Montana G, Romancino D P, di Carlo M D
Istituto di Biologia dello Sviluppo C.N.R., Palermo, Italy.
Mol Reprod Dev. 1996 May;44(1):36-43. doi: 10.1002/(SICI)1098-2795(199605)44:1<36::AID-MRD4>3.0.CO;2-U.
We have isolated and characterized a cDNA clone corresponding to a new member of bep (butanol, extracted, proteins) Paracentrotus lividus multigene family coding for cell surface proteins. The cDNA, called bep3, encodes a 370 amino acid protein and shares the same structural organization in the coding region with other members of the same gene family already characterized. Expression of this clone studied by Northern blot and by whole mount hybridization shows that the bep3 messenger is transcribed during oogenesis and utilized till the gastrula stage, whereas at the prism stage, unlike other members of the same gene family, new synthesis of messenger occurs. By whole mount hybridization spatial distribution of bep3 messenger in egg and embryos is established. This messenger appears located in the animal half of the unfertilized egg and moves to the cortical zone after fertilization; it is not present in the structures derived by the vegetal part of the embryo, such as the micromeres of the 16-cell stage, the primary mesenchyme cells of the blastula, and the primary intestine of the gastrula. At the prism stage instead, hybridization of bep3 messenger is restricted to the part of the embryo that will give origin to the oral region as successively confirmed by hybridization at the pluteus stage. The result of whole mount hybridization was confirmed by Northern blot hybridization of separated meso-macromere and micromere RNAs. A Southern blot experiment demonstrates that bep3 is codified by a single copy gene. Conservation of the bep multigene family in several Mediterranean and Japanese sea urchin species has also been analyzed.
我们已经分离并鉴定了一个与紫球海胆bep（丁醇提取蛋白）多基因家族的一个新成员相对应的cDNA克隆，该家族编码细胞表面蛋白。这个名为bep3的cDNA编码一个由370个氨基酸组成的蛋白质，并且在编码区域与已鉴定的同一基因家族的其他成员具有相同的结构组织。通过Northern印迹和整体杂交对该克隆的表达进行研究，结果表明bep3信使核糖核酸在卵子发生过程中被转录，并一直被利用到原肠胚阶段，而在棱柱幼虫阶段，与同一基因家族的其他成员不同，信使核糖核酸开始重新合成。通过整体杂交确定了bep3信使核糖核酸在卵子和胚胎中的空间分布。这种信使核糖核酸似乎位于未受精卵的动物极，受精后移至皮质区；它不存在于由胚胎植物部分衍生的结构中，如16细胞期的小分裂球、囊胚的初级间充质细胞和原肠胚的原肠。相反，在棱柱幼虫阶段，bep3信使核糖核酸的杂交仅限于胚胎中将来会发育成口区的部分，这一点在长腕幼虫阶段的杂交中得到了进一步证实。分离的中-大分裂球和小分裂球RNA的Northern印迹杂交证实了整体杂交的结果。Southern印迹实验表明bep3由单拷贝基因编码。我们还分析了bep多基因家族在几种地中海和日本海胆物种中的保守情况。
