E Di Mauro

Chromatin remodeling during Saccharomyces cerevisiae ADH2 gene activation.

We have analyzed at both low and high resolution the distribution of nucleosomes over the Saccharomyces cerevisiae ADH2 promoter region in its chromosomal location, both under repressing (high-glucose) conditions and during derepression. Enzymatic treatments (micrococcal nuclease and restriction endonucleases) were used to probe the in vivo chromatin structure during ADH2 gene activation. Under glucose-repressed conditions, the ADH2 promoter was bound by a precise array of nucleosomes, the principal ones positioned at the RNA initiation sites (nucleosome +1), at the TATA box (nucleosome -1), and upstream of the ADR1-binding site (UAS1) (nucleosome -2). The UAS1 sequence and the adjacent UAS2 sequence constituted a nucleosome-free region. Nucleosomes -1 and +1 were destabilized soon after depletion of glucose and had become so before the appearance of ADH2 mRNA. When the transcription rate was high, nucleosomes -2 and +2 also underwent rearrangement. When spheroplasts were prepared from cells grown in minimal medium, detection of this chromatin remodeling required the addition of a small amount of glucose. Cells lacking the ADR1 protein did not display any of these chromatin modifications upon glucose depletion. Since the UAS1 sequence to which Adr1p binds is located immediately upstream of nucleosome -1, Adr1p is presumably required for destabilization of this nucleosome and for aiding the TATA-box accessibility to the transcription machinery.

Hippocampal gene expression is modulated by hypergravity

We used the cDNA microarray technique to monitor simultaneously possible changes induced by hypergravity in the expression level of thousands of hippocampal genes. We tested the mRNA level of about 5000 genes in the hippocampus of mice subjected to 1.09 g (1g) or to 1.85 g (2g) for five repeated 1‐h daily rotations in a centrifuge (g = 9.81 m/s2). Data were compared with those obtained for mice kept stationary (C). The ratios 1g/C and 2g/C identified genes affected by rotation and rotation + hypergravity, respectively, whereas 2g/1g ratio identified those affected by hypergravity. We found that about 200 genes were affected by rotation and/or rotation + hypergravity. Almost all the genes affected by rotation + hypergravity were up‐regulated, only five being down‐regulated. The modulated genes code for proteins involved in a wide range of cellular functions (DNA/RNA metabolism, protein processing, intermediate metabolism, cytoskeleton and motility, cell cycle and apoptosis, signal transduction, neuronal structure/function), suggesting that rotation + hypergravity may affect several aspects of the hippocampal function in order to compensate for environmental changes. Six genes directly or indirectly involved in synaptic transmission and plasticity (proSAAS, neuroblastoma ras oncogene, ESTs moderately similar to thymosin beta‐10, syndet, inhibin beta E and Ngfi‐A binding protein 2) were found to be significantly modulated by hypergravity and unaffected or only slightly affected by rotation. The modulation by hypergravity of these genes suggests that this stimulus might induce plastic remodelling of the hippocampal circuits, possibly both at structural and functional level.

DNA topoisomerase I controls the kinetics of promoter activation and DNA topology in Saccharomyces cerevisiae.

Inactivation of the nonessential TOP1 gene, which codes for Saccharomyces cerevisiae DNA topoisomerase I, affects the rate of transcription starting at the ADH2 promoter. For both the chromosomal gene and the plasmid-borne promoter, mRNA accumulation is kinetically favored in the mutant relative to a wild-type isogenic strain. The addition of ethanol causes in wild-type yeast strains a substantial increase in linking number both on the ADH2-containing plasmid and on the resident 2 microns DNA. Evidence has been obtained that such an in vivo increase in linking number depends on (i) the activity of DNA topoisomerase I and of no other enzyme and (ii) ethanol addition, not on the release from glucose repression. A direct cause-effect relationship between the change in supercoiling and alteration of transcription cannot be defined. However, the hypothesis that a metabolism-induced modification of DNA topology in a eukaryotic cell plays a role in regulating gene expression is discussed.

