Isolation of poly(adenylic acid)-rich ribonucleic acid from mouse myeloma and synthesis of complementary deoxyribonucleic acid.
Isolation of poly(adenylic acid)-rich ribonucleic acid from mouse myeloma and synthesis of complementary deoxyribonucleic acid.
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从小鼠骨髓瘤中分离富含聚（腺苷酸）的核糖核酸并合成互补的脱氧核糖核酸。
DOI：
10.1021/bi00729a021
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发表时间：
1973
期刊：
影响因子：
2.9
通讯作者：
B. Mach
中科院分区：
文献类型：
--
作者：
C. Faust;H. Diggelmann;B. Mach
从小鼠骨髓瘤的膜结合多聚体中分离出一种含有多聚腺苷酸序列的RNA组分。退火到聚（胸苷酸）-纤维素柱，显著的RNA酶抗性（20%），以及RNA酶抗性片段（7S）的大小都表明该RNA部分含有大量富含聚（腺苷酸）的序列。在RNA酶抗性组分中发现80%的AMP证实了这一点。本文建立了聚胸苷酸-纤维素层析的温度和离子强度条件，使含聚腺苷酸的RNA得到最佳的回收，而核糖体RNA的污染最小。分离出的富含多聚腺苷酸的部分作为有效的RNA发挥作用。关于免疫应答的一个有趣的问题是抗体多样性的产生。到目前为止，还没有实验数据来决定提出的两个主要理论：生殖细胞理论和涉及体细胞突变和重组的理论（参见Smith et al.，1971年）。测量不同组织细胞中免疫球蛋白链的基因数量，包括可变部分的基因，可以回答这个问题。这样的测量可以通过DNA-DNA杂交来实现。来自瑞士日内瓦大学病理学系和瑞士洛桑实验癌症研究所。1972年9月/日收到。由瑞士国家科学基金会的赠款3，579，71和3，412，70资助。C. H. F.由美国癌症协会资助号PF-667 F。t日内瓦大学。瑞士实验癌症研究所（Swiss Institute of Experimental Cancer Research）用禽成髓细胞瘤病毒的RNA依赖性DNA聚合酶合成互补DNA的模板。该反应完全依赖于加入的RNA作为模板，寡胸苷酸作为引物。引发活性与寡胸苷酸的大小呈负相关。天然产物的密度平衡离心表明，一些放射性与RNA有关。然而，变性后，所有放射性条带均为DNA。用碱性蔗糖沉淀法测定的合成产物的平均大小取决于反应中所用脱氧核苷三磷酸的浓度。通过杂交证明了合成产物的特异性。在大量DNA过量的条件下，使用“Cot”分析（Britten和Kohne，1968; Gelb等，1971年）。这种方法的一个优点是，体外合成的DNA可以获得足够高的比活性，以提供有意义的数据。此外，与RNA杂交相比，DNA-DNA杂交在高温下不易降解。在肿瘤病毒中发现RNA依赖的DNA聚合酶后，（巴尔的摩，1970; Temin和Mizutani，1970），以及随后证明它可以使用9 S血红蛋白（Hb）1 mRNA作为补体1的模板。培养基A-100，50 mM Tris-HCl（pH 7.6）-100 mM KCl-5 mM MgCL; AMV，禽成髓细胞瘤病毒; SSC，标准柠檬酸盐（0.15 M NaCl-0.015 M柠檬酸钠）; DEP，焦碳酸二乙酯;聚（A），聚腺苷酸）。
An RNA fraction containing poly (adenylic acid) sequences has been isolated from membrane-bound poly-somes of mouse myeloma tumor. Annealing to a poly (thymidylic acid)-cellulose column, significant RNase-resistance (20%), and the size of the RNase-resistant fragment (7S) all suggest that this RNA fraction contains largetracts of poly-(adenylic acid)-rich sequences. This is confirmed by the find-ing of 80% AMP in the RNase-resistant fraction. Conditions of temperature and ionic strength for chromatography on poly (thymidylic acid)-cellulose are established for optimal recovery of RNA containing poly (adenylic acid) withmini-mal contamination by ribosomal RNA. The isolated poly-(adenylic acid)-rich fraction functions as an efficient RNA^^) ne of the intriguing questions concerning the immune response is the generation of antibody diversity. As yet, there are no experimental data to decide between the two main theories proposed: the germ line theory and the theories involving somatic mutation and recombination (for review see Smith et al., 1971). A measure of the number of genes for immunoglobulin chains in cells of different tissues, including the genes for the variable part, could answer that question. Such a measure could be achieved by DNA-DNA hybridizat From the Department of Pathology, University of Geneva, Geneva, Switzerland, and theSwiss Institute of Experimental CancerResearch, Lausanne, Switzerland. Received September/, 1972. Supported by Grants 3,579, 71 and 3,412, 70 from the Swiss National Science Foundation. C. H. F. was supported by American Cancer Society Grant No. PF-667F. t University of Geneva. § Swiss Institute of Experimental CancerResearch. template for the synthesis of complementary DNA with the RNA-dependent DNA polymerase of avian myeloblastosis virus. This reaction is completely dependent upon added RNA as template, and oligothymidylic acid as primer. Priming activity is inversely related to the size of the oligothymidylic acid. Densityequilibrium centrifugation of the native product shows that some of the radioactivity is associated with RNA. However, after denaturation, all the radioactivity bands as DNA. The averagesize of the product synthesized, as determined by alkaline sucrose sedimentation, depends on the con-centration of the deoxyribonucleoside triphosphates used in the reaction. The specificity of the product synthesized is demonstrated by hybridization. tion under conditions of large DNA excess, using a “Cot” analysis (Britten and Kohne, 1968; Gelb et al., 1971). One advantage of such an approach is that in vitro synthesized DNA could be obtained at high enough specific activities to give meaningful data. In addition, DNA-DNA hybridizations are less susceptible to degradation at high temperature when compared to those performedwith RNA. After the discovery of the RNA-dependentDNA polym-erase in tumor viruses (Baltimore, 1970; Temin and Mizutani, 1970), and the subsequent demonstration that it could use 9S hemoglobin (Hb) 1 mRNA as template for complemen-1 Abbreviations used are: Hb, hemoglobin; cDNA, complementary deoxyribonucleic acid; medium A-100, 50 mM Tris-HCl (pH 7.6)—100 mM KC1-5 mM MgCL; AMV, avian myeloblastosis virus; SSC, standard saline citrate (0.15 M NaCl-0.015 M sodium citrate); DEP, diethylpyrocarbonate; poly (A), polyfadenylic acid).