4214 Corrections Proc. Natl. Acad. Sci. USA 96 (1999)

Cell Biology. In the article “Identification and classification of Medical Sciences. In the article “CM101-mediated recovery of 16 new kinesin superfamily (KIF) in mouse genome” walking ability in adult mice paralyzed by spinal cord injury” by Terunaga Nakagawa, Yosuke Tanaka, Eiji Matsuoka, Sa- by Artur W. Wamil, Barbara D. Wamil, and Carl G. Heller- toru Kondo, Yasushi Okada, Yasuko Noda, Yoshimitsu Kanai, qvist, which appeared in number 22, October 27, 1998, of Proc. and Nobutaka Hirokawa, which appeared in number 18, Natl. Acad. Sci. USA (95, 13188–13193), the authors request September 2, 1997 of Proc. Natl. Acad. Sci. USA (94, 9654– that the following two corrections be noted. First, the following 9659), the authors request that the following correction be sentence on page 13188, “CM101 is an antipathoangiogenic noted. In Fig. 5, due to a technical error in the genetic mapping polysaccaride (20) derived from group B streptococcus (GBS) program, the cytogenetic map of the mouse kif5B was (21) that inhibits angiogenesis and subsequent infiltration of misidentified. The corrected Fig. 5 and legend are reproduced inflammatory cells and thereby formation of granulation below. tissue, which produces scarring (M. Neeman, R. Abramowitch, B.D.W., and C.G.H., unpublished data),” should read “CM101 is an antipathoangiogenic polysaccharide (20) derived from group B streptococcus (GBS) that inhibits angiogenesis (21) and scarring (B.D.W. and C.G.H., unpublished data). Gran- ulation tissue, which produces scarring, contains new capillar- ies and inflammatory cells [Rubin, E. & Farber, J. L., eds. (1994) Pathology (Lippincott, Philadelphia), 2nd Ed., p. 82].” Second, the reference to Dr. Michal Neeman on page 13192, “M. Neeman, personal communication,” should be deleted.

FIG. 5. Cytogenetic mapping was performed for some of the known KIFs. (A and B). Examples of the fluorescence in situ hybrid- ization (FISH) mapping (probed for kif5B). (A) The FISH signals on the (arrows). (B) The same mitotic figure stained with 4Ј,6-diamidino-2-phenylindole (DAPI) to identify chromosome 18. (C) The rest of the results are summarized. Downloaded by guest on September 27, 2021 Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9654–9659, September 1997 Cell Biology

Identification and classification of 16 new kinesin superfamily (KIF) proteins in mouse genome

TERUNAGA NAKAGAWA,YOSUKE TANAKA,EIJI MATSUOKA,SATORU KONDO,YASUSHI OKADA,YASUKO NODA, YOSHIMITSU KANAI, AND NOBUTAKA HIROKAWA*

Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan

Communicated by Setsuro Ebashi, National Institute for Physiological Sciences, Okazaki, Japan, June 30, 1997 (received for review March 25, 1997)

ABSTRACT KIF (kinesin superfamily) proteins are mi- kinesin superfamily remains elusive, but it appears that dif- crotubule-dependent molecular motors that play important ferent members are assigned specific cellular functions. In the roles in intracellular transport and cell division. The extent to mouse there are neuron-specific members such as KIF5A (12), which KIFs are involved in various transporting phenomena, nKHC (17), KIF1A (18), and KIFC2 (13) that are apparently as well as their regulation mechanism, are unknown. The not involved in cell division. [Note that Aizawa et al. (12) identification of 16 new KIFs in this report doubles the originally described KIF5. However, further careful studies existing number of KIFs known in the mouse. Conserved revealed that KIF5 consists of three members that are referred nucleotide sequences in the motor domain were amplified by to as KIF5A, -5B, and -5C in this paper. Henceforth, the name PCR using cDNAs of mouse nervous tissue, kidney, and small KIF5A is used for the sequence referred to as KIF5 in Aizawa intestine as templates. The new KIFs were studied with respect et al.] to their expression patterns in different tissues, chromosomal For neurons, our data suggest that certain classes of mem- location, and molecular evolution. Our results suggest that (i) branous organelles are transported separately down the axon there is no apparent tendency among related subclasses of by different motors. For example, KIF1A is associated with KIFs of cosegregation in chromosomal mapping, and (ii) organelles that contain synaptotagmin, synaptophysin, and according to their tissue distribution patterns, KIFs can be Rab3A, but not SV2, syntaxin 