Ariel Prunell

The mitochondrial genome of wild-type yeast cells

Abstract The organization of the mitochondrial genome of wild-type Saccharomyces cerevisiae cells has been investigated further, by degrading mitochondrial DNA with micrococcal nuclease. Under the conditions used, this enzyme very strongly degrades the A + T-rich stretches (spacers) whereas it only inflicts a limited number of breaks into the G + C-rich stretches (genes). The macromolecular fragments derived from the “genes” have been separated from the oligonucleotides originating from the “spacers” by gel filtration, and both sorts of products have been investigated. It has been shown (a) that the spacers are very homogeneous in base composition and have a G + C content lower than 5% (mitochondrial DNA has a G + C content of 18%); (b) that the genes are very heterogeneous in base composition, the G + C content ranging from about 25% to 50%, when the average size of the fragments is 1·2 × 10 5 ; smaller fragments, molecular weight 4 × 10 4 , having a G + C level as high as 65%, have been isolated in a yield of 10%; the average G + C content of genes is about 32%; (c) that genes and spacers are present in about equal amounts in the mitochondrial genome and that they have comparable average sizes.

The mitochondrial genome of wild-type yeast cells: VI. Genome organization

Chromatography on hydroxyapatite of HaeIII and HpaII digests of yeast mitochondrial DNA shows that 0·8×106 and 1×106 (molecular weight) of DNA per genome unit of 50×106, respectively, are present in the digests as short, single-stranded oligonucleotides, thus providing evidence for the clustering of both Hae sites and Hpa sites. An analysis of the double (Hae+Hpa) hydrolysate reveals that Hpa and Hae sites are also clustered with each other in about 60 (Hae, Hpa) site clusters. These “restriction site clusters” have a G+C-content in the 45 to 62% range and are about 35 base-pairs long on the average; “G+C-rich clusters”, which have a G+C-content of 60%, and contain no Hae or Hpa site, are possibly adjacent to the site clusters. In addition to the Hae and Hpa sites which are present in restriction site clusters, 40 “isolated” Hpa sites and very few “isolated” Hae sites also exist on the mitochondrial genome. The (Hae, Hpa) site clusters have nucleotide sequences similar to those found in bacterial promotors and operators, in that they consist of hyphenated palindromes in which short G-C and A-T sequences alternate with each other. Their localization is at the border of subsequent gene-spacer units, and their number may well be equal to that of mitochondrial genes and spacers. It is, therefore, very likely that the (Hae, Hpa) site clusters (and possibly, the G+C-rich clusters) are regulatory elements and that the mitochondrial genome of yeast is made up of genetic units, each one of which is formed by a regulatory sequence, a gene and a spacer.

The mitochondrial genome of wild-type yeast cells. V. Genome evolution.

When degraded with the restriction enzymes Hae III or Hpa II, the mitochondrial DNAs from one Saccharomyces carlsbergensis and three genetically unrelated S. cerevisiae wild-type strains yielded 71 to 113 fragments ranging in molecular weight from 10 4 to 4×10 6 . Genome unit sizes, calculated by adding up the molecular weights of all fragments produced by Hae III, Hpa II and, in some cases, Hind II+III and Eco RI, were in the 52 to 54×10 6 range for the three S. cerevisiae strains, whereas a value lower by about 10% was found for the S. carlsbergensis strain. These values are in agreement with the physical size of circular twisted yeast mitochondrial DNA, as determined by electron microscopy (Hollenberg et al. , 1970). Large differences in the electrophoretic patterns of Hae III and Hpa II fragments were found among the DNAs from different S. cerevisiae strains; S. cerevisiae and S. carlsbergensis DNAs showed only very few bands having the same mobility. Such differences appear to originate essentially from additions and deletions in the A+T-rich spacers and to be accompanied by a large preservation of gene order. Unequal crossing-over events at the spacers seem to be the source of additions and deletions and to underlie the evolution of the mitochondrial genome of yeast.

Photographic quantitation of DNA in gel electrophoresis.

Abstract A photographic procedure to quantitate the DNA in bands, obtained by gel electrophoresis after staining with ethidium bromide, is described. The relationship between the film darkening and the intensity of the light hitting the film was determined. The densities, measured in densitometric tracings of the negatives, were converted into fluorescence intensities. The fluorescence was found to be linearly proportional to the amount of DNA. Deviations due to gel overloading, to nonuniform electrophoretic migration and uv illumination, and to photodecomposition of ethidium bromide were investigated.

Variable center to center distance of nucleosomes in chromatin.

The length of linker DNA between nucleosomes may be determined by exonuclease digestion of nucleosome dimers and gel electrophoresis of the resulting DNA. This procedure must be applied to histone 1-containing nucleosomes to avoid rearrangement. The results show a highly variable length of linker DNA within a rat liver cell. One possible interpretation, in terms of a statistical distribution of nucleosomes, leads to a simple explanation of why the average length of linker DNA varies among tissues and organisms.

