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At least 19 recordsLinked to original sources

PubMed1992

An archipelago of CpG islands in Xq28: identification and fine mapping of 20 new CpG islands of the human X chromosome.

19 probes for CpG islands, mapping to Xq28, have been used as probes to construct a physical map of genes of this band of the human X chromosome. A total of 22 CpG islands have been precisely mapped in respect to known loci along the 9-10 Mb of Xq28. The fine mapping of such a large number of CpG islands has demonstrated that also in gene rich Giemsa light bands, like Xq28, gene distribution is non uniform: the CpG islands are clustered in the distal portion of the band in a 2 Mb region between the G6PD gene and the DXS15 locus. Moreover, 16 CpG islands were found between the G6PD and the RCP/GCP genes, a region of DNA of only about 300 kb. If this structural organization has a biological function it has yet to be determined. However, the isolation of large genomic regions enriched in gene sequences and the availability of cosmid or YAC contigs will provide the means to test the significance of such gene organization, as well as the material for large sequencing projects and gene search, for the identification of candidate genes for inherited disorders mapped to Xq28 and for comparative mapping.

Animals↗

PubMed1992

Methylation and sequence analysis around EagI sites: identification of 28 new CpG islands in XQ24-XQ28.

Thirty-two probes for CpG islands of the distal long arm of the human X chromosome have been identified. From a genomic library of DNA of the hamster-human cell hybrid X3000.1 digested with the rare cutter restriction enzyme EagI, 53 different human clones have been isolated and characterized by methylation and sequence analysis. The characteristic pattern of DNA methylation of CpG islands at the 5' end of genes of the X chromosome has been used to distinguish between EagI sites in CpG islands versus isolated EagI sites. The sequence analysis has confirmed and completed the characterization showing that sequences at the 5' end of known genes were among the clones defined CpG islands and that the non-CpG islands clones were mostly repetitive sequences with a non-methylated or variably methylated EagI site. Thus, since clones corresponding to repetitive sequences can be easily identified by sequencing, such libraries are a very good source of CpG islands. The methylation analysis of 28 different new probes allows to state that demethylation of CpG islands of the active X and methylation of those on the inactive X chromosome are the general rule. Moreover, the finding, in all instances, of methylation differences between male and female DNA is in very strong support of the notion that most genes of the distal long arm of the X chromosome are subject to X inactivation.

Amino Acid Sequence↗

PubMed1992

CpG islands as gene markers in the human genome.

CpG islands are short, dispersed regions of unmethylated DNA with a high frequency of CpG dinucleotides relative to the bulk genome. We have analyzed 375 genes and 58 pseudogenes from the human entries in the EMBL Database for the presence of CpG islands. All 240 islands identified are associated with genes, and almost all cover at least a part of one exon; i.e., they are useful landmarks in the genome for identifying genes. More than half of the genes analyzed were associated with islands. All housekeeping and widely expressed genes have a CpG island covering the transcription start, whereas 40% of the genes with a tissue-specific or limited expression are associated with islands. In this latter group of genes, the position of the islands was not biased toward the 5' end of the transcription unit.

Dinucleoside Phosphates↗

PubMed1992

Methylation analysis of CGG sites in the CpG island of the human FMR1 gene.

The fragile-X syndrome of mental retardation is associated with an expansion in the number of CGG repeats present in the FMR1 gene. The repeat region is within sequences characteristic of a CpG island. Methylation of CpG dinucleotides that are 5' to the CGG repeat has been shown to occur on the inactive X chromosome of normal females and on the X chromosome of affected fragile-X males, and is correlated with silencing of the FMR1 gene. The methylation status of CpG sites 3' to the repeat and within the repeat itself has not previously been reported. We have used two methylation-sensitive restriction enzymes, AciI and Fnu4HI, to further characterize the methylation pattern of the FMR1 CpG island in normal individuals and in those carrying fragile-X mutations. Our results indicate that: (i) CpG dinucleotides on the 3' side of the CGG repeat are part of the CpG island that is methylated during inactivation of a normal X chromosome in females; (ii) the CGG repeats are also part of the CpG island and are extensively methylated as a result of normal X-chromosome inactivation; (iii) similar to normal males, unaffected fragile-X males with small CGG expansions are unmethylated in the CpG island; for affected males, the patterns of methylation are similar to those of a normal, inactive X chromosome; (iv) in contrast to the partial methylation observed for certain sites in lymphocyte DNA, complete methylation was observed in DNA from cell lines containing either a normal inactive X chromosome or a fragile-X chromosome from an affected male.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Tissue-specific methylation of a CpG island in transgenic mice.

