Curr Genet (1989) 16:407-418 Current Genetics © Springer-Verlag 1989 DNA sequence analysis of the apocytochrome b gene of Podospora anserina: a new family of intronic open reading frame Donald J. Cummings l, Frangois Michel 2, and Kenneth L. McNally 1 Department of Microbiologyand Immunology,Universityof Colorado School of Medicine, Denver, CO 80262,USA 2 Centre de G6n6tiqueMol~culairedu CNRS, F-91190 Gif sur Yvette, France Summary. The 5,969 bp (base pair) DNA sequence of the apocytochrome b mitochondrial (mt) gene of race A Podospora anserina was located in a 8.5 Kbp region. This gene contained a 2,499 bp subgroup IB and a 1,306 bp subgroup ID intron as well as a 990 bp subgroup IB intron which is present in race A but not race s. The large subgroup IB intron and the race A specific IB intron both contained potential alternate splice sites which brought their open reading frames into phase with their upstream exon sequences. All three introns were compared with regard to their secondary structures and open reading frames to the other 30 group I introns in Podospora anserina, as well as to other fungal introns. We detected a new family of intronic ORFs comprising seven P. anserina introns, several N. crassa introns, as well as the T4td bacterio- phage intron. Sequence similarities to intron-encoded endonucleases were noteworthy. The DNA sequences reported here and in the accompanying paper complete the analysis of race s and race A mitochondrial DNA. Key words: Podospora mitochondrial DNA group 1 introns with alternate splice sites - Intron secondary structure-open reading frames. Introduction The DNA sequence of the apocytochrome b (cytb) gene from N. crassa (Burke et al. 1984; Collins et al. 1988), S. cerevisiae (Nobrega and Tzagoloff 1980) and A. nidulans (Waring et al. 1981) shows that all three genes are interrupted by introns, two, three and one, respectively. For the past four years we have focussed Offprint requests to: D. J. Cummings our attention on the DNA sequence of another asco- mycete, Podospora anserina (Cummings et al. 1985; Matsuura et al. 1986; Cummings et al. 1988; Cummings and Domenico 1988; Cummings et al. 1989a and 1989b; Cummings, manuscripts submitted). In parti- cular, we have compared the sequences of race A with race s. This was done primarily because these two races exhibit different rates of maternally inherited senescen- ce (Rizet 1957) and the two mitochondrial genomes differ in size (t01 Kbp for race A as compared to about 94 Kbp for race s, Cummings et al. 1979 and Kt~ck et al. 1985). We have shown that this difference in size is due to three additions to the race A genome, one in the COI gene (Matsuura et al. 1986) one in the ND4L gene (manuscript submitted) and the last shown here to be in the apocytochrome b gene. All three additions are introns, two group I and one group II (see Michel et al. 1982, Davies et al. 1982 and the accompanying paper for discussions of the secondary structure characteri- stics of group I and group II introns). In this report, we present the complete DNA sequence of the cyt b gene from Podospora anserina. This gene is 5,956 bp in length in race A but is only 4,966 bp in race s, due to the presence in race A of an additional subgroup IB intron, 990 bp in length. For both races, the cytb gene is interrupted by a 2,499 bp subgroup IB and a 1,306 bp subgroup ID intron. The sequences reported here and in the accompanying paper complete the analysis of 100,314 bp race A and 94,192 bp race s mitochondrial genomes. The entire continuous DNA sequence for the genomes will be reported separately. Materials and methods Plasmids utilized. Podospora anserina mitochondrial clones for fragments EcoRI-4 and 11from race s(+) matingtype and PstI-2 and 4b from raceA(+) were prepared and purifiedas before(Wrightet al.