[PMC free article] [PubMed] [Google Scholar] 46

[PMC free article] [PubMed] [Google Scholar] 46. to be KIF5C, we generated mice lacking the (Patel et al., 1993), squid (Kosik et al., 1990), ADU-S100 (MIW815) sea urchin (Wright et al., 1993), and fruit travel (Yang et al., 1988) have single standard KHC genes, KHC has diverged into a neuron-specific form and a ubiquitous form in vertebrates (Kato, 1991; Navone et al., 1992; Gudkov et al., 1994;Niclas et al., 1994; Vignali et al., 1996; Bost-Usinger et al., 1997;Meng et al., 1997). In mice, we have identified a distinct murine brain-specific KHC, KIF5A (5A) (Aizawa et al., 1992), in addition to the ubiquitous KHC (uKHC) or KIF5B (5B) (Gudkov et al., 1994;Meng et al., 1997). Here, we statement the cloning of KIF5C (5C), the third member of KHC in mouse brain. Specific antibodies revealed their specialized localization in the nervous system. Ubiquitous KIF5B showed a glial cell distribution pattern in general; however, interestingly, its expression was strongly upregulated in axon-elongating neurons, such as olfactory main neurons and mossy fibers. Although both KIF5A and KIF5C were expressed in neurons, they showed different patterns of distribution. Expression of KIF5A was observed in various kinds of neurons at comparable levels, whereas KIF5C was enriched in motor neurons. Upregulation of KIF5C in motor neurons was observed in the 2-week-old or older mice, which was developmentally increased, indicating the important functions of KIF5C in maintenance of motor neurons rather than in axonal formation. Because most of the KIF5 in the motor neurons of adult mice was suggested to be KIF5C, we disrupted the gene in mice to investigate the functions of KIF5C and the possible redundancy among the three KIF5s in neurons in living animals. Surprisingly, mutant mice were viable and did not show gross changes in the nervous system. The mutant mice LRRC15 antibody only showed smaller brain size and relative loss of motor neurons to sensory neurons. Three KIF5s showed high similarity, could rescue KIF5B mutant cells, and could form heterodimers. These data strongly show that there is functional redundancy among the three KIF5s. MATERIALS AND METHODS kif5C We obtained a cDNA clone of KIF5C from your mouse brain library in accordance with a standard cloning method (Kanai et al., 1989; Sambrook et al., 1989) using the motor domain sequence as the probe (Nakagawa et al., 1997). A full-length and several partial-length clones were isolated, transferred to pBluescript SK(+), and carefully sequenced. We constructed deleted fragments of the clones by the conventional 3 deletion method using Exonuclease III/mung bean nuclease (TaKaRa, Tokyo, Japan) to read the entire sequence (Kanai et al., 1992;Kanai and Hirokawa, 1995). All sequences were ADU-S100 (MIW815) go through bidirectionally at least four occasions with the ABI autosequencer 377 (Perkin-Elmer, Emeryville, CA). In expectation of the efficient expression of the three KIF5s in the ADU-S100 (MIW815) transfection study, we changed their sequences upstream of the ATG to the Kozak sequence (Kozak, 1987) using PCR as explained previously (Tanaka et al., 1998). The fragments were inserted into the cytomegalovirus (CMV) promoter-driven expression vector [pcDNA3.1/Zeo(?); Invitrogen, San Diego, CA], for the rescue study using cells. The full-length cDNA of each KIF5 was subcloned into the pET32a expression vector (Novagen, Madison, WI) to be expressed as thioredoxin/His-tag fusion proteins. Bacterial strain BL21(DE3) was transformed with these plasmids, and fusion protein expression was induced with 0.1 mmisopropyl–d-thiogalactopyranoside at 18C for 12 hr. The bacteria were disrupted with a French Press (Otake Corp.), and the fusion proteins were recovered on Ni-Chelating-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). The proteins were ADU-S100 (MIW815) conjugated to a cyanogen bromide (CNBr) Sepharose (Amersham Pharmacia Biotech) column to purify the antibodies or were used as requirements in the immunoblot analysis..

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