1A, or SNAP-25. Motors such divided into two classes–i.e., ubiquitous and specific tissue- as conventional kinesin or KIF3 were not associated with dominant. Further characterization of KIFs may elucidate KIF1A-containing organelle (18). This evidence suggests that unknown fundamental phenomena underlying intracellular different members of the KIF carry different cargos, even transport. Finally, we propose a straightforward nomencla- though the possibility of functional redundancy still remains. ture system for the members of the mouse kinesin superfamily. In neurons, at least, motor molecules other than KIF1A may exist that transport vesicles containing SV2, syntaxin 1A, and The dynamic aspect of intracellular organelle transport and SNAP-25. Thus with our present knowledge of KIF we may be cell division involve proper functioning of microtubule- unable to obtain the overall view of the complex intracellular dependent molecular motors such as kinesin and dynein transport system. These and other considerations strongly superfamily proteins. Since the biochemical identification (1) motivated us to search for the additional members of the KIF. and cDNA cloning (2) of kinesin heavy chain, cDNAs of Initially, based on PCR approach, our group has identified kinesin superfamily were genetically identified from seven KIFs—i.e., KIF1A (18), KIF1B (19), KIF2 (20), KIF3A various species (3–6). The deduced amino acid sequences of (21), KIF3B (22), KIF4 (23), and KIF5A (ref. 12; see notes in these members revealed the presence of a motor domain of the previous paragraph) in mouse brain. Three more KIFs high homology, and a more divergent nonmotor domain. The were identified by another PCR strategy (13). In the present latter comprises a large portion of the whole molecule, with study, the search for additional KIFs was intensified further by amino acid sequences that are characteristic for each members. an improved PCR strategy. We identified 16 new members of These findings support the observation by quick-freeze, deep- KIF, virtually doubling the number of known KIFs in the etch electron microscopy techniques that in nerve axons, many mouse. In this report the partial amino acid sequences of these different types of crossbridge structures are observed between new KIFs are presented with the classification based on cellular organelles and microtubules (7, 8). Some of these molecular evolutionary analysis and tissue distribution. We crossbridges were later identified to be kinesin (9). These and also present the chromosomal mapping for some of the known other findings suggest that the machinery for intracellular KIFs. Finally, we propose the use of a simplified nomenclature transport is highly sophisticated, and that a large family of as for the mouse kinesin superfamily. genes encoding the kinesin superfamily is to be expected. Molecular biological approaches were instrumental in iden- tifying members of the kinesin superfamily. Several amino acid MATERIALS AND METHODS motifs in the motor domain, such as IFAYGQT, DLAGSE, Isolation of mRNA. Mice were killed to obtain the following and HIPYR, are highly conserved among species. Fragments material: 0-day-old whole brain, 4-week-old hippocampus, flanked by these sequences can therefore be amplified by PCR 4-week-old olfactory bulb, 4-week-old kidney, and 4-week-old using degenerate oligonucleotide primers. This strategy iden- intestine. Each tissue was dissected from separate mice to tified many kinesin superfamily members in various organisms, such as Drosophila (10, 11), mouse (12, 13), Xenopus (14), fish Data deposition: The sequences reported in this paper have been (15), yeast (3), and rat (16). The reason for the diversity of the deposited in the GenBank database [accession nos. AB001456 (KIF1C), AB001433 (KIF3C), AB001434 (KIF6), AB001435 (KIF7), The publication costs of this article were defrayed in part by page charge AB001436 (KIF8), AB001437 (KIF9), AB001426 (KIF10), AB001427 (KIF11), AB001428 (KIF12), AB001429 (KIF13A), AB001430 payment. This article must therefore be hereby marked ‘‘advertisement’’ in (KIF13B), AB001431 (KIF14), AB001432 (KIF15), AB001425 accordance with 18 U.S.C. §1734 solely to indicate this fact. (KIF16A), AB001423 (KIF16B), AB001424 (KIF17), AB001457 © 1997 by The National Academy of Sciences 0027-8424͞97͞949654-6$2.00͞0 (KIFC3)]. PNAS is available online at http:͞͞www.pnas.org. *To whom reprint requests should be addressed.

