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F Antequera

Publications and source records attributed to F Antequera.

11 recordsLinked to original sources
PubMed1998

Initiation of DNA replication at CpG islands in mammalian chromosomes.

CpG islands are G+C-rich regions approximately 1 kb long that are free of methylation and contain the promoters of many mammalian genes. Analysis of in vivo replication intermediates at three hamster genes and one human gene showed that the CpG island regions, but not their flanks, were present in very short nascent strands, suggesting that they are replication origins (ORIs). CpG island-like fragments were enriched in a population of short nascent strands from human erythroleukaemic cells, suggesting that islands constitute a significant fraction of endogenous ORIs. Correspondingly, bulk CpG islands were found to replicate coordinately early in S phase. Our results imply that CpG islands are initiation sites for both transcription and DNA replication, and may represent genomic footprints of replication initiation.

Adenine Phosphoribosyltransferase↗
PubMed1993

Number of CpG islands and genes in human and mouse.

Estimation of gene number in mammals is difficult due to the high proportion of noncoding DNA within the nucleus. In this study, we provide a direct measurement of the number of genes in human and mouse. We have taken advantage of the fact that many mammalian genes are associated with CpG islands whose distinctive properties allow their physical separation from bulk DNA. Our results suggest that there are approximately 45,000 CpG islands per haploid genome in humans and 37,000 in the mouse. Sequence comparison confirms that about 20% of the human CpG islands are absent from the homologous mouse genes. Analysis of a selection of genes suggests that both human and mouse are losing CpG islands over evolutionary time due to de novo methylation in the germ line followed by CpG loss through mutation. This process appears to be more rapid in rodents. Combining the number of CpG islands with the proportion of island-associated genes, we estimate that the total number of genes per haploid genome is approximately 80,000 in both organisms.

Animals↗
PubMed1993

CpG islands.

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Animals↗
PubMed1990

High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines.

CpG islands are normally methylation free in cells of the animal, even when the associated gene is transcriptionally silent. In mouse NIH 3T3 and L cells, however, over half of the islands are heavily methylated. Near identity of the methylated subset in the two cell lines suggested that methylation is confined to genes that are nonessential in culture. In agreement with this, islands at several tissue-specific genes, but not at housekeeping genes, have become methylated in many human and mouse cell lines. At the chromatin level, methylated islands are Mspl resistant compared with their nonmethylated counterparts. We suggest that mutation-like gene inactivation due to CpG island methylation is widespread in many cell lines and could explain the loss of cell type-specific functions in culture.

Animals↗
PubMed1990

A nuclear protein that binds preferentially to methylated DNA in vitro may play a role in the inaccessibility of methylated CpGs in mammalian nuclei.

The effects of DNA methylation on gene expression and chromatin structure suggest the existence of a mechanism in the nucleus capable of distinguishing methylated and non-methylated sequences. We report the finding of a nuclear protein in several vertebrate tissues and cell lines that binds preferentially to methylated DNA in vitro. Its lack of sequence-specific requirements makes it potentially capable of binding to any methylated sequence in mammalian nuclei. An in vivo counterpart of these results is that methylated CpGs are inaccessible to nucleases within nuclei. In contrast, non-methylated CpG sites, located mainly at CpG islands, and restriction sites not containing this dinucleotide, are relatively accessible. The possibility that DNA methylation acts through binding to specific proteins that could alter chromatin structure is discussed.

Animals↗
PubMed1989

Specific protection of methylated CpGs in mammalian nuclei.

We have compared nuclear accessibility of methylated and nonmethylated sequences using restriction enzymes. MspI, which cuts CpG sites in naked DNA regardless of methylation, cut DNA in intact mouse liver or brain nuclei almost exclusively at CpG islands. Bulk chromatin was not significantly cleaved by MspI but was cleaved extensively by enzymes that do not recognize CpG. Quantitative analysis of limit digests showed that MspI and another methyl-CpG insensitive enzyme, Tth, have a strong bias against cutting methylated sites in these nuclei. Southern analysis confirmed this at three genomic loci. Our results suggest that resistance to nucleases is mediated by factors that are bound specifically to methylated CpGs. MeCP, a protein that binds to methylated DNA in vitro, may be one such factor, since nuclease resistance was significantly reduced in an MeCP-deficient cell line.

5-Methylcytosine↗
PubMed1988

Developmental characterization and chromosomal mapping of the 5-azacytidine-sensitive fluF locus of Aspergillus nidulans.

