Ion Induced Coil to Helix Transition of Dialyzed Poly d(A-T) from Cancer borealis

Abstract

Naturally occurring poly d(A-T) from testes of Cancer borealis was prepared starting with a fibrous DNA-RNA complex. The complex was chromatographed on a methylated albuminkieselguhr column, fractions with a high poly d(A-T) content were collected, treated with a large amount of pancreatic ribonuclease and rechromatographed on methylated albumin-kieselguhr.The purest preparations obtained had 0.74 mole ''lo of cytosine and 0.71 mole Controlled dialysis of the poly d(A-T) against distilled water resulted in a hyperchromic shift closely resembling thermal melting. Graded addition of bivalent metal (preferably Mg") to the dialyzed polymer at room temperature reversed the hyperchromic shift. The term "ionic freezing" is suggested for the transition from coil to helix induced by the addition of metal. The mitlpoint of this transition, designated I f , is as characteristic for a preparation of DNA as T,. I n the Mg++ to P ratio range of 0 to 0.5 both values are directly proportional, and the change in I f of 10 pM Mg++ corresponds to the change in T , of 5". I n this Mg" to P ratio range, the metal is bound stoichiometrically. Divalent cations differ in their efficiency of reforming helix and are arranged in the following series starting with the least effective (highest T m at 50 pM) : Zn>Mg> Ba>Ca>Mn>Co. The ion-induced coil to helix transition is pH dependent. The ion-induced transition may also be accomplished in 8 M urea provided the temperature is lowered to -0.5".From several considerations it seems probable that the bases participate in metal binding.Partial separation of the strands of poly d(A-T) after dialysis and their reunification by the added Mg" was confirmed by electron microscopy. Biological implications of this finding were discussed in terms of the possible role of Mg in tJhe initiation of strand separation. of guanine.

Fractionation of d(A-T) polymer from testes of Cancer borealis

“…In the previous procedure (Baranowska et al, 1968) the amounts of enzymes used to remove RNA were larger than required for a complete digestion of pure RNA, but rather moderate for the digestion of RNA/DNA hybrids. The reason for this moderation Trace Trace 0 Columns 4-7 refer to the d(A-T) portion which has been separated from each of the methylated albumin kieselguhr fractions by thermal chromatography on hydroxylapatite.6 As free bases, corrected for the loss of thymine, assuming T = A. figure 2: Elution pattern obtained from column chromatography of purified crab DNA on methylated albumin kieselguhr.…”

“…In view of this, no attempts were made to avoid scission of DNA. In fact, it was hoped that the previously observed i20,w value of 12 S would be reproduced (Baranowska et al, 1968). Such fragments would be large enough to preserve the character of the DNA and yet small enough to accentuate differences in C + G content, if these two bases are distributed nonuniformally.…”

“…In a previous paper of this series (Baranowska et al, 1968), a semimacro method of preparation of renatured poly d(A-T) from crab testes has been described. The preparation was slightly contaminated with RNA and had an unusually low C + G content of 1.5 mole % instead of the accepted 2.6-3.0 mole % (Sueoka and Cheng 1962a,b;Swartz et al, 1962;Goldberg et al, 1962;Smith, 1964;Nandi et al, 1965;Widholm and Bonner, 1966;Skinner, 1967; Hyman and Davidson, 1967).…”

Kinetic investigation of unfolding and partial refolding of a crab satellite (dA-dT)n

“…Isolation of Whole Crab DNA. The isolation procedure was essentially the technique of Marmur (1961) as modified by Baranowska et al (1968) and Brzezinski et al (1969); however, to minimize enzymatic degradation of the DNA, fresh tissue was used. The vas deferens and testes from 120 live male C. borealis were removed, suspended in a cold (~4°) solution of 0.1 M NaCl-0.1 M EDTA (pH 7.49), and homogenized.…”

Isolation and characterization of a neutral deoxyribonuclease from the testes of the crab Cancer pagurus

“…Bacterial DNAs were extracted by the method of Marmur (1961). Crab DNA was prepared following the technic of Baranowska et al (1968). Full deproteinization was achieved by repeated treatments with phenol.…”