9654 Cell Biology: Nakagawa et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9655 maintain freshness. The rest of the purification procedures are prime kit (Pharmacia). Quick Hyb (Stratagene) was used for performed as described (24). hybridization according to manufacturer’s protocol. The wash Synthesis of First-Strand cDNA. Two hundred units of schedule was 2ϫ SSC͞0.1% SDS at 65°C for 10 min (three Super Script II (GIBCO͞BRL) reverse transcriptase was used times). For some probes additional wash in 0.1ϫ SSC͞0.1% to transcribe 50–500 ng of poly(A)ϩ RNA per 20 ␮l reaction SDS for 10 min was needed to eliminate the cross- by an oligo(dT) primer (45°C for 50 min). Other reaction hybridization signal. conditions follow the manufacturer’s protocols. Sequence Alignment and Molecular Evolutional Analysis. A PCRs to Identify New KIFs. The oligonucleotide primers phylogenetic tree of the motor domain sequences of the KIF used in this study are listed in Table 1. The primers were was generated using the CLUSTALW program (28) based on the designed according to the conserved amino acid sequences neighbor joining (NJ) method (29). The confidence limits on IFAYGQT and VDLAGSE in the motor domain. For all the tree were calculated by performing bootstrap resampling PCRs, AmpliTaq DNA polymerase (Perkin–Elmer) and Ge- 1,000 times. The region between IFAYGQT and LAGSE was neAmp PCR system 9600 (Perkin–Elmer) were used. Primers used for the alignment. However, for some of the newly IFAYGQT and DLAGSE were used to amplify 0-day-old identified KIFs, only a partial sequence of the corresponding mouse whole brain first-strand cDNA; the reaction program domain was obtained due to the cloning artefacts. The accu- was as follows: 30 cycles at 94°C for 1 min, at 56°C for 1 min, racy of the tree obtained was confirmed by comparing the trees and at 72°C for 1 min. The reaction mixture contained 0.5 ␮l deduced by the NJ method and the maximum parsimony (MP) of cDNA solution (directly used from cDNA synthesis after method. MP method was performed according to the de- heat inactivation), 1 ␮l of 10 mM dNTP, 5 units of AmpliTaq scribed procedures (30). Sequences other than the newly DNA polymerase, 100 pmol of each primer, 10 mM Tris⅐HCl identified KIFs were obtained from the GenBank database. (pH 8.3) (at 25°C), 50 mM KCl, 1.5 mM MgCl2, and 0.001% The tree was drawn using the software TREEVIEW (31). (wt͞vol) gelatin, in a total volume of 50 ␮l. Multiple bands Cytogenetic Mapping. A ␭ EMBL3 library of genomic DNA appeared when separated by agarose gel electrophoresis. For derived from the mouse embryonic stem cell line J1 was most of the known kinesin superfamilies, the nucleotide screened with cDNA probes containing the motor domains of sequences between IFAYGQT and VDLAGSE correspond to KIF1A, -1B, -2, -3A, -3B, -5A, -5B, -5C, and -C2 by standard Ϸ500 bp. Thus, bands around 500 bp were cut out and the methods (27). Overlapping clones of about 20 kb long were DNAs were recovered by Gene CleanII (Bio 101). Purified isolated. Clones were proved to contain the correct genes by fragments were treated with T4 DNA polymerase and sub- partial sequencing. For fluorescence in situ hybridization cloned into EcoRV site of pBlueScript (Stratagene). Sequenc- mapping standard methods were used (32–34) [see DNA ing was done by Applied Biosystems auto sequencer models Biotech (Ontario, Canada) and Nippon Gene (Sendai, Ja- 373 and 377. For the other template cDNAs the remaining pan)]. primers were used for amplification. PCR schedule was as follows: 96°C for 45 sec, 55°C for 4 min, and 72°C for 3 min with RESULTS 6-sec extension per cycle for 35–40 cycles (25). Primers IFAY-RI, IFAY-BAM, and IFAY-CLA contain EcoRI, Identification of New KIFs. Fig. 1 shows the alignment of the BamHI, and ClaI sites, respectively. These sites were used to amino acid sequences of the PCR products of the new KIFs subclone the amplified fragments into pBlueScript. Calf intes- identified