Deoxyribonucleic Acid Base Composition of Cyanobacteria By MICHAEL HERDMAN , " MONIQUE JANVIER , JOHN B . WATERBURY , ? ROSMARIE
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چکیده
Strains. The strains examined are all maintained in the Pasteur Culture Collection (PCC) and havenow been deposited in the American Type Culture Collection (ATCC). They are identified here by both PCC and ATCC strain numbers. Full strain histories, media employed for cultivation and the explanation of generic terminology are given by Rippka et al. (1979). An additional strain (Oscillatoria agardhii) was isolated by Dr F. I. Kappers from the Veluwemeer, The Netherlands, and is now in pure culture as strain PCC 7805. Extraction of DNA. DNA for use in thermal denaturation experiments was purified by a method modified from that of Britten et al. (1968). Harvested cells (approximately 5 g wet wt) were suspended in 20 ml lysis mixture containing 8 M-urea, 1 M-NaClO,, 0.01 M-EDTA (disodium salt) and 1 % (w/v) sodium dodecyl sulphate (SDS) in 0.24 M-phosphate buffer (pH 7-0), disrupted by passage through a French press at 76 MPa, and partially deproteinized by shaking with an equal volume of chloroform/3-methylbutan-l-ol (24: 1, v/v) at room temperature for 30 min. Following centrifugation at 3000 g for 15 min the upper (aqueous) phase, containing DNA, was retained. DNA-grade hydroxyapatite (HAP; 2 g; Bio-Rad) was suspended in 20 ml 0.24 M-phosphate buffer (pH 7.0), boiled for 30 min, centrifuged at low speed, and washed twice with 15 ml MUP (Britten et al., 1968) containing 8 M-urea in 0.24 M-phosphate buffer (pH 7.0), being sedimented after each wash by brief low-speed centrifugation. The deproteinized nucleic acid solution was mixed with the washed HAP and stirred (5 min) to permit binding of DNA. The bound DNA was purified by washing the mixture (by centrifugation) seven times with 15 ml MUP, to remove RNA and residual protein, and then four times with 15 ml 0.014 M-phosphate buffer (pH 7.0), to remove urea. The purified DNA was eluted
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