Clustering of CpG dinucleotides in CpG-rich islands is a characteristic feature of mammalian genomes. Such CpG islands are frequently associated with genes and usually hypomethylated, regardless of the gene activity. This is the case for the CpG island of the murine Thy-1 gene. A transgenic line containing multiple copies of a truncated, concatemeric CpG island from the Thy-1.1 allele (Thy-1.2 background) showed that a stable fraction (approx. 0.20) became fully methylated in somatic tissues of homozygous mice with respect to testable restriction sites, while the remaining copies were methylation-free, i.e., this methylation appears to be an 'all-or-none' phenomenon. DNA from extraembryonic tissues (placenta and yolk sac) and epididymal sperm showed, however, an even higher degree of methylation in two distinct patterns. In the extraembryonic tissue, partial methylation of each copy was seen, whereas in sperm a high degree of 'all-or-none' methylation (greater than 0.35) was observed.

Animals↗

PubMed1992

Methylation of CpG-island-containing genes in human sperm, fetal and adult tissues.

The methylation of three human genes containing CpG islands and a CpG-depleted gene were measured in sperm, fetal and adult tissues. The c-Ha-ras was methylated extensively in the 3' region in sperm with a methylation-free region extending from the promoter to the third exon. The extent of methylation in the 3' region decreased in fetal cells, however, de novo methylation of sites closer to the island and within exon 1 were apparent. These sites were more completely methylated in adult lymphocytes and kidney. Essentially similar results were obtained with the CpG-island-containing genes, c-myc and HPRT (encoding hypoxanthine phosphoribosyl transferase), which showed that unmethylated sites near the CpG islands in sperm became methylated in fetal and adult cells. The variations in methylation seen in the non-island regions of the c-Ha-ras gene were mirrored in the insulin-encoding gene which does not contain a CpG island. The results show similar variations in methylation of non-island regions of DNA which occur independent of expression, and show that regions of extensive methylation in sperm may move closer to CpG islands in fetal and adult somatic cells.

Aging↗

PubMed1992

A cluster of CpG islands at D10S94, near the locus responsible for multiple endocrine neoplasia type 2A (MEN2A).

We report the characterization of a dense cluster of CpG islands at D10S94 in proximal 10q11.2. D10S94 is tightly linked to the gene responsible for multiple endocrine neoplasia type 2A (MEN 2A), a dominantly inherited tumor syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and/or parathyroid adenoma. To date, no recombinants between D10S94 and MEN2A have been identified. The gene(s) responsible for two additional dominantly inherited disorders involving cancer of the medullary thyroid, MEN 2B (MEN2B), and dominantly inherited MTC without additional clinical features (MTC1), also map to this region. The gene or genes responsible for these disorders may be located at or near the D10S94 locus. A 570-kb long-range restriction map has been generated by pulsed-field gel electrophoresis using probes developed during a 160-kb bidirectional cosmid walk at D10S94. Six CpG islands are clustered within a 180-kb region; five fall within a 145-kb NotI restriction fragment that is contained in its entirety in our cosmid contig. The SacII, SfiI, and NotI restriction maps for lymphoblast and cloned DNA are concordant. These CpG islands may represent the 5' ends of candidate genes for MEN2A, MEN2B, and/or MTC1. One gene designated mcs94-1, which is associated with one of the CpG islands in this cluster, has been isolated and characterized in detail.

Base Sequence↗

PubMed1992

CpG islands in mammalian gene promoters are inherently resistant to de novo methylation.

The CpG islands found at the 5' ends of many mammalian genes are typically unmethylated despite being both exposed to diffusible protein factors in nuclei and rich in CpG, the target site for DNA methyltransferase. We show here that the CpG islands associated with the human Thy-1 and profilin genes are inherently resistant to de novo methylation by purified murine DNA methyltransferase, and that the higher than expected tendency of CpG sites in islands to be flanked on both sides by G-C base pairs is the likely reason for the resistance. Several lines of evidence indicate that DNA methyltransferase does not make base-specific contacts with residues that flank CpG sites, and it is likely that CpG sites within islands are resistant to de novo methylation because of local conformational features such as ease of strand separation, minor groove dimensions, and alternative secondary structures. A role for minor groove contacts is consistent with the presence within a putative regulatory domain of numerous modified beta turn structural elements that can make minor groove contacts.

Antigens, Surface↗

PubMed1992

The hypervariable DXS255 locus contains a LINE-1 repetitive element with a CpG island that is extensively methylated only on the active X chromosome.