together with the previously identified ones (12, 29). tine alkaline phosphatase (Toyobo, Osaka) treatment of the The identification of the new KIFs are summarized in Table 2. subcloning vectors was done with less than 1 ␮g of DNA in 50 Tissue Distribution Patterns of the Newly Identified KIFs. ␮l volume. The buffer used for the reaction was 50 mM Some of the new KIFs turned out to be homologues of Tris⅐HCl (pH 9.0), 1 mM MgCl2, 0.1 mM ZnCl2,and1mM previously identified and characterized kinesin related pro- Spermidine (Sigma). Three units of enzyme was added to start teins in other species. Fig. 2 shows the tissue distribution the reaction (1 h at 37°C). An additional three units of enzyme patterns of the transcripts of some of the new KIFs as was added at the middle of the reaction. Competent cells were determined by Northern blot analysis. Twenty micrograms of prepared with DH5␣ bacteria as described (26). total RNA was loaded per lane unless otherwise specified. Northern Blot Analysis. Total RNA was purified as de- KIF5C turned out to be brain specific. The transcript of KIF1C scribed (24) from brain, lung, heart, liver, spleen, kidney, (Fig. 3A) was ubiquitously distributed and 4 bands were intestine, and testis tissue of 4-week-old mice. Twenty micro- detected, suggesting alternative splicing or multiple polyade- grams of total RNA was loaded on each lane of a 1% agarose nylaytion sites. Two micrograms of poly(A)ϩ RNA from gel. The concentration of RNAs was measured by light absor- 4-week-old mouse brain was blotted against KIF1C probe in bance as described (27). Probes were labeled with T7 Quick Fig. 3B. Four bands appear clearly in this Northern blot even

Table 1. Degenerate oligonucleotide primers used in this study Primer Sequence IFAYGQT 5Ј-AT(T͞C͞A),TT(T͞C),GCI,TA(T͞C),GGI,CA(A͞G),AC-3Ј DLAGSE 5Ј-(C͞T)TC,I(A͞C)(A͞T),ICC,IGC,IAG,(A͞G)TC-3Ј IFAY 5Ј-AT(A͞T͞C),TT(C͞T),(A͞G)CI,TA(C͞T),GGI,CA(A͞G),AC-3Ј IFAY-RI 5Ј-GGG,AAT,TCA,T(A͞T͞C)T,T(C͞T)(A͞G),CIT,A(C͞T)G,GIC,A(A͞G)A,C-3Ј IFAY-BAM 5Ј-CGG,GAT,CCA,T(A͞T͞C)T,T(C͞T)(A͞G),CIT,A(C͞T)G,GIC,A(A͞G)A,C-3Ј IFAY-CLA 5Ј-CCA,TCG,ATA,T(A͞T͞C)T,T(C͞T)(A͞G),CIT,A(C͞T)G,GIC,A(A͞G)A,C-3Ј LAGSE1 5Ј-CTC,(A͞G)CT,ICC,IGC,(C͞T)A(A͞G),(A͞G)TC,IA-3Ј LAGSE2 5Ј-CTC,(A͞G)CT,ICC,IGC,(A͞G)AG,(A͞G)TC,IA-3 LAGSE3 5Ј-CTC,IGA,ICC,IGC,(C͞T)A(A͞G),(A͞G)TC,IA-3Ј LAGSE4 5Ј-CTC,IGA,ICC,IGC,(A͞G)AG,(A͞G)TC,IA-3Ј LAGSE5 5Ј-TTC,(A͞G)CT,ICC,IGC,(C͞T)A(A͞G),(A͞G)TC,IA-3Ј LAGSE6 5Ј-TTC,(A͞G)CT,ICC,IGC,(A͞G)AG,(A͞G)TC,IA-3Ј LAGSE7 5Ј-TTC,IGA,ICC,IGC,(C͞T)A(A͞G),(A͞G)TC,IA-3Ј LAGSE8 5Ј-TTC,IGA,ICC,IGC,(A͞G)AG,(A͞G)TC,IA-3Ј 9656 Cell Biology: Nakagawa et al. Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 1. Amino acid sequence of the KIF motor domains. Sequences between IFAYGQT and DLAGSE, the highly conserved kinesin motor domain motif, were aligned using the program CLUSTALW. Arrows indicate the positions of the primers. Asterisks are identical amino acids. Dots are similar amino acids. For some new KIFs only a partial sequence within this domain is shown. This is due to the cloning artefact. The rest of the region was either lost by the restriction enzyme digestion or appeared as a hybrid molecule with another KIF which may be caused by low stringency PCR. Various cDNA templates were obtained from whole brain, hippocampus, olfatory bulb, kidney, and intestine of 4-week-old mice. after harsh wash condition. In Fig. 3C developmental changes KIF5C for the molecule identified by Kato (35). Using cDNA of the KIF1C transcript is shown by applying 2 ␮g of brain fragment of the motor domain kindly provided by K. Kato, we poly(A)ϩ RNA for each developmental stage. Northern blot- found that this was also specifically expressed in the brain (Fig. ting revealed KIF6 and KIF7 (data not shown) to be testis- 2). Thus there are three different kinesin heavy chains in specific. KIF8 was a mouse homologue of yeast KIP1 (37). mouse brain. KIF9 was testis-dominant with less signals detected from brain, Phylogenetic Analysis