938):287–297. [PubMed] [Google Scholar]
Sulston JE, Horvitz HR. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev Biol. 1977 Mar;56(1):110–156. [PubMed] [Google Scholar]
Swift GH, Hammer RE, MacDonald RJ, Brinster RL. Tissue-specific expression of the rat pancreatic elastase I gene in transgenic mice. Cell. 1984 Oct;38(3):639–646. [PubMed] [Google Scholar]
Szostak JW, Blackburn EH. Cloning yeast telomeres on linear plasmid vectors. Cell. 1982 May;29(1):245–255. [PubMed] [Google Scholar]
Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed] [Google Scholar]
Wagner EF, Stewart TA, Mintz B. The human beta-globin gene and a functional viral thymidine kinase gene in developing mice. Proc Natl Acad Sci U S A. 1981 Aug;78(8):5016–5020.[PMC free article] [PubMed] [Google Scholar]

Abstract