The mitochondrial genome of wild-type yeast cells: VIII. The spontaneous cytoplasmic “petite” mutation☆

The mitochondrial genomes of a number of spontaneous “petite” mutants of Saccharomyces cerevisiae were investigated by restriction enzyme analysis and by hybridization with restriction fragments from parental wild-type genomes. The nucleotide sequences forming the ends of the repeat units of the petite genomes were shown to be formed by GC clusters and, possibly, by AT spacers. These non-coding elements are characterized by the fact that they consist of, or contain, sequences which are repeated a number of times in the parental, wild-type genome and which are often symmetrical. The excision process leading to the formation of the spontaneous petite genomes appears to involve site-specific, illegitimate recombination events which take advantage of localized sequence homology, in agreement with a deletion model previously proposed. The same kind of excision process appears to be operative in the further deletions undergone by the mitochondrial genomes of spontaneous petite mutants. The genome organization and the excision mechanism appear to be largely different in spontaneous and ethidium-induced petite mutants.

Nucleosome conformational flexibility and implications for chromatin dynamics

The active role of chromatin in the regulation of gene activity seems to imply a conformational flexibility of the basic chromatin structural unit, the nucleosome. This review is devoted to our recent results pertaining to this subject, using an original approach based on the topology of single particles reconstituted on DNA minicircles, combined with their theoretical simulation. Three types of chromatin particles have been studied so far: a subnucleosome, that is, the (H3–H4)2 histone tetramer–containing particle, now known as the tetrasome; the nucleosome; and the linker histone H5/H1–bearing nucleosome (the chromatosome). All the particles were found to exist in two to three conformational states, which differ by their topological and mechanical properties. Our approach unveiled the molecular mechanisms of nucleosome conformational dynamics and will help to understand its functional relevance. A most surprising conclusion of the work was perhaps that DNA overall flexibility increases considerably upon particle formation, which might indeed be a requirement of genome function.

In vivo incorporation of cytosine arabinoside into Simian virus 40 DNA

Abstract Inhibition of Simian virus 40 DNA replication by cytosine arabinoside was found to be essentially irreversible. At high concentration of the inhibitor (20 μg/ml), cytosine arabinoside was incorporated into the growing viral DNA chains in internucleotide linkage. Use of lower concentration of the same inhibitor (2 μg/ml) allowed its recovery into supercoiled viral DNA component I.

Flexibility Of Nucleosomes On Topologically Constrained DNA

The nucleosome plays an ever increasing role in our comprehension of the regulation of gene activity. Here we review our results on nucleosome conformational flexibility, its molecular mechanism and its functional relevance. Our initial approach combined both empirical measurement and theoretical simulation of the topological properties of single particles reconstituted on DNA minicircles. Two types of particles were studied in addition to the conventional nucleosome: a subnucleosome consisting of DNA wrapped around the (H3-H4)2 histone tetramer, now known as a tetrasome, and the linker histone H5/H1-bearing nucleosome, or chromatosome. All particles were found to thermally fluctuate between two to three conformational states, which differed by their topological and mechanical characteristics. These findings were confirmed for the nucleosome and the tetrasome by the use of magnetic tweezers to apply torsions to single arrays of these particles reconstituted on linear DNA. These latter experiments further revealed a new structural form of the nucleosome, the reversome, in which DNA is wrapped in a right-handed superhelical path around a distorted octamer. This work suggests that the single most important role of chromatin may be to considerably increase overall DNA flexibility, which might indeed be a requirement of genome function.

Random arrangement of nucleosomes on DNA in chromatin

The location of nucleosomes on the bulk of the DNA [l] as well as on 5 S [2] and ovalbumin genes [3] and on polypyrimidine/polypurine stretches [4] was found to be random. This has led to the suggestion that the packaging of DNA in nucleosomes is unlikely to bear directly on the control of gene activity but may serve primarily a structural purpose [ 11. It has been observed [5], however, that this randomness could be, at least in part, a consequence of a rearrangement of histones induced by micrococcal nuclease digestion. This difficulty is obviated only in the particular cases of SV40 [6-81 and polyoma virus minichromosomes [8] where the same conclusion was obtained using a different method based on restriction endonuclease digestions. The possibility for such a rearrangement is indicated by the observation that extensive digestion of chromatin with micrococcal nuclease generates compact dimers [9,10]. Some insight into the way compact dimers are formed comes from an experiment in which dimers from an early micrococcal digest of chromatin are trimmed with exonuclease III from E. coli [ 111: no sliding is detected as long as dimers retain their full complement of HI (A. P., R. D. Kornberg, submitted); later in the digestion, HI is released and nucleosomes slide towards each other, generating compact dimers in high yield (A. P., unpublished data). The lower yield of compact dimers obtained with micrococcal nuclease may not reflect a lower ability of this enzyme to cause sliding but is due to the susceptibility of dimers to cleavage into monomers.