of Known Kinesin Superfamily Pro- lung, and kidney. KIF10 was a mouse homologue of CENP-E teins. To classify the newly identified KIFs, molecular evolu- (38). KIF11 was a mouse homologue of Xenopus Eg5 (39). tionary analysis was performed. Phylogenetic trees were ob- KIF12 was kidney-dominant. KIF13A transcript was overall tained by both neighbor joining (Fig. 4A) (29) and maximum ubiquitous but dominant in brain, lung, heart, kidney, and parsimony methods (Fig. 4B) (40). Both trees are unrooted. By testis. The transcript of KIF13B was distributed ubiquitously. comparing the two trees, some of the branching patterns of the KIF14 transcript was detected in brain and kidney. KIF15 peripheral branches are nearly identical. For example, the transcript was dominantly detected in spleen and testis. branching patterns of the KIF1, KIF13, and KIF16 families are KIF16A was overall ubiquitous but dominant in lung, heart, identical in both methods. In other cases differences are seen. kidney, and testis. KIF16B was lung-dominant. For example, KIF14 locates in a completely different branch. As seen from the Northern blot results, the source of the The reliability of each branch point was evaluated by calcu- cDNA material does not necessarily correlate with transcript lating the bootstrap values. distribution pattern. For example, most of the testis-dominant These results allow us to assess whether a newly identified KIFs were identified with nervous tissue as a starting material. KIF is a homologue of previously identified motors in other Our results suggest that we can roughly classify the KIFs by organisms. For example, KIF11 is highly homologous to Eg5 in their transcript distribution patterns into two classes—i.e., Xenopus. However, careful interpretation is needed. First, only ubiquitous and tissue-dominant. KIF1C, -13B, and -16A are those that are shown to be evolutionarily close by the two the ubiquitously distributed, whereas the others (KIF5C, -6, -9, tree-generating methods may be considered to be highly -12, -13A, -14, -15, and -16B) have the biased tissue distribu- homologous. Second, even if two genes from different species tion. appear evolutionarily close in both trees, there still remains the Although the washing procedure of the Northern blot possibility that the two are not species homologues but rather membranes were done with care, the possibility of cross- orthologues. One good example is seen in the branching hybridization still remains. However, considering the differ- patterns for KIF13A, KIF13B, and CELF56E3 (a sequence ence of the molecular weight of each transcript, the cross- obtained from the Caenorhabditis elegans genome project). hybridization may not be between the different families. KIF13A can be a species homologue of CELF56E3 just as Two Brain-Specific Kinesin Heavy Chains Exist in the likely as KIF13B. Thus, the information obtained from the Mouse. In the previous search, we identified KIF5 as a phylogeny tree should be interpreted with caution. Full cDNA brain-specific type of conventional kinesin heavy chain (12). sequences of each of the molecules are required to finally Another kinesin heavy chain identified by Kato (35) was determine whether two molecules are species homologues or similar to both KIF5 (12) and to ubiquitous kinesin (36). Thus not. In any case, by neglecting the details of molecular to avoid confusion in nomenclature, we decided to use the evolution we were able to name the newly identified molecules. name KIF5A for the sequence referred to as KIF5 in Aizawa The numbers were given according to the chronological orders et al. (12), KIF5B for the ubiquitous kinesin heavy chain, and of identification. However, some of the names were later changed when we noticed that the molecule can be considered Table 2. Summary of searching the new KIFs to consist a subfamily with another molecule. Thus, the No. of numbering itself has no more meaning than each molecules are clones different. Library sequenced Identification Chromosomal Mapping of the KIFs and KIFCs. To examine the distribution of the KIF genes on the chromosome, we 0 day whole brain 83 KIF3C, KIF1C, KIF8, KIF11 performed fluorescence in situ hybridization using the 4 week hippocampus 50 KIF6, KIF7, KIF9, KIF10 genomic sequences as probes. The results are shown in Fig. 5. 