Search PubMed⌕ Search

Biomedical subjects

D J Cummings

Publications and source records attributed to D J Cummings.

At least 37 records · Page 2Linked to original sources
PubMed1987

A novel family of mitochondrial plasmids associated with longevity mutants of Podospora anserina.

We have identified a novel family of plasmids, each containing very short monomeric units, in Podospora anserina longevity mutants. These plasmids, termed small mitochondrial DNAs (sMt-DNAs), are derived from a highly ordered 368-base pair region of the mitochondrial genome. A total of five direct repeat sequences and seven significant regions of dyad symmetry (i.e. palindromes) were found within a 434-base pair mitochondrial sequence, which includes this 368-base pair region. Mitochondrial DNA rearrangements accompany the formation of these small plasmids indicating their derivation from a plastic region of the mitochondrial genome. A possible relationship between the direct repeat sequences, the palindromic regions, and the excision process is discussed.

Ascomycota↗
PubMed1987

Podospora anserina does not senesce when serially passaged in liquid culture.

A procedure was developed for the prolonged growth of the ascomycete fungus Podospora anserina in liquid culture to determine the effects of such growth on the senescence phenotype. Senescence in P. anserina, which is maternally inherited and associated with the excision and amplification of specific mitochondrial plasmids, occurs when this species is grown on solid medium. In two independent experiments no evidence of senescence was observed as mycelia were serially passaged in liquid culture. Further, when separable mycelial masses, termed puff balls, from the liquid cultures were plated on solid medium, a significant increase in their average longevity was observed. The apparent immortality of P. anserina in liquid culture was not dependent upon mitochondrial DNA rearrangements, nor was it affected by the presence of a previously described senescence plasmid, alpha senDNA. Evidence was obtained indicating that growth in liquid culture exerts selective pressure to maintain the wild-type mitochondrial genome.

Ascomycota↗
PubMed1987

A Podospora anserina longevity mutant with a temperature-sensitive phenotype for senescence.

A Podospora anserina longevity mutant was identified with a temperature-sensitive phenotype for senescence. This mutant, termed TS1, grew for over 3 m at 27 degrees C, but when shifted to 34 degrees C, it underwent senescence between 10 and 18 cm. A previously described senescence-associated plasmid, alpha senDNA, derived from the mitochondrial genome, was not detected in TS1 at 27 degrees C but was present in senescent cultures at 34 degrees C. A similar result was observed in progeny strains obtained by crossing the TS1 mutant with a wild-type strain. Other mitochondrial excision-amplification DNAs in addition to alpha senDNA were also observed in the senescent cultures. Most were derived from a specific region of the mitochondrial genome. These results provide evidence that alpha senDNA is involved in TS1 senescence and suggest that this plasmid may play a role in the formation of other mitochondrial excision-amplification plasmids.

Aging↗
PubMed1986

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

Senescence in Podospora anserina is maternally inherited and the parameters of senescence are race specific. We have compared the restriction enzyme 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 alpha senDNA, a class II intron, near the 5' end of the COI gene, separated from alpha senDNA by two class I introns. DNA sequence analysis showed that fragment A is also a class II intron, but with only 10% DNA sequence homology to alpha senDNA. Like alpha 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.

Amino Acid Sequence↗
PubMed1986

A DNA polymerase activity with characteristics of a reverse transcriptase in Podospora anserina.

In the filamentous ascomycete fungus Podospora anserina, senescence is associated with dramatic changes of the juvenile mitochondrial genome. During senescence specific regions of the non-senescent genome are excised, ligated and amplified as a plasmid. In this paper, we report our studies on nuclear-mitochondrial extracts for the presence of RNA-dependent DNA polymerase activity (reverse transcriptase). In so-called middle aged mycelia of race A, we detected a DNA polymerase possessing properties of a reverse transcriptase. It prefers the ribopolymer templates poly(rA)-oligo(dT)12-18 and poly(rCm)-oligo(dG)12-18 over the deoxypolymer template poly(dA)-oligo(dT)12-18. It also uses natural rabbit globin mRNA as a template. The enzymatic activity can clearly be distinguished from a second DNA polymerase activity found in the same extracts of both young and middle aged mycelia.

Aging↗
PubMed1986

Paramecium mitochondrial DNA sequences and RNA transcripts for cytochrome oxidase subunit I, URF1, and three ORFs adjacent to the replication origin.

