Curr Genet (1989) 16:187-194 CurrentGenetics © Springer-Verlag 1989 Characterization of the termini of linear plasmids from Nectria haematococca and their use in construction of an autonomously replicating transformation vector Deborah A. Samac 1 and Sally A. Leong 1, 2 IDepartment of Plant Pathology and 2Plant Disease Resistance Research Unit, USDA, Agricultural Research Service University of Wisconsin-Madison, 1630 Linden Drive, Madison, WI 53706, USA Summary. The mitochondria of isolate FS37 from Nectria haematococca mating population I (Fusarium solani f. sp. cucurbitae) contain two linear plasmids, pFSC1 and pFSC2, of 9.2 and 8.3 kbp, respectively. Evidence for a protein blocking the 5' termini of these plasmids was obtained from exonuclease digestion experiments. A single protein band with an apparent Mr of 80 K was labeled when the DNA-protein com- plex of either plasmid was reacted with [125I] Bolton- Hunter reagent and then digested with DNase I. DNA sequence analysis of the termini of both plasmids revealed long inverted repeats of 1,211 bp (pFSC1) and 1,027 bp (pFSC2). No sequence similarity was found between the terminal inverted repeats (TIRs) ofpFSC1 and pFSC2, nor was any similarity identified between the TIRs of the these plasmids and sequences of TIRs from other linear DNAs. A restriction fragment con- taining the TIR of pFSC1 conferred autonomous replication when incorporated into an integrative transformation vector of Ustilago maydis. Key words: Fusarium solani - Mitochondrial plasmids - Terminal inverted repeats - Terminal proteins Introduction Linear double-stranded DNAs require specialized ter- minal structures to replicate in a cell. All DNA polymerases require a DNA or RNA primer with a free 3'-OH to initiate DNA replication. Replication of the linear double stranded DNA genomes of adenovirus and the Bacillus subtilis bacteriophage ci)29 initiates at the termini and requires the presence of terminal inverted repeat sequences (TIRs) and a terminal prot- ein (Escarmis et al. 1984; Garcia et al. 1983; Sussen- bach 1984; Yoshikawa and Ito 1981). The terminal Offprint requests to: S. Leong protein, covalently linked to the first 5' nucleotide, is a primer for DNA polymerase (Challberg et al. 1980; Mellado et al. 1980). The role of TIRs in DNA replication is less clear. The terminal sequences may be necessary for recognition of the origin of replication by DNA polymerase or by DNA binding proteins (Guti- errez et al. 1988; Sussenbach 1984). Interestingly, linear plasmids found in plants (Erickson et al. 1985; Kemble and Thompson 1982; Levings and Sederoff 1983; Paillard et al., 1985; Turpen et al. 1987), bacteria (Hirochika et al. 1984), and fungi (Duvell et al. 1988; Hishinuma et al. 1984; Kikuchi et al. 1984; Kistler and Leong 1986; Meinhardt et al. 1986) also have putative terminal proteins and TIRs. Very little is known about the terminal proteins from linear plasmids. In most cases, terminal proteins have been identified on the basis of protection of DNA from 5' specific exonucleases. It has been proposed, on the basis of similarity with adenovirus and (I)29, that plasmid terminal proteins play a role in DNA replica- tion (Kemble and Thompson 1982). Terminal inverted repeats of plasmids have been identified primarily by electron microscopy of intra- molecular homoduplexes; the DNA sequence is known for only a few plasmid TIRs (Duvell et a1.1988; Hishinuma et al. 1984; Levings and Sederoff 1983; Paillard et al. 1985). The role of the TIRs in linear plasmids, and whether they are required for replica- tion, is not known. If TIRs function as origins of replication, they may be useful in constructing autono- mously replicating transformation vectors. Mitochondria of an isolate of Nectria haematococ- ca mating population I, a fungal pathogen of cucur- bits, contain two linear plasmid DNAs (Samac and Leong 1988), pFSC1 (9.2kbp) and pFSC2 (8.3kbp). Here we report the physical properties of the termini of pFSC1 and pFSC2 and the construction of an autonomously replicating vector incorporating DNA from pFSC1.