4 week olfactory bulb 100 KIF16B There seems to be little relation between closely related KIFs 4 week kidney 200 KIF12, KIF13A, KIF13B, KIF14, and their chromosomal localization. For example KIF1A and KIF16A, KIF17 1B, KIF3A and -3B, KIF5A, -5B, and -5C, respectively, are 4 week intestine 100 KIF15 closely related members. However, their chromosomal loca- Cell Biology: Nakagawa et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9657

FIG. 2. Our first approach to elucidate the function of each new KIFs is to examine the tissue distribution. We performed Northern blot analyses for the new KIFs that have almost no functional implication from the updated database. Twenty micrograms of total RNA from various mouse tissues (4 week old) was loaded on each lanes. Probes used are from the PCR amplified and subcloned fragment of the motor domain listed in Fig. 1. Controls were taken by probing the membrane with rat ␤- probe. The arrowhead with the number indicates the molecular weight in kilobases. The numbers in the left of each membrane is the name of the molecule (KIF is abbreviated). ACT is the beta actin control blot. Lanes: 1, testis; 2, intestine; 3, kidney; 4, spleen; 5, liver; 6, heart; 7, lung; 8, brain. tions differ and no apparent segregation seems to exist. These mouse KIF molecules. The total number of the known KIFs in data implies that the evolution of the KIF genes may be mouse has now reached more than 30. Although 3 (KIF8, different from those genes that are clustered in certain regions KIF10, and KIF11) are likely to be the homologues of the of the genome, such as globin genes. previously characterized genes in other species, still 13 are new. In a PCR with a mixture of degenerate oligonucleotide prim- DISCUSSION ers, the outcome of the amplified cDNA fragment is strongly biased by the annealing efficiency between the template and The improved PCR strategy lead to the identification of 16 new the primers. Thus, the probability of discovering the new KIFs KIFs in the mouse, virtually doubling the number of the known does not necessarily correlate with the level of the transcript of that particular KIF in the cDNA template. Our findings does not give any information about the actual size of the KIF family in the mouse genome, and there are reasons to believe there may be even more unknown KIFs. What Are the Functions of the New KIFs? When focusing on microtubule-dependent intracellular transport, highly differ- entiated cells such as neurons and epithelial cells are good model systems to study. However, can we actually distinguish whether the new KIFs are for intracellular transport or for cell division on the basis of the information we have at this moment? Even if the two molecules from different species are shown to be evolutionarily very close, it does not necessarily mean that the two are the species homologues. This is because the phylogenetic analysis performed by us uses only partial amino acid sequences of the motor domain. From our previous study, KIF1A and KIF1B are highly homologous in the region that covers the entire motor domain, but the cargos they carry are completely different. However, there seems to be some relationship between molecular evo- lution and function. For example, the branching pattern in the phylogenetic tree deduced by two different methods matches for KIF8, KIP1, CIN8, BimC, Eg5, and Cut7. This is the so FIG. 3. Northern blot analysis of KIF1C. Twenty micrograms of called the BimC͞Eg5 family (8), and most of the molecules in total RNA from various mouse tissues (4 week old) was loaded on each this class are concerned in cell division. The