A 2-kb region adjacent to the replication origin (ori) and a 3-kb region located between the small and large ribosomal RNAs of Paramecium mitochondrial (mt) DNA have been sequenced and the locations of their transcripts determined. The ori segment contains four transcripts, some of which are overlapping, which encode a known protein and two other open reading frames. The other segment encodes, on separate transcripts, the cytochrome c oxidase subunit one gene (COI) and the URF1 gene (ND1) common to most mt genomes. All these genes have the same orientation and do not contain introns. The COI gene is the most divergent of those known and has an internal 108 amino acid 'insert' not found in COI genes from other organisms. With these data it is possible to define a probable Paramecium mt genetic code. With the exception that TGA codes for tryptophan and the use of different start codons, Paramecium mtDNA appears to follow the universal code. GTA possibly can be used as a start codon.

Amino Acid Sequence↗
PubMed1986

Identification of Paramecium mitochondrial proteins using antibodies raised against fused mitochondrial gene products.

Paramecium aurelia mitochondrial (mt) DNA fragments carrying the coding regions for two proteins, P1 (in the region adjacent to the origin of replication) and COII (subunit II of cytochrome oxidase), were used to study mt gene expression. The sequence for the portion of mtDNA containing P1 has already been described [Pritchard et al., Gene 44 (1986) 243-253]. The complete nucleotide sequence of the portion containing the COII gene is presented here. An 18.5-kDa protein was produced in maxicells when a fragment containing a major portion of the sequence coding for P1 was used. This fragment and a fragment carrying the COII gene were cloned into the expression vector pTRPLE', and antibodies were raised against the resulting fusion proteins in an Escherichia coli lysate. Western blots of Paramecium mt extracts identified two proteins, one 21 kDa (COII) and the other 23.5 kDa (P1). The size of the P1 protein is in agreement with the size of the open reading frame in that region of mitochondrial DNA. Based on extensive amino acid homology to the Paramecium gene and limited homology to COII genes from other organisms, the COII gene in another ciliate, Tetrahymena pyriformis, was identified just upstream of the small subunit rDNA in previously published sequences (Schnare et al., 1986). The size of the COII gene and the homology with the COII gene from Tetrahymena suggest that ATC, ATT, GTG and GTC could be used as translational initiators in Paramecium mitochondria.

Amino Acid Sequence↗
PubMed1985

Excision-amplification of mitochondrial DNA during senescence in Podospora anserina. DNA sequence analysis of three unique "plasmids".

During senescence in the filamentous fungus Podospora anserina, specific regions of the mitochondrial genome, termed senDNA are excised, ligated and amplified. We have cloned in their entirety three such autonomously replicating plasmids, alpha, beta and epsilon senDNA. None of these plasmids displayed cross-hybridization nor did we detect any significant DNA homology by computer analysis. The complete DNA sequence of the 2.5 kb alpha, the 5.5 kb epsilon and about 3.4 kb of the 9.8 kb beta senDNA is presented (kb = 10(3) base-pairs). These sequences were analyzed for the presence of consensus sequences common to introns, and it was found that alpha senDNA has the characteristics of a group II intron, epsilon senDNA contains three group I introns, and beta senDNA did not show relevant sequences in the 3.4 kb examined. Comparison of the 5' and 3'-flanking sequences of alpha senDNA with oxi 3 (Co I) amino acid sequences from Neurospora crassa and Saccharomyces cerevisiae revealed significant homology and provided strong support that the excised alpha senDNA itself consists entirely of an intron. Upstream from the oxi 3 gene a transfer RNA cysteine sequence was detected. beta senDNA contained four tRNA sequences, aspartic acid, serine, valine and tryptophan, and sequences homologous to URFC (untranslated reading frame C) as well as two new URFs. epsilon senDNA contained sequences homologous to ATPase 8 and URFl; URFl was interrupted by three group I introns. The excision site sequences, as located by S1 nuclease mapping were unique for each senDNA. Analysis for repeated units showed that each plasmid contained elements which could be involved in secondary structure required for the alignment of distal ends preparatory to excision. These results are interpreted in terms of the structural requirements of mobile elements including the possible involvement of reverse transcriptase in the excision-ligation-amplification process.

