Mencarelli C, Lupetti P, Rosetto M, Mercati D, Heuser J E, Dallai R
Dipartimento di Biologia Evolutiva, Università di Siena, Siena, Italy.
Cell Motil Cytoskeleton. 2001 Nov;50(3):129-46. doi: 10.1002/cm.10004.
The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species, Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but they were unable to undergo processive movement in vitro. They propagated a three-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and molecular data are also discussed.
双翅目昆虫鲁氏茎蜂蝇（Asphondylia ruebsaameni）独特的精子轴丝的特征是微管双联体数量异常多（多达2500个），呈双平行螺旋排列。每个螺旋内排的双联体是倾斜的，因此它们的外臂指向外排中下一个双联体的B微管。双联体仅具有外臂，不存在与中央微管对/辐条复合体相关的结构。通过快速冷冻、深度蚀刻电子显微镜分析表明，动力蛋白臂的结构与其他生物体中描述的结构相同；然而，它似乎比之前在相关双翅目物种黄足单节蜂（Monarthropalpus flavus）中发现的结构更为复杂，因为臂的足部区域显示出一个球状额外结构域，该结构域插在相邻的臂之间。用ATP和钒酸盐处理去膜精子会诱导动力蛋白臂发生构象变化。SDS-聚丙烯酰胺凝胶电泳表明存在一条单一的高分子量动力蛋白条带，或者在电泳分辨率最佳的凝胶中存在两条间隔非常近的条带。该多肽与针对位于动力蛋白催化位点磷酸结合环中的特定氨基酸序列产生的多克隆抗体发生阳性反应。通过逆转录-聚合酶链反应（RT-PCR）扩增出了与催化磷酸结合区域相对应的动力蛋白重链相关DNA序列。鉴定出了两个编码轴丝动力蛋白序列的不同片段（Asph-ax1和Asph-ax2）。使用这些序列作为探针在基因组DNA上进行的Southern印迹分析表明，它们是不同基因的一部分。在Asph-ax1片段中对应于黄足单节蜂假定假基因中核苷酸缺失位点的位置鉴定出了一个内含子。鲁氏茎蜂蝇精子在体内输精管和受精囊中都表现出旋转运动，但在体外无法进行前进运动。只有当通过某种机械手段将其限制在弯曲构型时，它们才会传播三维波。基于这些生化和分子数据，还讨论了黄足单节蜂和鲁氏茎蜂蝇这两种双翅目物种之间的系统发育关系。
