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Isolation of CpG islands from large genomic clones.

ABSTRACT: Positional cloning is a powerful method for the identification of genes. Using genetic and physical mapping methods the genomic region within which a particular gene is located can relatively easily be narrowed down to a comparatively small area contained within cosmid, PAC or BAC clones. It is then a matter of identifying genes within these clones. Here we describe the appli-cation of a technique, which has been successfully used for the bulk purification of CpG islands from whole genomes, to the isolation of CpG island sequences from such clones. As CpG islands overlap transcription units they can be used to isolate full-length cDNAs for associated genes, either by probing cDNA libraries or by searching databases. CpG islands are linked with approximately 60% of human genes and because their isolation is independent of the expression profile of these genes this approach would complement other expression-based methods of gene identification. By applying this technique to a cosmid clone known to contain the PAX6 gene we successfully isolated the CpG island for this gene along with other CpG island-like sequences. Closer examination revealed that an extensive genomic region around the 5'-end of PAX6 is unusual with regard to methylation and GC content. CpG island sequences were also successfully isolated from a PAC clone carrying the MBD1 gene. These included the complete CpG island containing the first exon and regulatory sequences from MBD1.

SUBMITTER: Cross SH

PROVIDER: S-EPMC148429 | biostudies-other | 1999 May

REPOSITORIES: biostudies-other

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Isolation of CpG islands from large genomic clones.

Cross S H SH Clark V H VH Bird A P AP

Nucleic acids research 19990501 10

Positional cloning is a powerful method for the identification of genes. Using genetic and physical mapping methods the genomic region within which a particular gene is located can relatively easily be narrowed down to a comparatively small area contained within cosmid, PAC or BAC clones. It is then a matter of identifying genes within these clones. Here we describe the appli-cation of a technique, which has been successfully used for the bulk purification of CpG islands from whole genomes, to t ...[more]

PMID: 10219082

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Project description:BackgroundMammalian CpG islands (CGIs) normally escape DNA methylation in all adult tissues and developmental stages. However, in our previous study we unexpectedly identified many methylated CGIs in human peripheral blood leukocytes. Methylated CpG dinucleotides convert to TpG dinucleotides through deaminization of their cytosine bases more frequently than hypomethylated CpG dinucleotides. Therefore, we wondered how methylated CGIs in germline or non-germline cells maintain their CpG-rich sequences. It is known that events such as germline hypomethylation, CpG selection, biased gene conversion (BGC), and frequent CpG fixation can contribute to the maintenance of CpG-rich sequences in methylated CGIs in germline or non-germline cells. However, it has not been investigated which of the processes maintain CpG-rich sequences of methylated CGIs in each genomic position.ResultsIn this study, we comprehensively examined the contribution of the processes described above to the maintenance of CpG-rich sequences in methylated CGIs in germline and non-germline cells which were classified by genomic positions. Approximately 60-80% of CGIs with high methylation in H1 cell line (H1-HM) in all the genomic positions showed a low average CpG→TpG/CpA substitution rate. In contrast, fewer than half the numbers of CGIs with H1-HM in all the genomic positions showed a low average CpG→TpG/CpA substitution rate and low levels of methylation in sperm cells (SPM-LM). Furthermore, a small fraction of CGIs with a low average CpG→TpG/CpA substitution rate and high levels of methylation in sperm cells (SPM-HM) showed CpG selection. On the other hand, independent of the positions in genes, most CGIs with SPM-HM showed a slightly higher average TpG/CpA→CpG substitution rate compared with those with SPM-LM.ConclusionsRelatively high numbers (approximately 60-80%) of CGIs with H1-HM in all the genomic positions preserve their CpG-rich sequences by a low CpG→TpG/CpA substitution rate caused mainly by their SPM-LM, and for those with SPM-HM partly by CpG selection and TpG/CpA→CpG fixation. BGC has little contribution to the maintenance of CpG-rich sequences of CGIs with SPM-HM which were classified by genomic positions.

Dataset Information

Isolation of CpG islands from large genomic clones.

Publications

Isolation of CpG islands from large genomic clones.

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