Whitehead J L, Wang S Y, Bost-Usinger L, Hoang E, Frazer K A, Burnside B
Department of Molecular and Cell Biology, University of California, 335 Life Sciences Addition, Berkeley, CA, 94720-3200, USA.
Exp Eye Res. 1999 Nov;69(5):491-503. doi: 10.1006/exer.1999.0724.
The heterotrimeric microtubule motor kinesin II has been shown to be required for morphogenesis and maintenance of both motile flagella and immotile sensory cilia. Recently, we showed that the KIF3A subunit of kinesin II is concentrated in the inner segment and connecting cilium of fish photoreceptors. Here we report the gene structure of human KIF3A (HsKIF3A) and describe its localization in human and monkey retina. We also describe the localization of both KIF3A and KIF3B kinesin II subunits in Xenopus retina. Using a portion of HsKIF3A we had amplified from adult human retinal cDNA, we found by a GenBank database search that an identical sequence had already been obtained by the Human Genome Center at Lawrence Berkeley National Laboratories in a direct sequencing analysis of 680 kb of human chromosome 5q31. By comparing the genomic sequence of HsKIF3A to the open reading frame (ORF) of the highly homologous mouse Kif3A, we determined that the HsKIF3A gene has 17 exons and an ORF of approximately 2.1 kb, predicting a protein of 80.3 kDa. Antibodies against sea urchin KRP85, a KIF3A homologue, bound to a single band of approximately 85 kDa in immunoblots of total retina protein from human, monkey and Xenopus. In these same samples, a single band of approximately 95 kDa is recognized by antibodies against Xklp3, a Xenopus KIF3B homologue. In sections of Xenopus retina, both antibodies strongly labelled photoreceptor inner segments and the outer limiting membrane. Both antibodies also labelled photoreceptor axonemes. The axonemal localization of kinesin II subunits suggests that kinesin II may play a role in transport of materials from the photoreceptor cell body to the outer segment.
异源三聚体微管马达驱动蛋白II已被证明是运动鞭毛和非运动感觉纤毛形态发生及维持所必需的。最近，我们发现驱动蛋白II的KIF3A亚基集中在鱼类光感受器的内段和连接纤毛中。在此，我们报告人类KIF3A（HsKIF3A）的基因结构，并描述其在人类和猴视网膜中的定位。我们还描述了非洲爪蟾视网膜中KIF3A和KIF3B驱动蛋白II亚基的定位。利用我们从成人视网膜cDNA中扩增得到的HsKIF3A片段，通过GenBank数据库搜索发现，劳伦斯伯克利国家实验室的人类基因组中心在对人类5号染色体5q31区域680 kb的直接测序分析中已经获得了相同序列。通过将HsKIF3A的基因组序列与高度同源的小鼠Kif3A的开放阅读框（ORF）进行比较，我们确定HsKIF3A基因有17个外显子，ORF约为2.1 kb，预测编码一个80.3 kDa的蛋白质。针对海胆KRP85（一种KIF3A同源物）的抗体，在人、猴和非洲爪蟾全视网膜蛋白免疫印迹中与一条约85 kDa的单带结合。在这些相同样本中，针对非洲爪蟾KIF3B同源物Xklp3的抗体识别出一条约95 kDa的单带。在非洲爪蟾视网膜切片中，两种抗体都强烈标记光感受器内段和外限制膜。两种抗体也标记光感受器轴丝。驱动蛋白II亚基在轴丝上的定位表明，驱动蛋白II可能在从光感受器细胞体向外段运输物质中发挥作用。