The DXS255 locus at Xp11.22 is highly polymorphic due to a 26-bp variable number of tandem repeats (VNTR) motif. In previous studies, one of the MspI sites flanking the VNTR manifested a correlation between methylation and X chromosome inactivation. Here we show, by DNA sequence analysis, that this MspI site is located within the CpG island at the 5' end of a LINE-1 element, which is 2.5 kb from the VNTR. The methylation status of the CpG island was assessed in Southern blotting experiments using the methylation-sensitive enzymes HpaII, HhaI, and BssHII. All these sites were completely methylated on active X chromosomes, consistent with previously reported findings of full methylation of LINE-1 elements throughout the genome. However, on inactive X chromosomes these sites were predominantly unmethylated, although patterns were found to be heterogeneous. The results suggest that LINE-1 elements on the inactive X chromosome are not suppressed by full methylation of their CpG islands. The differential methylation of the DXS255 CpG island provides the basis for a highly informative X inactivation analysis system.

Base Sequence↗

PubMed1992

An X chromosome inactivation assay based on differential methylation of a CpG island coupled to a VNTR polymorphism at the 5' end of the monoamine oxidase A gene.

A CpG island has been identified just upstream of the first exon of the human monoamine oxidase A (MAOA) gene, localized to Xp11.4-Xp11.23. Southern blotting following digestion with the methylation sensitive restriction endonucleases SmaI, HpaII and HhaI, indicated that CpG dinucleotides within the CpG island were unmethylated on the active X chromosome and extensively methylated on the inactive X chromosome. These sites of differential methylation were close to a polymorphic GT-dinucleotide/VNTR region, which is located 1 kb 3' of the first exon and has a heterozygosity value of 75%. PCR primers were designed for amplification of 1.2-1.3 kb DNA fragments, encompassing both the hypervariable region and a cluster of six HpaII sites within the CpG-rich region. Cleavage of HpaII sites was found to be restricted to active X chromosomes. Therefore, following HpaII digestion, DNA fragments were exclusively amplified from inactive X chromosomes. The resulting PCR products were digested with SacI, which reduced the size of the DNA fragments containing the hypervariable region to 230-330 bp, and were subsequently analyzed on denaturating polyacrylamide gels. Because amplified fragments were exclusively derived from the inactive X chromosome, the relative densities of the two allelic fragments should reflect the proportions of cells that have either of the two X chromosome inactivated. The results of this PCR-based X chromosome inactivation assay were fully concordant with Southern blotting methylation analyses at the PGK locus. It therefore provides a rapid and informative method in tumour clonality analysis and carrier detection in X-linked diseases.

Base Sequence↗

PubMed1992

The gene encoding the palmitoylated erythrocyte membrane protein, p55, originates at the CpG island 3' to the factor VIII gene.

A CpG island 30 kb 3' to the human factor VIII gene in Xq28 is associated with a 2-kb transcript. This gene encodes a previously described palmitoylated membrane protein, p55, containing a src homology motif, SH3. Although originally described in reticulocytes, the transcript is expressed in a wide variety of human tissues. The gene is also present in the mouse and expressed in all mouse tissues examined. No known factor VIII gene deletions extend into the p55 gene. Since the function of the p55 protein is not known, the p55 gene is formally a candidate for any of the 19 or more disease genes that have not been isolated but are closely linked genetically to the factor VIII gene.

Amino Acid Sequence↗

PubMed1990

5-Azacytidine-induced reactivation of the human X chromosome-linked PGK1 gene is associated with a large region of cytosine demethylation in the 5' CpG island.

Hamster-human cell hybrids containing an inactive human X chromosome were treated with 5-azacytidine and derived clones were examined for phosphoglycerate kinase activity and cytosine methylation in the human PGK1 (X chromosome-linked phosphoglycerate kinase) gene. Comparisons between expressing and nonexpressing clones indicated that demethylation of several methylation-sensitive restriction sites outside of the 5' CpG island were unnecessary for expression. High-resolution polyacrylamide gel analysis of 25 Hpa II, Hha I, and Tha I sites revealed that all clones expressing PGK1 were unmethylated in a large region of the CpG island that includes the transcription start site and 400 base pairs upstream. Many nonexpressing clones had discontinuous patterns of demethylation. Remethylation was often observed in subclones of nonexpressing hybrids. These data suggest that a specific zone of methylation-free DNA within the PGK1 promoter is required for transcription. In addition, the presence of neighboring methylcytosines appears to decrease the heritable stability of unmethylated CpGs in this region.

5-Methylcytosine↗

PubMed1992

Choice of enzymes for mapping based on CpG islands in the human genome.

The frequencies of sites for rare-cutting restriction enzymes in 2.9 million bp of human genomic DNA sequence in the EMBL database have been determined and compared with the expected frequencies. Rare cutters can be divided into four groups based on certain features of their recognition sites. Mlu, I, Nru I, Spl I, and Pvu I are predicted to cleave genomic DNA most infrequently, which is borne out by the fragment lengths observed for Mlu I and Nru I. Thus, these four enzymes are ideal for making long-range maps based on pulsed-field electrophoresis. Other enzymes like Not I are useful for making more detailed maps. Finer maps for identification of CpG islands and associated genes should involve several rare cutters including Eag I, Sac II and Bss HII. A cluster of sites for at least two such enzymes is a good indicator of a CpG island, and 78% of the island-associated genes can be located in this way.

