cytidylyl-3--5--guanosine and Chromosome-Deletion

Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methylation in 30 cases of sporadic cutaneous melanoma. The methylation status of the 5' CpG island of p16 was initially determined by Southern analysis and then reevaluated (in a blinded manner) using methylation-specific PCR, methylation-sensitive single nucleotide primer extension, and bisulfite genomic sequencing. All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. Although methylation-associated gene silencing does not represent a common mechanism for p16 inactivation in sporadic melanoma, our findings provide support that PCR-based techniques, such as methylation-specific PCR and methylation-sensitive single nucleotide primer extension, can be reliably used for the accurate detection and quantitation of aberrant levels of DNA methylation in tumor specimens.

Topics: Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 9; Dinucleoside Phosphates; DNA Methylation; DNA Primers; DNA, Neoplasm; Genes, p16; Humans; Melanoma; Mutation; Polymerase Chain Reaction; Promoter Regions, Genetic; Restriction Mapping; Skin Neoplasms; Tumor Cells, Cultured

The recently identified cyclin-dependent kinase inhibitor p15INK4B is localized to a region on chromosome 9p21 frequently deleted in human tumors. Previous evidence has pointed to a related gene, p16INK4A, as the principal target of this deletion. We report that in gliomas and, to a striking degree, in leukemias, the p15 gene is commonly inactivated in association with promoter region hypermethylation involving multiple sites in a 5'-CpG island. In some gliomas and all of the primary leukemias, this event occurs without alteration of the adjacent gene, p16INK4A. In other tumors, including lung, head and neck, breast, prostate, and colon cancer, inactivation of p15INK4B occurs only rarely and only with concomitant inactivation of p16. Aberrant methylation of p15INK4B is associated with transcriptional loss of this gene. Treatment with the demethylating agent 5-aza-2'-deoxycytidine leads to re-expression of p15 mRNA. In selected leukemia cell lines, p15 inactivation correlates with known resistance to the growth-suppressive effects of transforming growth factor-beta. These results suggest that p15INK4B is inactivated selectively in leukemias and gliomas and seems to constitute an important tumor suppressor gene loss in these neoplasms.

Topics: Adult; Breast Neoplasms; Carrier Proteins; Cell Cycle Proteins; Cell Line; Child; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 9; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Dinucleoside Phosphates; DNA; DNA, Neoplasm; Female; Genes, Tumor Suppressor; Glioma; Homozygote; Humans; Leukemia; Leukemia, Myeloid, Acute; Lung Neoplasms; Lymphocytes; Male; Methylation; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prostatic Neoplasms; Reference Values; Restriction Mapping; Tumor Cells, Cultured; Tumor Suppressor Proteins

The H19 gene produces an abundant developmentally regulated transcript of unknown function in normal embryos. In the mouse it lies on chromosome 7 and is subject to transcriptional regulation by parental imprinting, which results in the maternally inherited gene being expressed and the paternally inherited gene being repressed. Embryos carrying maternal duplication/paternal deficiency for distal chromosome 7 (MatDi7) therefore express a double dose of H19. Here we examine the parental-origin-specific epigenetic modifications that may be involved in this regulation by comparing CpG methylation and nuclease sensitivity of chromatin in MatDi7 embryos with normal littermates. We show that specific sites in the CpG island promoter and 5' portion of the gene are methylated only on the paternal allele. Furthermore, active maternal alleles in chromatin of MatDi7 embryos are more sensitive and accessible to nucleases. Therefore hypermethylation and chromatin compaction in the region of the H19 promoter is associated with repression of the paternally inherited copy of the gene. Most, but not all, of these sites are unmethylated in sperm, with methylation of the paternal promoter occurring after fertilization. These results contrast with our findings for the closely linked and reciprocally imprinted gene encoding insulin-like growth factor II (ref. 4).

Topics: Animals; Chromatin; Chromosome Deletion; Deoxyribonucleases; Dinucleoside Phosphates; Dosage Compensation, Genetic; Embryo, Mammalian; Fathers; Female; Gene Expression Regulation; Genes; Insulin-Like Growth Factor II; Male; Methylation; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Mothers; Promoter Regions, Genetic; Restriction Mapping; RNA, Messenger; Spermatozoa

We have identified a previously unrecognized missense mutation in a patient with severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID). The mutation is a G646-to-A transition at a CG dinucleotide and predicts a glycine-to-arginine substitution at codon 216. Computer analysis of secondary structure predicts a major alteration with loss of a beta-pleated sheet in a highly conserved region of the protein. The basepair substitution also generates a new site for the restriction enzyme BstXI in exon 7 of the genomic DNA. Digestion of genomic DNA from the patient and from his parents revealed that he was homozygous for the mutation and that his mother and father were carriers. This mutation in homozygous form appears to be associated with very severe disease, since the patient had perinatal onset of clinical manifestations of SCID, the highest concentration of the toxic metabolite deoxyATP in nine patients studied, and a relatively poor immunologic response during the initial 2 years of therapy with polyethylene glycol-adenosine deaminase. Analysis of DNA from 21 additional patients with ADA-SCID and from 19 unrelated normals revealed that, while none of the normal individuals showed the abnormal restriction fragment, two of the 21 patients studied were heterozygous for the G646-to-A mutation.

