Curr Genet (1995) 28:429-436 9 Springer-Verlag 1995 Jennifer E. Graham 9 Jonathan G. Spanier 9 Jonathan W. Jarvik Isolation and characterization of Pioneer1, a novel Chlamydomonas transposable element Received: 2 February / 21 April 1995 Abstract During the course of this study a novel family of Chlamydomonas mobile elements has been identified in natural isolate strain 224. The first member of this class to be characterized, a 2.8-kb element named Pioneerl, was trapped in an intron of the nitrate reductase structural gene, NIT1. This element has been cloned and completely se- quenced and found to be unusual in structure. Pioneer elements are present in a very low-copy number of three per genome in strain 224. The copy number increased by one upon transposition of Pioneerl. Hybridization of Pioneerl to a variety of Chlamydomonas strains confirmed that this element differed from previously described Chla- mydomonas transposons. It also indicated that related ele- ments are present in low-copy number in natural isolate strains 356 and S 1D2, but not in the most commonly used laboratory strains 137c and 21 gr. For these reasons, mem- bers of the Pioneer family might prove useful as insertional mutagens. Key words Chlamydomonas reinhardtii 9 Transposon 9 Nitrate reductase Introduction Transposable elements affect the regulation of gene ex- pression and are thought to play a role in shaping genomes through evolution. They are also useful as molecular ge- netic tools. A transposon tagging system in Chlamydomo- J. E. Graham ([])l. j. G. Spanier 1 . J. W. Jarvik Department of Biological Sciences, Carnegie Mellon University 4400 Fifth Avenue, Pittsburgh, PA 15213, USA i Present address: Department of Microbiology and Immunology, Box 9037, University of North Dakota School of Medicine, Grand Forks, ND 58202, USA Communicated by K. P. Van Winkle-Swift nas reinhardtii would facilitate the isolation of genes re- sponsible for a variety of cellular processes such as the bi- ogenesis and function of the flagellar apparatus, photore- ception, mating, and cell differentiation. Two different families of transposons have been iden- tified in C. reinhardtii: Gulliver and TOC1. Gulliver, a 12-kb element discovered by Ferris (1989), appears to be a class-II (DNA-mediated) element since it has 15-bp im- perfect inverted repeats at the ends. It resembles the maize transposon Ac in that it produces an 8-bp target-site dupli- cation upon insertion, and in the similarity of its 7-bp ter- minal sequence to that of Ac. Approximately 12 copies of Gulliver are present per genome in C. reinhardtii, with dif- ferent laboratory strains exhibiting numerous restriction fragment length polymorphisms (RFLPs). Day et al. (1988) have isolated members of a second family of C. reinhardtii transposons, the prototype of which is called TOCI.1. TOCI.1 was first detected as a 5.7-kb insertion in an intron of the OEE1 (oxygen-evolv- ing enhancer 1) gene of mutant strain FUD44 (Mayfield et al. 1987). It resembles retrotransposons in that it has long terminal repeats (LTRs) (Day et al. 1988). However, they are arranged unusually in that only part of an LTR is present on the left end of TOC1.1. The missing part resides on the right end of the element. The copy number of TOC 1.1 in various strains ranges from two to more than 30 per ge- nome. Gulliver has recently been used to tag NIT2, a Chlamy- domonas nitrate reductase regulatory gene (Schnell and Lefebvre 1993). However, it is not clear at this time whether Gulliver or TOC1.1 are suitable elements for the development of a generally useful transposon tagging system since it has not been demonstrated that they move randomly throughout the genome at a sufficiently high fre- quency. We wished to identify new Chlamydomonas trans- posons which might be used to develop a transposon tagg- ing system. In this study, we used the nitrate reductase structural gene, NIT1, to trap Chlamydomonas transpo- sons in the new natural isolate strain 224 (CC-2343; Spa- nier et al. 1992) and have isolated and characterized a novel mobile element which we name Pioneerl.