Donald J. Cummings

Are mitochondrial structural genes selectively amplified during senescence in Podospora anserina

Genetic and transcriptional maps have been constructed for the mitochondrial genome of the Ascomycete Podospora anserina. These data have been plotted on the restriction maps for Sal I, Xho I, Bam HI, Eco RI, BgI II and Hae III. We have characterized and cloned a new and unique senescent mitochondrial DNA (beta-event senDNA) and have organized all of the recognized senDNAs on the genomic maps. We make the observation that all of the known and characterized senDNAs are derived from specific genes or gene regions of the young mitochondrial genome. We have unambiguously assigned the alpha-event senDNA (the 2.6 kb monomer) to the oxi3 gene locus and the beta-event senDNA to the oxi2 gene locus.

An additional class II intron with homology to reverse transcriptase in rapidly senescing Podospora anserina

SummarySenescence in Podospora anserina is maternally inherited and the parameters of senescence are race specific. We have compared the restriction enyzme fragment maps of race A, the most rapidly senescing race, with race s and have found three inserts in race A which are not present in race s mitochondrial DNA. Fragment A was mapped and found to be located downstream of the so-called α senDNA, a class II intron, near the 5′ end of the COI gene, separated from a senDNA by two class I introns. DNA sequence analysis showed that fragment A is also a class 11 intron, but with only 10% DNA sequence homology to a senDNA. Like α senDNA, intron A contains significant amino acid homology with known reverse transcriptases. The importance of this additional class II intron in the mitochondrial genome with the relative rate of senescence in race A is discussed.

An unusual region of Paramedum mitochondrial DNA containing chloroplast-like genes

Based on DNA and amino acid comparisons with known genes and their products, a region of the Paramecium aurelia mitochondrial (mt) genome has been found to encode the following gene products: (1) photosystem II protein G (psbG); (2) a large open reading frame (ORF400) which is also found encoded in the chloroplast (cp) DNA of tobacco (as ORF393) and liverwort (as ORF392), and in the kinetoplast maxicircle DNA of Leishmania tarentolae (as ORFs 3 and 4); (3) ribosomal protein L2 (rpl2); (4) ribosomal protein S12 (rps12); (5) ribosomal protein S14 (rps14); and (6) NADH dehydrogenase subunit 2 (ndh2). All of these genes have been found in cp DNA, but the psbG gene has never been identified in a mt genome, and ribosomal protein genes have never been located in an animal or protozoan mitochondrion. The ndh2 gene has been found in both mitochondria and plastids. The Paramecium genes are among the most divergent of those sequenced to date. Two of the genes are encoded on the strand of DNA complementary to that encoding all other known Paramecium mt genes. No gene contains an identifiable intron. The rps12 and psbG genes are probably overlapping. It is not yet known whether these genes are transcribed or have functional gene products. The presence of these genes in the mt genome raises interesting questions concerning their evolutionary origin.

Altered genetic code in Paramecium mitochondria: Possible evolutionary trends

SummaryThe sequence and presumptive structure of a tRNA trp gene from Paramecium tetraaurelia are given. The gene is located 1,500 bp downstream from the 13S rRNA gene, in about the middle of the genome. Paramecium tRNA trp has a completely normal TψC loop and stem, however its anticodon (UCA) constitutes an alteration in the “universal” genetic code, similar to those seen in fungal and mammalian mitochondria. Most features of Paramecium tRNA trp resemble other mitochondrial counterparts; however, its sequence is more homologous to the “unaltered” tRNA trp (anticodon CCA) from E. coli. Paramecium mitochondria may resemble a primitive stage of organelle evolution.

Transcription of a mitochondrial plasmid during senescence in Podospora anserina.

SummaryIn the ascomycete fungus Podospora anserina, cellular senescence is characterized by the excision, circularization, and amplification of specific segments of the non-senescent mitochondrial genome. During senescence, various plasmids can be found in the mitochondria, and different senescent events produce different plasmid populations. In this paper we have examined the transcriptional activity of one mitochondrial plasmid (α-sen DNA) and have contrasted this with the non-senescent mitochondrial genome of rapidly (A+) and slowly (s+) senescing races. In non-senescent and senescent mitochondria we observe two RNAs which are homologous to α-sen DNA and to the parental locus on the native genome. These are 2.4 and 2.5 kb long and have different 5′ ends while overlapping throughout most of their lengths. They may represent different transcripts for α-sen DNA and the parental genome and indicate that excision of the plasmid begins 450 bp from the 5′ end of the genomic coding sequence. Transcription of the α-sen DNA plasmid appears to be active in both senescent and in non-senescent mycelia.

Structural and functional analysis of the origin of replication of mitochondrial DNA from Paramecium aurelia. II: A-T rich repeat units serve as autonomously replicating sequences

SummaryReplication of mitochondrial DNA in Paramecium aurelia involves the formation of a covalent crosslink at one end of this linear molecule and proceeds unidirectionally, producing a dimer consisting of two head to head monomers. The initiation regions within the dimer molecules have been sequenced and shown to be palindromic except for a central nonpalindromic A+T rich sequence, arranged in direct tandem repeats. This nonpalindromic region (see accompanying paper) has been identified as the cross-link which converts the initiation terminus into a continuous sequence. In this study, yeast transformation was used to assay the dimer initiation regions of P. aurelia mtDNA for the presence of autonomously replicating sequences. P. aurelia mtDNA fragments from species 1 and 4 were cloned into the yeast vector YIP5 and the hybrid plasmids (YPaM) were used to transform yeast. The dimer initiation regions from both species promoted high frequency transformation and extrachromosomal maintenance of YPaM plasmids. Subcloning analysis of the ARS-containing mtDNA fragments indicates, specifically, that the nonpalindrome, repetitive sequences are responsible for the autonomously replicating properties of YPaM plasmids.