Base Composition↗

PubMed1992

Binding of proteins from embryonic and differentiated cells to a bidirectional promoter contained within a CpG island.

We have analysed binding sites of nuclear protein factors to a CpG island (HTF9), which contains the promoter for a pair of overlapping, divergently-transcribed "housekeeping" genes. Using DNaseI protection assays with extracts from a range of differentiated and undifferentiated cell lines, including mouse embryonic stem (ES) and embryonal carcinoma (EC) cells, we located multiple protein binding sites on HTF9. Most of the sites were outside the defined core promoter and could bind to previously identified transcription factors. These included constitutive, inducible and apparently tissue-specific factors in an extremely asymmetric array relative to the transcription start sites of the two genes. A number of sites showed different binding specificities or affinities in different cell types, including ES cells. However, we found no factors that were specific for both ES and EC cells, and no protein-binding site protected exclusively in undifferentiated embryonic cells.

3T3 Cells↗

PubMed1992

Identification of novel RFLPs in the vicinity of CpG islands in Xq28: application to the analysis of the pattern of X chromosome inactivation.

Probes for CpG islands were cloned from the distal long arm of the human X chromosome; three of them were found to be polymorphic. A HindIII RFLP was identified by the probe 2-25 (DXS606), and it was mapped to the Xq27-Xq28 boundary. Probes 2-19 (DXS605) and 2-55 (DXS707), which identify EcoRI and MspI polymorphisms, respectively, have been mapped to the distal part of Xq28, in the G6PD-RCP/GCP gene region. Probe 2-19 has been further localized about 16 kb from the 3' end of the G6PD gene. The new RFLPs may be useful for the precise mapping of the many disease genes localized in this part of the human X chromosome. Probe 2-19 is highly informative, and it has been studied in greater detail. Using the methylation-sensitive rare-cutter enzyme EagI in conjunction with the polymorphic EcoRI site, we were able to demonstrate that the RFLP may be used both to study randomness of X chromosome inactivation and for carrier detection in X-linked syndromes where nonrandom X inactivation occurs. It is conceivable that the combined use of 2-19 and of the probes described so far (pSPT-PGK and M27 beta) will make analysis of X inactivation feasible in virtually every female.

Alleles↗

PubMed1990

Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation.

A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found that HpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is less than or equal to 10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days; about the time of X-chromosome inactivation of the inner cell mass.

Animals↗

PubMed1991

Revised genomic consensus for the hypermethylated CpG island region of the human L1 transposon and integration sites of full length L1 elements from recombinant clones made using methylation-tolerant host strains.

Efficient recovery of clones from the 5' end of the human L1 dispersed repetitive elements necessitates the use of deletion mcr- host strains since this region contains a CpG island which is hypermethylated in vivo. Clones recovered with conventional mcr+ hosts seem to have been derived preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site duplication in the insertion site as commonly observed with truncated L1 elements. These data would be consistent with two mechanisms of integration of transposing L1 elements with different mechanisms predominating for full length and truncated elements.

Base Sequence↗

PubMed1992

Isolation and sequence of two genes associated with a CpG island 5' of the factor VIII gene.

Many disease loci have been linked to the telomeric end of the long arm of the human X-chromosome, Xq28. We have isolated and sequenced cDNA sequences corresponding to two novel genes that map to Xq28. These genes, c6.1A and c6.1B, are transcribed in opposite directions from a CpG island that lies approximately 70 kilobases (kb) upstream (5') of the factor VIII locus. One of these genes, c6.1A, is highly conserved between species and expressed abundantly in many human and mouse tissues, whereas, c6.1B is moderately conserved and has a restricted tissue distribution of expression. The Xq28 gene c6.1A has an autosomal homologue that is transcriptionally inactive in B-cell lines. An open reading frame (ORF) predicting a peptide of 293 amino acids is observed for c6.1A but c6.1B does not possess a single long ORF. No striking homologies to existing genes could be found for either of the two new loci. Expressed sequences that are physically close to the factor VIII gene are candidates for disease loci that map to this region of Xq28. The relevance of these genes to disease loci was investigated using DNA and RNA from hemophilia A patients bearing deletions that extend in a 5' direction away from factor VIII. The results imply that neither of these genes are primarily responsible for the development Xq28-linked diseases. However, c6.1A and c6.1B define a region of Xq28 that is deleted in two brothers that suffer from mental handicap and dysmorphism as well as hemophilia A. Thus, this region is likely to contain loci that are important for physical and mental development.

Amino Acid Sequence↗