The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfiteinduced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by derming the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.Cytosine methylation has long been recognized as an important factor in the silencing of genes in mammalian cells. Recent studies have shown that cytosine methylation at single CpG dinucleotides within the recognition sites of a number of transcription factors is sufficient to block binding of the factors to DNA (1-6) and to inhibit transcription (3-5). Therefore, to determine the role of cytosine methylation in specific regulatory mechanisms in vivo, it has become important to know the methylation status of individual CpG dinucleotides in genomic DNA. Genomic sequencing protocols, which have been developed to ascertain the methylation status of selected regions within genes, utilize the Maxam and Gilbert chemical cleavage reactions carried out on genomic DNA (7) with various additional procedures to enhance the signal from the sequence under investigation (8, 9). These protocols are versatile in that they can be adapted for identification of protein-binding sites on genomic DNA in vivo (8, 10) but have two major drawbacks with respect to the identification of 5-methylcytosine residues. First, 5-methylcytosine is identified by the lack of a band in all tracks of a sequencing gel; any background cleavage ladder or close spacing of bands can result in difficulties of interpretation. Second, the sequence obtained represents a population average for many DNA molecules, so that the protocols cannot be adapted for sequencing s...

No abstract

The detection of 5'-methylcytosine by the bisulphite-mediated genomic sequencing method has considerably aided study of the role of methylation in areas such as X chromosome inactivation, genomic imprinting and cancer research. However on occasion difficulty has been experienced in obtaining complete conversion of cytosine to uracil in regions of the target DNA. We report here a simple improvement to the method involving addition of urea to the bisulphite reaction, a step which greatly improves the reaction efficiency, presumably by maintaining the target DNA in single stranded form, thereby allowing complete and reliable conversion.

No abstract

SummaryThe morphology of the fowl spermatozoon, as revealed by use of the electron microscope and such techniques as partittl enzymic digestion and disruption with distilled water, is described in detail, and compared with that observable by light microscopy.The sperm head carries at its anterior extremity a spindle-shaped body, the apical spine, which normally is closely covered by a conical membranous cap. The apical cap, which has been overlooked by previous workers, may be detached by dilution of the semen with distilled water. It seems likely that these structures are intimately concerned in the penetration of the vitelline membrane during fertilization.The axial filament, which contains nine L fibrils arising from the al}terior distal centriole and two M fibrils, passes through the mid-piece and continues the full length of the tail, approaching 100 Il. in length. It is surrounded in the mid-piece by a number of granules, presumably of mitochondrial origin, which are arranged to give an appearance of bilaterally symmetrical segmentation. The mid-piece is externally surrounded by a delicate membrane easily disrupted in distilled water. There is no evidence for the presence of a spireme or other helically-wound structure in the mid-piece.In the tail, the axial filament is encased in an amorphous sheath, which decreases in thickness towards the tip of the tail and is easily disrupted by distilled water, allowing the axial filament to fray into eleven fibrils. Two of these fibrils are differentiated from the remaining nine by their dimensions and greater susceptibility to distilled water. It is suggested that the nine L fibrils constitute the locomotor organ of the sperm. It is possible that the two M fibrils function as a rudimentary nervous system.In direct contrast with the state prevailing in mammalian sperm, there is no helically-wound cord surrounding the axial filament in the tail. This seems to explain why the tails of fowl and of certain other sperm fray easily in distilled water, while those of mammalian sperm do not.Certain dilution phenomena are explained by the presence of an adsorbed layer of colloidal material, which is removable by great dilution or repeated washing of the sperm. The layer greatly modifies the rate of osmosis in hypotonic solutions. There is no trace of a lipoid or other capsule external to the cell-wall, as has been postulated to explain similar protective phenomena occurring with other sperm .