FRACTIONATION OF D(A-T) POLYMER FROM TESTES OF CANCER BOREALIS

A fibrous complex composed of d(A-T) polymer, major deoxyribonucleic acid, ribonucleic acid, and histone has been prepared from the testes of Cancer borealis as described previously (Baranowska, B., Baranowski, T., and Laskowski, M., Sr. (1968), European J. Biochem, 4, 345). The complex was treated with massive doses of enzymes (pancreatic ribonuclease, T1 ribonuclease, and pronase) to remove contaminants; the purified deoxyribonucleic acid (containing only satellite and major deoxyribonucleic acid component) was isolated and chromatographed on methylated albumin kieselguhr by stepwise elution with increasing concentrations of NaCl. Each fraction from methylated albumin kieselguhr was chromatographed on hydroxylapatite with constant salt concentration but varying temperature. This procedure yielded renatured poly d(A-T) which was clearly separated from denatured major component. The relative amount of poly d(A-T) in each methylated albumin kieselguhr fraction increased with increasing concentration of eluting salt, reached a maximum at 0.75 M NaCl, and then decreased. The poly d(A-T) from methylated albumin kieselguhr fractions eluted with lower NaCl concentrations had lower s20,w and higher C + G values than d(A-T) originating from fractions eluted with higher salt concentrations. The reversed procedure in which purified crab deoxyribonucleic acid was first fractionated on hydroxylapatite then on methylated albumin kieselguhr yielded several fractions of renatured poly d(A-T). The s20,w values increased in fractions eluted with higher salt concentrations. A high C + G content was found in the low molecular weight fraction, whereas fractions in the range of 4.8-9.9 S had lower but comparatively constant C + G values. These results strongly support the assumption of a nonuniform distribution of C + G in the d(A-T) polymer. An alternative procedure for the preparation of renatured poly d(A-T) is direct thermal chromatography of the fibrous complex on hydroxylapatite. This step removes ribonucleic acid and histone as efficiently as the more laborious use of enzymes and repeated shaking with chloroform. Hydroxylapatite chromatography may yield a nondenatured major component of crab deoxyribonucleic acid, if upon reaching 80° the temperature is brought back to 60° and the strength of the eluting phosphate buffer increased to 0.5 M. The use of thermal chromatography on hydroxylapatite as an analytical method for determination of the amount of satellite in a mixture is also described. The method is as simple as the determination of melting temperature profile, and more convenient for quantitative work. © 1969, American Chemical Society. All rights reserved.