Eppard M, Rhiel E
AG Geomikrobiologie, ICBM, Carl-von-Ossietzky-Universität Oldenburg, Germany.
Mol Gen Genet. 1998 Nov;260(4):335-45. doi: 10.1007/s004380050902.
Total RNA was isolated from the diatom Cyclotella cryptica and separated into poly(A)+ and poly(A)- fractions. These fractions were subjected to in vitro translation/immunoprecipitation experiments using an antiserum directed against the predominant light-harvesting complex of Cy. cryptica (ccry antiserum) and a heterologous antiserum raised against the light-harvesting complex of the cryptophyte Cryptomonas maculata (cmac antiserum). From translation reactions programmed with poly(A)+ RNA the ccry-antiserum immunoprecipitated polypeptides with relative molecular weights (Mr) of 27000, 25000, 23000 and 21000, while the cmac-antiserum precipitated proteins with Mrs of 32500 and 27000, respectively. Subsequent cDNA synthesis and immunological screening of the cDNA library with both antisera resulted in the isolation of six cDNA clones encoding light-harvesting subunits. Full-length precursors were 199-210 amino acids in length and had Mrs of 20000-23000. The lengths of the putative signal peptides were 29 or 30 amino acids. Pairwise comparison revealed that the similarity between the clones ranged from 54-99% on the nucleotide level and from 36-99% at the amino acid level. In agreement with the data from the screens with the two antisera, the genes clustered into two groups. The data provide evidence that the genes constitute a heterogeneous multigene family and that the light-harvesting system of Cy. cryptica might be as complex as that of higher plants and green algae.
从硅藻小环藻(Cyclotella cryptica)中分离出总RNA,并将其分为聚腺苷酸加(poly(A)+)和聚腺苷酸减(poly(A)-)组分。使用针对小环藻主要捕光复合体的抗血清(ccry抗血清)和针对隐藻黄斑隐球藻(Cryptomonas maculata)捕光复合体产生的异源抗血清(cmac抗血清),对这些组分进行体外翻译/免疫沉淀实验。在用聚腺苷酸加RNA编程的翻译反应中,ccry抗血清免疫沉淀出相对分子质量(Mr)为27000、25000、23000和21000的多肽,而cmac抗血清分别沉淀出相对分子质量为32500和27000的蛋白质。随后的cDNA合成以及用两种抗血清对cDNA文库进行免疫筛选,导致分离出六个编码捕光亚基的cDNA克隆。全长前体长度为199 - 210个氨基酸,相对分子质量为20000 - 23000。推定信号肽的长度为29或30个氨基酸。成对比较显示,克隆之间在核苷酸水平上的相似性范围为54 - 99%,在氨基酸水平上为36 - 99%。与用两种抗血清筛选的数据一致,这些基因聚为两组。这些数据提供了证据,表明这些基因构成一个异质多基因家族,并且小环藻的捕光系统可能与高等植物和绿藻的捕光系统一样复杂。