Topics: Adenosine Deaminase; Base Composition; Cell Line, Transformed; Chromosome Deletion; Deoxyribonucleases, Type II Site-Specific; Dinucleoside Phosphates; Erythrocytes; Exons; Heterozygote; Homozygote; Humans; Immunologic Deficiency Syndromes; Infant, Newborn; Male; Mutation; Polymorphism, Restriction Fragment Length; Protein Conformation

The mouse CpG-rich island HTF9 harbours the divergent RNA initiation sites shared by two genes that are both expressed in a housekeeping fashion. In this work we have analyzed the architecture of the HTF9 promoter. Gel shift assays were first employed to locate nuclear factor-binding sites within HTF9. Multiple protein-binding sites were identified across a 500 bp-long region, two of which appear to interact with novel factors. Deletion analysis was used to determine the requirements for the different sites in transient expression of a CAT reporter gene. Although multiple elements contributed to the overall promoter strength in each orientation, extensive deletions failed to affect the basal level of transcription from HTF9 in either direction. Thus, only a subset of elements is necessary to activate transcription from HTF9. Functional redundancy may be a general feature of housekeeping CpG-rich promoters.

Topics: Animals; Base Sequence; Binding, Competitive; Blotting, Southern; Blotting, Western; Chloramphenicol O-Acetyltransferase; Chromosome Deletion; Dinucleoside Phosphates; DNA Fingerprinting; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Mice; Molecular Sequence Data; Promoter Regions, Genetic; RNA; Transcription, Genetic

The endogenous retrovirus, intracisternal A-particle (IAP), is expressed at unique stages during murine embryogenesis and is also activated during the in vitro differentiation of F9 cells. We have examined the DNA elements and protein factors that control IAP expression during F9 differentiation. In the present study an IAP upstream enhancer (IUE) is identified by transient transfection assays and found to be active in both undifferentiated and differentiated cells. Further analyses reveal that a ubiquitous 65 kDa protein factor, the IUE binding protein (IUEB), binds with the IUE. Site-specific methylation within the IUEB binding site strongly inhibits both IUEB binding and IUE transcriptional activity, suggesting that methylation may regulate IUE function and IAP expression.

Topics: Animals; Base Sequence; Cell Differentiation; Chromosome Deletion; Dinucleoside Phosphates; DNA Mutational Analysis; DNA-Binding Proteins; DNA-Cytosine Methylases; Enhancer Elements, Genetic; Gene Expression Regulation, Viral; Genes, Intracisternal A-Particle; HeLa Cells; Humans; Methylation; Mice; Molecular Sequence Data; Repetitive Sequences, Nucleic Acid; Retroviridae; Transcription Factors; Transcription, Genetic

The use of optimally methylation-tolerant mcrA- mcrB- strains has been shown to produce an over tenfold increase in the plating efficiencies of mammalian genomic libraries, compared to a superior conventional phage host strain LE392 which is mcrB+. However, there is an even more significant effect of mcr restriction. Amongst the recombinants recovered with an mcrB+ host, we have found that there is an additional 30-fold reduction in the frequencies of clones containing the heavily methylated 5'-CpG island sequences of both the human and rat L1 repetitive elements. The mcrA product was also found to restrict clones of these methylated genomic segments, but not as strongly as mcrB. However, the use of packaging extracts made from mcrA+ lysogens did not result in convincing reductions in the recoveries of these dispersed methylated elements. The magnitude of mcr restriction during plating due to methylated dispersed elements is sufficient to make a significant proportion of mammalian genomes unclonable from genomic libraries constructed previously using conventional mcr+ hosts.

Topics: Animals; Bacteriophages; Chromosome Deletion; Cloning, Molecular; Deoxyribonucleases; Dinucleoside Phosphates; DNA Transposable Elements; Escherichia coli; Genes, Bacterial; Genetic Vectors; Genomic Library; Genotype; Humans; Methylation; Rats; Repetitive Sequences, Nucleic Acid

Adenomatous polyposis coli (APC), a dominantly inherited disorder, has been mapped to chromosome 5q15-q21 by family linkage studies. Cells from patients with deletions in this region, in one case associated with polyposis in a family, have been used to construct human hamster hybrid cell lines that retain either the normal or deleted chromosome 5. These lines have been used to identify markers from the region of the polyposis gene obtained by cloning the ends of 0.5- to 2-megabase BssHII fragments purified by pulsed-field gel electrophoresis. Three markers are described that map within the deletions and must therefore be close to the APC gene.

Topics: Adenoma; Adenomatous Polyposis Coli; Base Sequence; Blotting, Southern; Cell Line; Cell Transformation, Viral; Chromosome Deletion; Chromosomes, Human, Pair 5; Cloning, Molecular; Dinucleoside Phosphates; Gene Library; Genes, Dominant; Herpesvirus 4, Human; Humans; Karyotyping; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Oligonucleotide Probes; Restriction Mapping