Imbalance in dosage of the genes for the heterochromatin components Sir3p and histone H4 results in changes in the length and sequence organization of yeast telomeres

Abstract Telomeric heterochromatin plays an essential role in telomere function, including the regulation of telomere length. We observe that in Saccharomyces cerevisiae an imbalance in the dosage of genes for two protein components of heterochromatin (namely Sir3p and histone H4) causes modifications in telomere length and telomere sequence organization. The effects of Sir3p/H4 imbalance were analyzed in yeast strains in which the wild-type SIR3 gene (normally a single-copy gene) was either absent or present in 20–30 copies, and both histone H4 genes (HHF1 and HHF2) were present or HHF1 was deleted, thus covering a wide range of viable gene-dosage combinations. Modifications of telomeres and of subtelomeric regions were identified by analyzing both the overall telomere population and by focusing on two single telomeric regions: the left telomere of chromosome III (LIII) and the right telomere of chromosome XI (RXI). The modifications induced by alteration of the Sir3p/H4 ratio consist of a reduction in the length and an increase in the instability of the terminal block of (C1–3A)n repeats and in susceptibility to insertion of Y′ elements into this repeat element. Restoration of the wild-type gene ratio (by removal of the extra copies of SIR3 or by complementation with the missing second copy of HHF) restored the original telomere organization, both with respect to the length of the (C1–3A)n repeat stretch and the absence of Y′ elements. This behavior shows that the stability of the wild-type sequence organization requires maintenance of the normal structure of telomeric heterochromatin.

Telomere-based neo-Darwinian selection of yeast clonal subpopulations.

Abstract In Saccharomyces cerevisiae, imbalance of the genes coding for the heterochromatin components Sir3p and histone H4 (namely, overdosage of SIR3 and lack of one of the two genes coding for H4) causes modifications in telomere length and telomere sequence organization, favoring the insertion of Y′ elements into a stably shortened (C1–3A)n repeat tract. We report here that the newly inserted Y′ elements are unstable and are lost with high frequency, generating clonal subpopulations with short telomeres, as revealed by the analysis of a specific telomere (LIII) and of the overall population of telomeres. Moreover, the growth rates of the subpopulations with and without Y′ elements on LIII are different, the Y′-less individuals reproducing 20% more slowly than individuals bearing Y′ elements. When grown together with Y′-bearing individuals, the subpopulations with the normal LIII telomere (which are viable and genetically stable if grown alone) are rapidly competed out. Hence, genetic imbalance for the structural components of heterochromatin results in a complex and rapidly changing mixture of subpopulations in such cultures. Thus, in situations where subpopulations are allowed to compete, heterochromatin-based differential growth rates result in neo-Darwinian clonal selection.

Detection of human genomic mutations by chemical single-reaction DNA sequencing

▼The ideal DNA sequencing method should yield complete and unambiguous information in a single electrophoretic pattern and should be simple, rapid and economical. A single-lane sequencing procedure is available, based upon the dideoxy Sanger methodology. This method cannot a priori be compressed further. Chemical DNA sequencing offers the potential of absolute compression: if one could obtain unambiguous and complete sequence information in a single pattern, then several different DNAs, each labeled with a different fluorochrome, could be analyzed in the same electrophoretic lane. This procedure could be quite relevant for mass genetic screenings. We have developed a chemical method (Ref. 1, 2) that yields a one-reaction one-lane sequence determination, thus allowing fast, multiple, simultaneous sequencing and direct sequence comparisons in the same electrophoretic lane. The method is based on the base-selective degradation (G>A>C>T) of DNA by N-methyl formamide (see Fig. 1). The chemical rationale of this method is as follows. Amides (as determined for formamide, N-methyl formamide and Ndimethyl formamide) react with DNA according to a multistep mechanism. (a) Degradation of the purine and pyrimidine bases: for purines, degradation occurs by nucleophilic attack in C-8, leading to degradative C-8-ring opening of the imidazole ring; for pyrimidines by nucleophilic attack in C-6, leading to degradative C-6-ring opening of the pyrimidine ring. (b) β-Elimination of the sugar-protons with consequent cleavage of the phosphate bonds. N-Methyl formamide has proved to be the best compound to carry out the sequencing reaction because of its efficiencies in base