Kravariti L, Thomas J, Sourmeli S, Rodakis G C, Mauchamp B, Chavancy G, Lecanidou R
Department of Biochemistry and Molecular Biology, University of Athens, Panepistimioupolis, 15701, Athens, Greece.
Insect Biochem Mol Biol. 2001 Mar 15;31(4-5):473-9. doi: 10.1016/s0965-1748(00)00152-1.
Bombyx mori unpaired early chorion gene copies 6F6.1,.2 and.3 are exceptions to the typical organization and distribution pattern of known early ErA/ErB, middle A/B and late HcA/HcB divergently transcribed gene pairs. Contrary to such pairs, the boundaries of the 6F6 regulatory sequences are not easily defined; moreover, they share common sequence elements with the regulatory sequences of middle and late genes. In order to perform a functional study of the tissue and temporal specificity of the 6F6 putative promoter region, we decided to apply biolistics. In the present work, use of a region from the 6F6.2 5' untranslated sequence, spanning nucleotides -138 to the cap site, gave an expected expression pattern of a lacZ reporter gene. Temporal specificity was further verified by control experiments using the cloned intergenic sequence of the late gene pair HcA/B.12, which resulted in lacZ expression in late choriogenic follicles. At present, despite the recent successful germinal transgenesis of Bombyx mori, the biolistic transient expression system seems to be the most rapid technique to pursue the functional study of the promoter region of early chorion genes, including the three unconventional early 6F6 genes.
家蚕未配对的早期绒毛膜基因拷贝6F6.1、.2和.3是已知的早期ErA/ErB、中期A/B和晚期HcA/HcB反向转录基因对典型组织和分布模式的例外情况。与这些基因对不同，6F6调控序列的边界不易界定；此外，它们与中期和晚期基因的调控序列共享共同的序列元件。为了对6F6假定启动子区域的组织和时间特异性进行功能研究，我们决定应用生物弹道学方法。在本研究中，使用6F6.2 5'非翻译序列中从核苷酸-138到帽位点的区域，得到了lacZ报告基因的预期表达模式。通过使用晚期基因对HcA/B.12的克隆基因间序列进行对照实验进一步验证了时间特异性，该实验导致在晚期成绒毛卵泡中表达lacZ。目前，尽管最近家蚕成功实现了生殖系转基因，但生物弹道学瞬时表达系统似乎是进行早期绒毛膜基因启动子区域功能研究（包括三个非常规早期6F6基因）的最快速技术。