Amino Acid Sequence↗
PubMed1985

Analysis of class I introns in a mitochondrial plasmid associated with senescence of Podospora anserina reveals extraordinary resemblance to the Tetrahymena ribosomal intron.

Recently, the nucleotide sequences for three "mitochondrial plasmids" associated with senescence of Podospora anserina were determined (Cummings et al. 1985). One of these sequences, corresponding to the plasmid termed epsilon senDNA, contains three class I introns, all within a protein coding sequence equivalent to the mammalian "URF1" gene. Here, we present primary and secondary structure analyses for two of these introns as well as a partial analysis for the third, which extends beyond the DNA sequence determined. With regard to both primary and secondary structure, the closest known relative of intron 1 is the self-splicing intron in the large ribosomal RNA gene of Tetrahymena. One secondary structure domain at the periphery of intron 1 and Tetrahymena models is also present in intron 2. The latter intron is the longest known class I member and contains remnants of two protein-coding sequences, one of which is split by the other. Evolutionary processes that might be responsible for the unusual structure of introns 1 and 2 are discussed.

Animals↗
PubMed1984

Paramecium mitochondrial genes. I. Small subunit rRNA gene sequence and microevolution.

The sequences of the small subunit mitochondrial rRNA genes from two divergent species of Paramecium (primaurelia and tetraurelia) were determined. The gene lies near the center of the linear mitochondrial genome, on the same strand as are all other currently identified genes. The sequences generally resemble their counterparts found in cytoplasmic, procaryotic, and other mitochondrial sources. The rDNA gene boundaries were located by nuclease S1 protection. Small subunit rDNA spans about 1680 nucleotides, including an extraneous 83-base pair sequence very near the 3' end which is unique to Paramecium mitochondria. This "insert" occurs at the apex of the highly variable in length penultimate helix, according to proposed models for small subunit rRNA secondary structure. A discontinuity occurs in isolated rRNA near the start of the insert, resulting in a stable 13 S RNA species and a small segment containing the remaining 3' portion of the gene. The overall rRNA gene sequence was 94% conserved between the two species, and the nucleotide differences consisted of 53% transitions, 37% transversions, and 9% insertions plus deletions. These substitutions were somewhat clustered, and the two most divergent regions coincided with the gene boundaries. The sequence was aligned with Escherichia coli 16 S rRNA for direct comparison of sequence and structure.

Animals↗
PubMed1984

Paramecium mitochondrial genes. II. Large subunit rRNA gene sequence and microevolution.

Mature Paramecium mitochondrial large subunit rRNA consists of two stable segments: a 20 S segment described previously and a unique 283-base segment similar to 5.8 S rRNAs typically found in eucaryotic cytoplasmic RNA. pBR325 clones of both gene regions from both Paramecium primaurelia and Paramecium tetraurelia were sequenced and aligned. The gene segments lie adjacent to each other very near the replicative terminal end of the linear Paramecium mitochondrial genome and are transcribed from a common 23 S precursor. The precise gene ends were determined using nuclease S1 protection; the large subunit rRNA gene complex (consisting of "5.8 S-like" rRNA, a 19-26-base excised region, and 20 S rRNA) spans about 2654 base pairs. The gene complex is preceded by a 15-base poly(T) tract and terminates randomly within a 20-base A + T-rich segment immediately preceding the tRNATyr gene. The sequences from the two species were 4% divergent, the changes consisting of 59% transitions, 38% transversions, and 3% insertions or deletions. The sequences were aligned with Escherichia coli 23 S rRNA, and a secondary structure model is presented for the entire molecule based on structures proposed for E. coli 23 S rRNA.

Animals↗
PubMed1983

DNA sequence of the excision sites of a mitochondrial plasmid from senescent Podospora anserina.

