The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to down-weight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.

/pdf/clustal-w-improving-the-sensitivity-of-progressive-multiple-2na2s9sq2h.pdf

Clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice

Repetitive DNA sequences form a large portion of the genomes of eukaryotes. The 'selfish DNA' hypothesis proposes that they are maintained by their ability to replicate within the genome. The behaviour of repetitive sequences can result in mutations that cause genetic diseases, and confer significant fitness losses on the organism. Features of the organization of repetitive sequences in eukaryotic genomes, and their distribution in natural populations, reflect the evolutionary forces acting on selfish DNA.

The evolutionary dynamics of repetitive DNA in eukaryotes

Extrachromosomal rDNA circles--a cause of aging in yeast.

In nearly all eukaryotes, at least some individuals inherit mitochondrial and chloroplast genes from only one parent. There is no single mechanism of uniparental inheritance: organelle gene inheritance is blocked by a variety of mechanisms and at different stages of reproduction in different species. Frequent changes in the pattern of organelle gene inheritance during evolution suggest that it is subject to varying selective pressures. Organelle genes often fail to recombine even when inherited biparentally; consequently, their inheritance is asexual. Sexual reproduction is apparently less important for genes in organelles than for nuclear genes, probably because there are fewer of them. As a result organelle sex can be lost because of selection for special reproductive features such as oogamy or because uniparental inheritance reduces the spread of cytoplasmic parasites and selfish organelle DNA.

https://www.pnas.org/content/pnas/92/25/11331.full.pdf

Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution.

Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging?

Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 mu) were present; instead, smaller circles characteristic for each mutant studied were found. Eco R1 digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type. The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 mu. These circles ranged in size from 0.89 mu to greater than 20 mu; only one molecule out of some 200 molecules was thought to be of full length (31 mu). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm3) and a second at 1.699 g/cm3. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23 x 10(6) daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20 x 10(6) daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length. These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho- mutation in the yeast, S. cerevisiae.

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Mitochondrial (Mt) DNA from Podospora anserina was isolated and characterized with respect to density in CsCl, contour length and endonuclease restriction enzymes. The density of Mt DNA for four races examined was 1.694 g/cm3, compared with 1.712 g/cm3 for nuclear DNA. Extraction in the presence of a nuclease inhibitor, aurintricarboxylic acid and isolation in DAPI CsCl gradients allowed us to isolate high molecular weight DNA. Mt DNA isolated by total DNA extraction contained ca. 1% of circular molecules, 31 μm in contour length; Mt DNA isolated from purified mitochondria contained 2–4% of these 31 μm circles. Analysis with Eco RI restriction endonuclease revealed that each of the four races examined, s, A, T and E had a characteristic fragment pattern. Races s and A Mt DNA differed by only one fragment after Eco RI enzymatic digestion; similarly, these two DNA differed by only one or two fragments after Hae III digestion.

Mitochondrial DNA from Podospora anserina. I. Isolation and characterization.

AMONG mobile genetic elements, self-splicing introns are of particular interest. They belong to either group I or group II depending on their three-dimensional structure1& ndash;3. Homing, the systematic intron invasion of an intronless gene when it encounters its homologous intron-bearing allele, is the only means for intron mobility so far demonstrated4. It depends on the activity of the intron-encoded protein and is very specific for the acceptor site4& ndash;7. Intron transposition, the transfer of an intron to a novel site, predicted on the basis of phylogenetic studies8,9 and in vitro reverse-splicing experiments, has been proposed to be responsible for evolutionary intron spreading2,10& ndash;14. Here we present results from polymerase chain reaction experiments consistent with transposition of a group II intron. This event is proposed to account for the site-specific deletion in the mitochondria! chromosome of the fungus Podospora anserina that is associated with the premature death syndrome15 and might also be involved in the senescence process16 affecting this species.

Transposition of a group II intron

Senescence in podospora anserina: Amplification of a mitochondrial DNA sequence

The non-nuclear DNA of a number of independent senescent cultures of Podospora anserina was extracted and studied. In all cases, a specific repetitive DNA (SEN-DNA) arranged in multimeric sets of circular molecules, was identified. Depending on the senescent culture, the SEN-DNA was found either in a band of about same density as the mitochondrial DNA from young mycelia (1.694 g/cm3) or in a band of higher density (1.699 g/cm3). Electron microscopy, restriction enzyme analysis and Southern hybridization experiments allowed us to establish that: (1) SEN-DNAs obtained from independent senescent cultures, both from the same strain and from different strains, can differ in the size of their monomer unit (from 2.5 to 6.3 kb). (2) All SEN-DNAs hybridize with mitochondrial DNA of a young culture and not with nuclear DNA. (3) These SEN-DNAs belong to two classes which hybridize with two non-overlapping regions of the mitochondrial chromosome.

Léon Belcour

Papers

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Mitochondrial DNA from Podospora anserina. I. Isolation and characterization.

Transposition of a group II intron

Senescence in podospora anserina: Amplification of a mitochondrial DNA sequence

Mitochondrial DNA amplification in senescent cultures of Podospora anserina : Variability between the retained, amplified sequences