A complex of histones H2A, H2B, H3 and H4 has been isolated from purified rat liver nuclei by a method which is both gentle and rapid. Nuclei were homogenised in 0.25 M sucrose and the residual nuclear material obtained after centrifligation was adsorbed on calcium phosphate gel. After removing histone H1 from the adsorbed material by washing with 1M NaCl in 25 mM sodium phosphate buffer, pH 6.0, histones H2A, H2B, H3 and H4 were eluted together, with 2 M NaCl in 25 mM sodium phosphate buffer, pH 7.0. The core histones so obtained migrated as a single sharp band on polyacrylamide gel electrophoresis under non-denaturing conditions. Fractionation of the freshly prepared core histones on a Sephadex G-100 column yielded two major protein peaks. The peak having the larger elution volume contained histones H2A and H2B in equal amounts while the peak with the smaller elution volume contained all the four histones. Histones H3 and H4 were present in larger proportions in the second peak.

We propose a molecular mechanism for the intra-cellular measurement of the ratio of the number of X chromosomes to the number of sets of autosomes, a process central to both sex determination and dosage compensation inDrosophila melanogaster. In addition to the two loci,da andSxl, which have been shown by Cline(Genetics, 90, 683, 1978)and others to be involved in these processes, we postulate two other loci, one autosomal (Ω) and the other, X-linked (π). The product of the autosomal locusda stimulates Ω and initiates synthesis of a limited quantity of repressor.Sxl and π ,both of which are X-linked, compete for this repressor as well as for RNA polymerase. It is assumed thatSxl has lower affinity than π for repressor as well as polymerase and that the binding of polymerase to one of these sites modulates the binding affinity of the other site for the enzyme. It can be shown that as a result of these postulated interactions transcription from theSxl site is proportional to the X/A ratio such that the levels ofSxl⁺ product are low in males, high in females and intermediate in the intersexes. If, as proposed by Cline, theSxl^- product is an inhibitor of X chromosome activity, this would result in dosage compensation. The model leads to the conclusion that high levels ofSxl⁺ product promote a female phenotype and low levels, a male phenotype. One interesting consequence of the assumptions on which the model is based is that the level ofSxl⁺ product in the cell, when examined as a function of increasing repressor concentration, first goes up and then decreases, yielding a bell-shaped curve. This feature of the model provides an explanation for some of the remarkable interactions among mutants at theSxl, da andmle loci and leads to several predictions. The proposed mechanism may also have relevance to certain other problems, such as size regulation during development, which seem to involve measurement of ratios at the cellular level.

The methylation status of the nuclear DNA from a mealybug, aPlanococcus species, has been studied. Analysis of this DNA by High Performance Liquid Chromatography and Thin Layer Chromatography revealed the presence of significant amounts of 5-—methylcytosine. Since analysis of DNA methylation using the Msp I/Hpa II system showed only minor differences in susceptibility of the DNA to the two enzymes, it seemed possible that 5-methylcytosine (5mC) occurred adjacent to other nucleotides in addition to its usual position, next to guanosine. This was verified by dinucleotide analysis of DNA labelledin vitro by nick translation. These data show that the total amount of 5-methylcytosine in this DNA is slightly over 2.3 mol %, of which 0.61% occurs as the dinucleotide 5mCpG, 0.68% as 5mCpA, 0.59% as 5mCpT and 0.45% as 5mCpC. 5mCpG represents approximately 3.3% of all CpG dinucleotides. The experimental procedure would not have permitted the detection of 5mCp5mC, if it occurs in this system. Unusually high amounts of 6-methyladenine (approximately 4 mol %) and 7-methylguanine (approximately 2 mol %) were also detected, 6-methyladenine and 7-methylguanine occurred adjacent to all four nucleotides. The total G+C content was 33.7% as calculated from dinucleotide data and 32.9% as determined from melting profiles.