Analysis of the total base composition of DNA from seven different normal human tissues and eight different types of homogeneous human cell populations revealed considerable tissue-specific and cell-specific differences in the extent of methylation of cytosine residues. The two most highly methylated DNAs were from thymus and brain with 1.00 and 0.98 mole percent 5-methylcytosine (m5C), respectively. The two least methylated DNAs from in vivo sources were placental DNA and sperm DNA, which had 0.76 and 0.84 mole percent m5C, respectively. The differences between these two groups of samples were significant with p less than 0.01. The m5C content of DNA from six human cell lines or strains ranged from 0.57 to 0.85 mole percent. The major and minor base composition of DNA fractionated by reassociation kinetics was also determined. The distribution of m5C among these fractions showed little or no variation with tissue or cell type with the possible exception of sperm DNA. In each case, nonrepetitive DNA sequences were hypomethylated compared to unfractionated DNA.

The over-all 5-methylcytosine (m5C) content of DNA from normal tissues varies considerably in a tissue-specific manner. By high-performance liquid chromatography, we have examined the m5C contents of enzymatic digests of DNA from 103 human tumors including benign, primary malignant and secondary malignant neoplasms. The diversity and large number of these tumor samples allowed us to compare the range of DNA methylation levels from neoplastic tissues to that of normal tissues from humans. Most of the metastatic neoplasms had significantly lower genomic m5C contents than did most of the benign neoplasms or normal tissues. The percentage of primary malignancies with hypomethylated DNA was intermediate between those of metastases and benign neoplasms. These findings might reflect an involvement of extensive demethylation of DNA in tumor progression. Such demethylation could be a source of the continually generated cellular diversity associated with cancer.

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Functional 30S ribosomes were reconstructed from total Escherichia coli 30S ribosomal proteins and 16S ribosomal RNA synthesized in vitro by T7 RNA polymerase. Up to 700 mol of RNA/mol of template could be obtained. The transcript lacked all ten normally modified bases and had three additional 5' G residues, an A----G change at position 2, and, in 22% of the molecules, one or two extra 3' residues. The synthetic 16S RNA could be assembled into a particle that cosedimented with authentic 30S and was indistinguishable from 30S by electron microscopy. When supplemented with the 50S subunit, the particles bound tRNA to the 70S P site in a codon- and Mg2+-dependent manner. The specific binding activity was 94% that of particles reconstituted with natural rRNA and 52% that of native 30S. Cross-linking to P site bound tRNA was also preserved. Changing C-1400, the residue known to be close to the anticodon of P site bound tRNA, to A had little effect on reconstitution, but the C----G substitution caused a marked inhibition of assembly. tRNA could bind to both reconstituted mutants, but cross-linking was greatly reduced. These results show that none of the modified bases of 16S RNA are essential for P site binding and that position 1400 may be more important for ribosome assembly than for tRNA binding. Base-specific in vitro mutagenesis can now be used to explore in detail the functional properties of individual residues in ribosomal RNA.

Sodium bisulfite is a mutagen which can specifically deaminate more than 96% of the cytosine residues in single-stranded DNA via formation of a 5,6-dihydrocytosine-6-sulfonate intermediate. Under the same reaction conditions, only 2-3% of the 5-methylcytosine (m5Cyt) residues in single-stranded XP-12 DNA, which has 34 mole% m5Cyt, was converted to thymine (Thy) residues. In contrast, at the deoxynucleoside and free base levels, the same treatment with bisulfite and then alkali converted 51% and > 95%, respectively, of the m5Cyt to the corresponding Thy derivatives. However, the rate of reaction of m5Cyt and its deoxyribonucleoside was much slower than that of the analogous quantitative conversion of cytosine or deoxycytidine to uracil or deoxyuridine, respectively. The much lower reactivity of m5Cyt and its derivatives compared to that of the unmethylated analogs is primarily due to a decrease in the rate of formation of the sulfonate adduct.

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The heat-induced conversion of 5-methylcytosine (m5C) residues to thymine residues and of cytosine to uracil residues in single-stranded DNA was studied. The calculated rates for deamination at 37 degrees C and pH 7.4 were approximately 9.5 X 10(-10) and 2.1 X 10(-10) sec-1, respectively. N4-Methyldeoxycytidine, which is in the DNA of certain thermophilic bacteria, was more heat-resistant than was deoxycytidine and much more than was 5-methyldeoxycytidine. Thermophilic bacteria which contain N4-methylcytosine rather than m5C in their genomes may thereby largely avoid heat-induced mutation due to deamination, which is incurred by the many organisms that contain m5C in their DNA.