During senescence in Podospora anserina, specific gene regions of the mitochondrial genome are excised and amplified. The most prevalent, termed alpha-event senDNA, is a 2600 bp circular molecule which is excised from the contiguous Hae III fragments 23,14 region of the mitochondrial DNA restriction map. We have cloned alpha-DNA plasmid from races s+ and A+ as well as the genomic fragments Hae III 23,14 and have sequenced those regions which constitute the alpha-junction sites. We have found that one excision site (J1) is located 24 bp from the proximal Hae III 23 restriction site and the other (J2) 172 bp from the distal Hae III 14 site. Flanking the alpha-DNA sequences on the mitochondrial genome, there are 10 bp palindromic sequences: CAATATATTG, ending 3 bases from the J1 site, and ATTATATAAT which starts 8 bases from the J2 site. Neither of these 10 bp palindromes are present on the alpha-DNA plasmid. Abutting the J1 site on the alpha-DNA there is a 5 bp sequence (GTGCT) which is repeated 8 bp downstream. In joining the two distal J1 and J2 sites, a 7 bp repeat (ACGTGCG) is produced. These results are discussed within the context of site-specific recombination.

Ascomycota↗
PubMed1983

Integration of mitochondrial gene sequences within the nuclear genome during senescence in a fungus.

Cellular senescence in the ascomycete fungus Podospora anserina is associated with the appearance of an altered mitochondrial genome. Discrete mitochondrial DNA sequences are excised and amplified and isolated as multimerically arranged, head-to-tail repetitions. We have referred to the most frequently observed excision/amplification product as alpha-event senDNA. It is a 2.6-kilobase pair (kbp) monomeric unit (see refs 1, 3, 7) and is often found in senescent mitochondria in conjunction with other excision products. At the final stage of senescence these plasmids constitute virtually all of the DNA present in senescent mitochondria; they have replicated to high copy number at the expense of the young native genome. Because P. anserina is characterized by race-specific timing of senescence (that is, a programme of senescence), we have begun to contrast rapidly and slowly senescing races in terms of senDNA. Here we present evidence that young mitochondria of the rapidly senescing race, A+, possess an extremely high copy number of alpha-event senDNA plasmid in contrast to the more slowly senescing races s+ or s-. Moreover, we observe that during senescence the alpha-event senDNA and the beta-event senDNA (a 9.8-kbp monomer) are transposed to the nucleus and integrated into nuclear DNA. These plasmids contain the coding information for subunits I and III (respectively) of the mitochondrial cytochrome c oxidase. This constitutes the first clear evidence for the active mobilization of genetic elements from the mitochondrion to the nucleus.

Aging↗
PubMed1983

Inter-species sequence diversity in the replication initiation region of Paramecium mitochondrial DNA.

Mitochondrial DNA from Paramecium aurelia is a linear molecule. Replication is initiated at a unique cross-linked molecular terminus. During replication dimer length molecules, consisting of two head-to-head monomers, are generated. We have cloned the head-to-head dimer initiation region from five different species and several stocks (or races) within species and determined its DNA sequence. For all species, this dimer initiation region consists of a central non-palindromic sequence containing almost exclusively A and T, arranged in an array of direct tandem repeats. In an intra-species comparison, the sequences of the repeat units are relatively homogeneous; inter-species comparisons, however, show diversity except for a conserved "Goldberg-Hogness box", T-A-T-A-A-A-T-A. The size of a repeat unit and the number of repeats within a molecule can vary over a wide range, even in an intra-species comparison. Because of these wide inter-species variations observed, it is likely that the function of this region imposes few constraints on the sequence other than its high A + T content and possibly a Goldberg-Hogness box. The array of direct tandem repeats may have arisen from unequal recombination or crossover within this region. Adjacent to the non-palindromic region is a transcribed sequence which is highly conserved for all species and presumably represents a gene coding region.

Animals↗
PubMed1983

Antigenic variation during cellular aging in Paramecium tetraurelia.

Clones of Paramecium tetraurelia undergoing cellular aging were exposed to two antisera, anti-32 and anti-25, raised against surface antigen isolated from cells grown at 32 degrees C or 25 degrees C. When young clones were exposed to the two antisera about 50% were immobilized by anti-32 while others were not immobilized by either antisera. These clones were grouped as group A or B depending upon their sensitivity or nonsensitivity to the two antisera. Young cells of group A were immobilized by anti-32 and during aging these cells either maintained the same antigenic type or transformed to another. Young group B clones were not immobilized by either antisera but gradually became sensitive to anti-32. Some clones from both groups showed sensitivity to both antisera. The possible mechanism of antigenic variation during fission age is discussed.

Animals↗
PubMed1982

Altered genetic code in Paramecium mitochondria: possible evolutionary trends.

The 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 TpsiC 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.

Base Sequence↗
PubMed1982

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.

Ascomycota↗