In Aspergillus nidulans, a fungus that possesses negligible, if any, levels of methylation in its genome, low concentrations of 5-azacytidine (5-AC) convert a high percentage of the cell population to fluffy phenotypic variants through a heritable modification of a single nuclear gene (M. Tamame, F. Antequera, J. R. Villanueva, and T. Santos, Mol. Cell. Biol. 3:2287-2297, 1983). This new 5-AC-altered locus, designated here fluF1, was mapped as the closest marker to the centromere that has been identified so far on the right arm of chromosome VIII. Of all mutagens tested, only 5-AC induced the fluffy phenotype with a significant frequency. Furthermore, we determined that the wild-type, dominant allele of the fluF gene was primarily accessible to modification by 5-AC at the initial stages of fungal vegetative growth. These results indicated that 5-AC does not act through random mutagenic action but, rather, that fluF constitutes a specific target for this drug during a well-defined period of fungal development. Alteration of fluF by 5-AC resulted in a dramatic modification of the developmental program of A. nidulans. The resulting fluffy clones were characterized by massive, uncontrolled proliferation of undifferentiated hyphae, a drastic delay in the onset of asexual differentiation (conidiation), and colonies with an invasive nature. These features are reminiscent of the malignant properties of tumor cells. We propose that the locus fluF plays a primary role in the control of cell proliferation in A. nidulans and that its alteration by 5-AC produces pleiotropic modifications of the developmental program of this fungus.

Aspergillus nidulans↗
PubMed1985

Developmental modulation of DNA methylation in the fungus Phycomyces blakesleeanus.

DNA methylation is a rather sparse event among fungi. Phycomyces blakesleeanus seems to be one of the few exceptions in this context. 5-Methylcytosine represents 2.9% of the total cytosine in spore DNA and is located in approximately the same amount at any of the four CA, CT, CC or CG dinucleotides. A progressive and gradual drop in total 5-methylcytosine parallels the development of the fungus. This demethylation is non random but sequence specific and is not accounted for equally by the four different methylated dinucleotides, CG being much less affected (20% demethylated) than CA, CT and CC (more than 90% demethylated at the same time). "De novo" methylation to restore the initial pattern probably takes place during spore maturation. By using specific hybridization probes we have been able to show that the rRNA genes are not significantly methylated at any stage of development, regardless of their transcription status.

5-Methylcytosine↗
PubMed1984

DNA methylation in the fungi.

A systematic study on the incidence and patterns of cytosine methylation in the fungi has been carried out by restriction and nearest-neighbor analysis of DNAs isolated from undifferentiated cells of several fungal species. With respect to DNA modification, the fungi appear to be a heterogeneous group, with a 5-methylcytosine content ranging from undetectable levels (less than or equal to 0.1% of cytosine residues methylated in 18 out of 20 species tested) to low but detectable levels (e.g. congruent to 0.2 and congruent to 0.5% of the total cytosines methylated in Sporotrichum dimorphosporum and Phycomyces blakesleeanus, respectively). In the species where it has been detected, 5-methylcytosine is located mostly at CpG doublets, and the methylated sites are clustered in long tracts (10-30 kilobase pairs) separated from essentially unmethylated regions. This methylated compartment, which comprises a small fraction (1-11%) of the total DNA, contains at least a specific set of repetitive sequences. These results contrast with the higher 5-methylcytosine content found in the fungus Physarum polycephalum and in vertebrates and higher plants.

5-Methylcytosine↗
PubMed1983

High-frequency conversion to a "fluffy" developmental phenotype in Aspergillus spp. by 5-azacytidine treatment: evidence for involvement of a single nuclear gene.

Transient exposure of mycelia from Aspergillus niger and Aspergillus nidulans to the cytidine analog 5-azacytidine, leading to no more than 0.3 to 0.5% substitution for cytosine by 5-azacytosine in A. nidulans DNA, resulted in the conversion of a high fraction of the cell population (more than 20%) to a mitotically and meiotically stable "fluffy" developmental phenotype. The phenotypic variants are characterized by the developmentally timed production of a profuse fluffy network of undifferentiated aerial hyphae that seem to escape signals governing vegetative growth. Genetic analysis with six different fluffy clones reveals that this trait is not cytoplasmically coded, is recessive in heterozygous diploids but codominant in heterokaryons, and exhibits a 1:1 Mendelian segregation pattern upon sexual sporulation of heterozygous diploids. Complementation and mitotic haploidization studies indicated that all variants are affected in the same gene, which can be tentatively located on chromosome VIII of A. nidulans. Molecular analysis to search for modified bases showed that DNA methylation is negligible in in both A. niger and A. nidulans and that no differences could be detected among DNAs from wild-type cells, fluffy clones, or mycelia exposed to 5-azacytidine. It thus appears that high-frequency conversion of fungal mycelia to a stable, variant developmental phenotype by 5-azacytidine is the result of some kind of target action on a single nuclear gene and that this conversion can occur in organisms virtually devoid of DNA methylation.

Aspergillus nidulans↗