Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila.

CpG methylation is essential for mouse development as well as gene regulation and genome stability. Many features of mammalian DNA methylation are consistent with the action of a de novo methyltransferase that establishes methylation patterns during early development and the post-replicative maintenance of these patterns by a maintenance methyltransferase. The mouse methyltransferase Dnmt1 (encoded by Dnmt) shows a preference for hemimethylated substrates in vitro, making the enzyme a candidate for a maintenance methyltransferase. Dnmt1 also has de novo methylation activity in vitro, but the significance of this finding is unclear, because mouse embryonic stem (ES) cells contain a de novo methylating activity unrelated to Dnmt1 (ref. 10). Recently, the Dnmt3 family of methyltransferases has been identified and shown in vitro to catalyse de novo methylation. To analyse the function of these enzymes, we expressed Dnmt and Dnmt3a in transgenic Drosophila melanogaster. The absence of endogenous methylation in Drosophila facilitates detection of experimentally induced methylation changes. In this system, Dnmt3a functioned as a de novo methyltransferase, whereas Dnmt1 had no detectable de novo methylation activity. When co-expressed, Dnmt1 and Dnmt3a cooperated to establish and maintain methylation patterns. Genomic DNA methylation impaired the viability of transgenic flies, suggesting that cytosine methylation has functional consequences for Drosophila development.

DNA methylation: biology and significance.

The modification of DNA by cytosine methylation is crucial for normal development. DNA methylation patterns are distinctive between tissues and are maintained with high fidelity during cell division. DNA methylation probably exerts its effects through alterations in chromatin structure, with a resultant effect on genetic transcription. 5-methylcytosine is also prone to spontaneous hydrolytic deamination to thymine. Whilst most G:T mismatches so produced are repaired, failure of mismatch repair leads to established mutation. Indeed, mutations that are the result of 5-methylcytosine transitions account for a disproportionate number of genetic mutations described in malignant and non-malignant disease. There is also evidence for substantial deregulation of DNA methylation in malignancy. Whether this deregulation is crucial for the transformation process, or simply an epiphenomenon associated with it, is still not established.

Measurement of genome wide DNA methylation by reversed-phase high-performance liquid chromatography.

A method to determine the extent of cytosine methylation in DNA is described. The technique involves the enzymatic hydrolysis of DNA to its deoxyribonucleotide components and subsequent separation and quantification of the nucleotides by isocratic reversed-phase high-performance liquid chromatography (HPLC). The system gives highly reproducible results and, under suitable conditions, is capable of measuring 5-methylcytosine levels in as little as 1 microg of DNA.

Restriction endonuclease isoschizomers ItaI, BsoFI and Fsp4HI are characterised by differences in their sensitivities to CpG methylation.

BsoFI , ItaI and Fsp4HI are isoshizomers of Fnu4HI (5'-GC NGC-3'). Both Fnu4HI and BsoFI have previously been shown to be inhibited by cytosine-specific methylation within the recognition sequence. Fnu4HI is inhibited if either the internal cytosine at position 2 or the external cytosine at position 5 of the restriction sequence is methylated, but the precise nature of the methylation sensitivity of BsoFI is unclear from the literature. The methylation sensitivities of ItaI and Fsp4HI have not previously been reported. By methylating the plasmid pUC18 with M.SssI (a DNA cytosine-5'-methyltransferase with a specificity for CpG), we have determined that ItaI is sensitive only to methylation of internal CpG sites within the restriction sequence. The methylation sensitivity of Fsp4HI is identical to that of Fnu4HI, being inhibited by methylation of either internal CpG sites or overlapping CpG sites. BsoFI , like the other isoschizomers tested, is sensitive to a combination of internal and overlapping CpG methylation. BsoFI is also sensitive to overlapping CpG methylation (in the absence of internal CpG methylation) if CpG overlap with both sides of the recognition sequence. Sites containing one overlapping CpG (in the absence of internal CpG) are cut when methylated but show marked individual variation in their rates of cleavage. Considerable variation in the rate of cleavage by BsoFI is also observed at sites containing only internal methylated CpG. Some sites are cut slowly, whilst others fail to cut even after prolonged incubation with excess of enzyme.

Nucleic acid composition of bone marrow mononuclear cells in cobalamin deficiency.

Recent studies using anion exclusion chromatography have suggested that uracil is misincorporated into the DNA of patients with megaloblastic anemia to levels detectable by nonradioactive methods. We have investigated the nucleotide composition of DNA from the bone marrow mononuclear cells of eight patients with cobalamin deficiency and compared this with that found in normal subjects. The median level of uracil in the megaloblastic group was 0.082 mol% of cytosine (approx. 0.02 mol% of all bases in DNA), which was similar to that found in the control group (median 0.085 mol% of cytosine) and may be attributable, at least in part, to artefactual deamination of deoxycytidine monophosphate during the DNA hydrolysis. Our findings give no support for the view that, by overwhelming the uracil N-glycosidase mechanism, the degree of uracil misincorporation in megaloblastic anemia is sufficient to increase the steady state level of uracil in the DNA by amounts detectable by nonradioactive methods. Using high performance liquid chromatography, we have also demonstrated normal levels of methylcytosine in the DNA of megaloblastic subjects.

Obstetric management in von Willebrand's disease: a report of 24 pregnancies and a reivesw of the literature.

The case records of 13 patients (24 pregnancies) with von Willebrand's disease (vWD) were studies rettospectively. The overall incidence of primary and secondary post-partum haemorrhage (PPH) was 15.8% and 25% respectively, all primary PPH occurring in tyre 2 discase (3/14 deliveries, 21.4%). The risk of primary PPH in type 2 patients who did not receive prophylactic factor VIII was 37.5% (3/8 deliveries). Factor VIII coagulant activity (VIII:C) and von Willebrand factor antigen (vWF:Ag) rose above bascline values by a factor of at least 1.5 during the pregnancy in most case. More severely affected patients were less likely to benefit significatntly. A baseline VIII:C of <15 iu/dl (4/14 cases) was predictive of a third trimester level of <15 iu/dl. Improvements in the von Willebrand factor activity were less marked. The baseline von Willebrand factor activity was <15 iu/dl in all patients with serial data, none of whom achieved a third-trimester von Willebrand factor activity of >50 iu/dl. The bleeding times were unaltered significantly in all but one of the cases, reflecting a general failure of the primary haemostatic defect to improve with pregnancy. The findings demonstrate that coagulation parameters do not universally improve in pregnancy in vWD, especially when preconception levels are low. The risk of primary PPH is generally higher in type 2 diseases. The level of factor VIII:C is not a good predictor of the risk of primary PPH in type 2 patients. Secondary PPH is a significatnt risk in both type 1 and type 2 patients.

Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a.

Current evidence indicates that methylation of cytosine in mammalian DNA is restricted to both strands of the symmetrical sequence CpG, although there have been sporadic reports that sequences other than CpG may also be methylated. We have used a dual-labeling nearest neighbor technique and bisulphite genomic sequencing methods to investigate the nearest neighbors of 5-methylcytosine residues in mammalian DNA. We find that embryonic stem cells, but not somatic tissues, have significant cytosine-5 methylation at CpA and, to a lesser extent, at CpT. As the expression of the de novo methyltransferase Dnmt3a correlates well with the presence of non-CpG methylation, we asked whether Dnmt3a might be responsible for this modification. Analysis of genomic methylation in transgenic Drosophila expressing Dnmt3a reveals that Dnmt3a is predominantly a CpG methylase but also is able to induce methylation at CpA and at CpT.