Welsh R S, Vyska K
Institut für Medizin Kernforschungsanlage Jülich GmbH, Federal Republic of Germany.
Basic Appl Histochem. 1987;31(3):281-98.
Highly purified DNA obtained from calf thymus nuclei was found to cleave after reaction with a chelating agent and subsequent dialysis against 0.01 M phosphate. During the cleavage release of proteineous material into the dialysate was observed. By means of anion exchange resin column chromatography, this material was separated into 9 main fractions. Two of these fractions P1 and P5) were found to contain the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by Chelex 100 treatment. The proportion of phosphoserine was much greater in P5 than in P1. P1 and P5 contained essentially no nucleotide material. All other fractions (P2, P3, P3a, P4, P5a, P6, P7, P8, P6a, P9) were found to contain ribonucleotides and deoxynucleotides. The deoxynucleotide content was about 10% of total nucleotide content. After a deionizing treatment with Chelex, the amounts of nucleotides were extensively reduced to a level corresponding to about 1 nucleotide of 10 amino acids. In separate experiments, commercial DNA (S-DNA) was ultrasonicated, and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. From DEAE Sephacel chromatography of this material the fraction obtained having the highest proportion of protein aceous material was hydrolyzed with Pronase and again chromatographed on DEAE Sephacel. From this fractionation a single fraction containing deoxynucleotides and amino acids was found. The mixture obtained by hydrolysis of this fraction with snake venom diesterase and was again rechromatographed, which revealed two peaks, one corresponding to deoxynucleotide material and a second one to a mixture of 4 amino acids, phosphoserine, asp, glu, and gly. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between their deoxynucleotide and amino acid portions. The results indicate that in purified S-DNA phosphopeptides are linked through covalent bonds to the terminal deoxynucleotide residues.
从小牛胸腺细胞核中获得的高度纯化的DNA，在与螯合剂反应并随后用0.01M磷酸盐透析后被发现会发生裂解。在裂解过程中，观察到有蛋白质物质释放到透析液中。通过阴离子交换树脂柱色谱法，该物质被分离成9个主要组分。发现其中两个组分（P1和P5）含有磷酸丝氨酸、天冬氨酸、苏氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸、缬氨酸、异亮氨酸、亮氨酸和精氨酸等氨基酸，以及磷酸丝氨酸的金属离子络合物。这些络合物通过Chelex 100处理而解离。P5中磷酸丝氨酸的比例比P1中高得多。P1和P5基本上不含核苷酸物质。发现所有其他组分（P2、P3、P3a、P4、P5a、P6、P7、P8、P6a、P9）都含有核糖核苷酸和脱氧核苷酸。脱氧核苷酸含量约占总核苷酸含量的10%。在用Chelex进行去离子处理后，核苷酸的量大幅减少到相当于每10个氨基酸约1个核苷酸的水平。在单独的实验中，将商业DNA（S-DNA）超声处理，并用胰DNA酶、外切核酸酶III和S1核酸酶消化。对该物质进行DEAE Sephacel色谱分析，将获得的蛋白质物质比例最高的组分用链霉蛋白酶水解，然后再次在DEAE Sephacel上进行色谱分析。从这次分级分离中发现了一个含有脱氧核苷酸和氨基酸的单一组分。用蛇毒二酯酶水解该组分得到的混合物再次进行色谱分析，显示出两个峰，一个对应脱氧核苷酸物质，另一个对应4种氨基酸（磷酸丝氨酸、天冬氨酸、谷氨酸和甘氨酸）的混合物。由此得出结论，用于二酯酶消化的组分由脱氧核苷酸-氨基酸组成，其脱氧核苷酸和氨基酸部分之间存在共价二酯键。结果表明，在纯化的S-DNA中，磷酸肽通过共价键与末端脱氧核苷酸残基相连。
