We washed cells in PBS and lysed them in radioimmunoprecipitation assay buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40, 1% Nadeoxycholate, and 0.1% SDS) with added protease inhibitors. We resolved Empirical Relationship between Intra-Purine and Intra-Pyrimidine Differences in Conserved Gene Sequences DNA sequences seen in the normal character-based representation appear to have a formidable mixing of the four nucleotides without any apparent order. Nucleotide frequencies and distributions in the sequences have been studied extensively, since the simple rule given by Chargaff almost a century ago that equates the total number of purines to the pyrimidines in a duplex DNA sequence. While it is difficult to trace any relationship between the bases from studies in the character representation of a DNA sequence, graphical representations may provide a clue. These novel representations of DNA sequences have been useful in providing an overview of base distribution and composition of the sequences and providing insights into many hidden structures. We report here our observation based on a graphical representation that the intra-purine and intra-pyrimidine differences in sequences of conserved genes generally follow a quadratic distribution relationship and show that this may have arisen from mutations in the sequences over evolutionary time scales. From this hitherto undescribed relationship for the gene sequences considered in this report we hypothesize that such relationships may be characteristic of these sequences and therefore could become a barrier to large scale sequence alterations that override such characteristics, perhaps through some monitoring process inbuilt in the DNA sequences. Such relationship also raises the possibility of intron sequences playing an important role in maintaining the characteristics and could be indicative of possible intron-late phenomena. The apparent lack of pattern of composition and distribution of bases in DNA sequences have been one of the enduring problems of molecular biology. Chargaff's rule provided a clear relationship between the numbers of guanines and cytosines, and between adenines and thymines in duplex DNA sequences, understood well by the Watson-Crick model. However, no clear relationship has been found as yet between the occurrences of these four bases in an individual strand of a gene sequence, although much work have been done on understanding nucleotide frequencies and base distributions in DNA sequences. To cite a few examples, Sueoka [1] proposed a unitary theory based on genetic and evolutionary considerations which attempted to account for the main characteristics of the distribution of DNA base composition in nature. The author considered the GC content of DNA sequences of organisms and mutations of GC base pairs to AT base pairs and vice versa over many generations from which a distribution function of the GC content of DNA molecules at equilibrium was obtained based on assumptions of rather uniform mutation and selection pressures affecting base pair conversions.