Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.

https://www.pnas.org/content/pnas/93/18/9821.full.pdf

Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands

https://www.cell.com/cell/pdf/S0092-8674(00)81656-6.pdf

DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

https://www.cell.com/cell/pdf/0092-8674(87)90579-4.pdf

Dystrophin: The protein product of the duchenne muscular dystrophy locus

Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.

It is likely that most vertebrate genes are associated with 'HTF islands'--DNA sequences in which CpG is abundant and non-methylated. Highly tissue-specific genes, though, usually lack islands. The contrast between islands and the remainder of the genome may identify sequences that are to be constantly available in the nucleus. DNA methylation appears to be involved in this function, rather than with activation of tissue specific genes.

CpG-rich islands and the function of DNA methylation

Mice carrying mutations at the W locus located on chromosome 5 are characterized by severe macrocytic anaemia, lack of hair pigmentation and sterility1. Mutations at this locus appear to affect the proliferation and/or migration of cells during early embryogenesis and result in an intrinsic defect in the haematopoietic stem cell hierarchy1,2. An understanding of the molecular basis of the complex and pleiotropic phenotype in W mutant mice would thus provide insights into the important developmental processes of gametogenesis, melanogenesis and haematopoiesis. Here we show that the mouse mutant W19H has a deletion of the c-kit proto-oncogene. Interspecific backcross analysis demonstrates that the W locus is very tightly linked to c-kit and that the two loci cannot be segregated at this level of analysis, c-kit is the cellular homologue of the oncogene v-kit of the HZ4 feline sarcoma virus3 and encodes a transmembrane protein tyrosine kinase receptor that is structurally similar to the receptors for colony-stimulating factor-1 (CSF-1) and platelet derived growth factor4,5. The co-localization of c-kit with W provides a molecular entry into this important region of the mouse genome. In addition, these observations provide the first example of a germ-line mutation in a mammalian proto-oncogene and implicate the c-kit gene as a candidate for the W locus.

The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus.

https://www.cell.com/cell/pdf/0092-8674(77)90151-9.pdf

Preferential expression of the maternally derived X chromosome in the mouse yolk sac.

Thirteen biochemical groups of wild mice from Europe, Asia, and Africa belonging to the genus Mus are analyzed at 22–42 protein loci. Phylogenetic trees are proposed and patterns of biochemical evolution are discussed, as well as the possible contribution of wild mice to the genetic diversity of laboratory stocks.

Biochemical diversity and evolution in the genus Mus.

Fluorescence in situ hybridization (FISH) is an effective technique for localizing cloned DNA probes directly onto metaphase chromosomes. Human genome mapping using FISH has been significantly enhanced by the development of new techniques, especially high-resolution gene mapping with direct R-banding FISH and physical gene ordering with multi-color FISH. By contrast, FISH techniques have not been put to practical use for the analysis of the mouse genome compared with the human. We have developed and modified FISH techniques for use in mouse genome analysis. In this article we summarize and review our recent results with FISH analyses in the following studies: (i) high-resolution gene mapping with the direct R-banding FISH, (ii) analysis of chromosomal rearrangement with multi-color FISH, (iii) establishment of centromere mapping with the major satellite DNA probe, (iv) analysis of chromatin structure in meiotic cells, and (v) application of FISH in cytogenetic studies of genetic variation in the mouse, showing that these applications of FISH are very useful for mouse genome analysis.

Application of fluorescence in situ hybridization in genome analysis of the mouse

We have used elevated levels of plasma creatine phosphokinase activity to identify muscular dystrophy phenotypes in mice and to screen the progeny of chemical mutagen-treated male mice for X chromosome-linked muscular dystrophy mutations. We were not successful in identifying heterozygous carriers of these induced muscular dystrophy mutations in greater than 8000 progeny. However, we were highly successful in identifying three additional alleles of the characterized mdx locus. These alleles of mdx were recovered from various mutagen-treated males and they occur on an X chromosome that carries flanking markers that allow us to follow the mutations in genetic crosses and in the development of corresponding mutant stocks. These alleles have been designated as mdx2Cv, mdx3Cv, and mdx4Cv. Preliminary data show that mice with mdx2Cv and mdx3Cv mutations have muscular dystrophic phenotypes that do not grossly differ from the characterized mdx mutation. These additional mdx mutations expand the value of mouse models of X chromosome-linked muscular dystrophy and potentially define additional sites of mutation that impair dystrophin expression.

https://www.pnas.org/content/pnas/86/4/1292.full.pdf

Verne M. Chapman

Papers

The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus.

Preferential expression of the maternally derived X chromosome in the mouse yolk sac.

Biochemical diversity and evolution in the genus Mus.

Application of fluorescence in situ hybridization in genome analysis of the mouse

Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice.