Oliva R, Mezquita J, Mezquita C, Dixon G H
Department of Physiology and Biochemistry, Faculty of Medicine, University of Barcelona, Spain.
Dev Biol. 1988 Feb;125(2):332-40. doi: 10.1016/0012-1606(88)90216-3.
cDNA clones were prepared from poly(A)+ mRNA isolated from a population enriched in postmeiotic rooster testes spermatogenic cells. A series of clones was sequenced at random and two partial sequences corresponding to the C-terminal coding and 3' untranslated region of the chicken protamine mRNA were obtained. The deduced amino acid sequence of this C-terminal coding region corresponds to the sequence previously described at the protein level for the chicken protamine, galline [Nakano, M., Tobita, T., and Ando, T. (1976), Int. J. Peptide Prot. Res. 8, 565-578]. To study the expression of this protamine gene, RNA was prepared from chicken testes at different stages of development, electrophoresed in formaldehyde-agarose gels, transferred to a nylon membrane, and hybridized with a rooster protamine cDNA probe. Two populations of mRNA of sizes ranging between 420 and 465 bases are expressed in postmeiotic rooster testis cells. To determine if there was a differential expression of the two populations of mRNA in the final postmeiotic haploid stages of spermatogenesis, RNA was purified from adult rooster cells separated at unit gravity according to their differences in size by the Staput technique. The RNA was similarly analyzed by Northern blots. The results indicate that round spermatids are enriched in the 465-nucleotide mRNA species, whereas in the final stage of elongated spermatids the 420-nucleotide species is the only one present, suggesting either post-transcriptional processing, the presence of two different sets of genes that are differentially expressed, or a single set of genes with differential promoter usage.
从富含减数分裂后公鸡睾丸生精细胞的群体中分离出的聚腺苷酸加尾mRNA制备了cDNA克隆。随机对一系列克隆进行测序，获得了与鸡鱼精蛋白mRNA的C末端编码区和3'非翻译区相对应的两个部分序列。该C末端编码区推导的氨基酸序列与先前在蛋白质水平上描述的鸡鱼精蛋白加林的序列相对应[中野，M.，户田，T.，安藤，T.（1976年），《国际肽与蛋白质研究杂志》8，565 - 578]。为了研究该鱼精蛋白基因的表达，从不同发育阶段的鸡睾丸中制备RNA，在甲醛 - 琼脂糖凝胶中进行电泳，转移到尼龙膜上，并用公鸡鱼精蛋白cDNA探针进行杂交。在减数分裂后公鸡睾丸细胞中表达了两个大小在420至465个碱基之间的mRNA群体。为了确定在精子发生的最终减数分裂后单倍体阶段这两个mRNA群体是否存在差异表达，根据细胞大小差异通过斯塔普特技术从成年公鸡细胞中以单位重力分离出RNA并进行纯化。同样通过Northern印迹法对RNA进行分析。结果表明，圆形精子细胞中富含465个核苷酸的mRNA种类，而在延长精子细胞的最终阶段，420个核苷酸的种类是唯一存在的，这表明可能存在转录后加工、存在两组差异表达的不同基因，或者是一组具有不同启动子使用方式的基因。