branching patterns lanes. Probes used are from the PCR amplified and subcloned for KIF1A, -1B, -1C, unc104, CELF56E3, KIF13A, -13B, fragment of the motor domain. KIF1C appears as four bands in the KIF16A, and -16B are also same between the two different Northern blot. (A) Tissue distribution of KIF1C transcripts. (B) Two analysis method. If the relationship between molecular evo- micrograms of mRNA from 4-week-old mouse brain was resolved and blotted likewise. The molecular weights are marked in the right side lution and function found for the BimC͞Eg5 family holds true in kb. (C) Two micrograms of mRNA was resolved to examine the for this family, the five new KIFs in this KIF1, -13, and -16 developmental transcription pattern of KIF1C in brain. Br, brain; Lu, branch may be involved in intracellular organelle transport. lung; H, heart; Lv, liver, Sp, spleen; K, kidney; Int, intestine; Tes, testis. Because KIF12 and KIF14 are put in different branches in the 9658 Cell Biology: Nakagawa et al. Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 4. Phylogenetic tree was constructed using the new sequence data of the KIFs listed in Fig. 1. Other sequences were available from the database and added to generate the tree. The amino acid sequence between IFAYGQT and DLAGSE was used to align the sequences. The tree is unrooted. We confirmed the accuracy of the resultant phylogenetic tree by comparing the trees deduced via the neighbor joining (NJ) method (A) and maximum parsimony (MP) method (B). In A, branching that gives bootstrap values higher than 90 are marked. In B, bootstrap values are listed in the tree. The GenBank accession numbers of the sequence data used are as follows: KIF1A, D29951; KIF1B, D17577; KIF1C, AB001456; KIF2, D12644; KIF3A, D1264; KIF3B, D26077; KIF3C, AB001433; KIF4, D12646; KIF5A, C44259; KIF5B(MMuKHC), L27153; KIF5C(MMnKHC), X61435; KIF6, AB001434; KIF7, AB001435; KIF8, AB001436; KIF9, AB001437; KIF10, AB001426; KIF11, AB001427; KIF12, AB001428; KIF13A, AB001429; KIF13B, AB001430; KIF14, AB001431; KIF15, AB001432; KIF16A, AB001425; KIF16B, AB001423; KIF17, AB001424; KIFC1, D49545; KIFC2, D49544; KIFC3, AB001457; unc104, M58582; CELF56E3, U41536; CHO1, X67155; CHO2, X83576; Kid, D38751; SMY1, M69021; nod, M94188; MCAK, U11790; XKCM1, U36485, U36486; ncd, X57475; KAR3, M31719; Eg5, X54002; Cut7, X57513; BimC, M32075; Cin8, M90522; KIP1, Z11962; CENP-E, Z15005; XKLP1, X82012; Chromokinesin(Chromokine), U18309; NKIN, L47106; unc116, L19120; DmKHC, M24441; HsnKHC, U06698; HsuKHC, X65873; KLP68D, U15974; FLA10, L33697; KRP85(KinII), L16993; KRP95(KinII), U00996. two trees, we interpret these are unclassified for the moment. take place in a vectorial manner from the basement to the KIF12 is dominantly expressed in kidney and KIF14 in brain apical lumen (directional intracellular transport). However, and kidney. Taking into account the tissue distribution pat- the sperm cells thus formed also have active flagella which terns, the evidence is enough to encourage us to further include a lot of KIFs and dyneins. Thus it is difficult to say characterize these two molecules too. anything at this moment about the function of KIF6, -7, and -9. What Can Be Said from the Tissue Distribution Northern On the other hand, the KIFs that are neither testes- nor Blotting Data? We classified the newly identified KIFs into spleen-dominant, such as KIF12, -13A, -14, and -16B, are ubiquitous and tissue-dominant types according to the North- potentially interesting as motors for specific intracellular trans- ern blotting data. In general, one might suggest that molecules ports. that are ubiquitously expressed in the entire organism may What Can We Learn from the New KIFs. Much of the function in some sort of fundamental processes of cells in studies aimed toward intracellular transport are done in neu- general. KIF1B, for example, is a ubiquitous molecule and rons and polarized epithelial cell systems. The involvement of functions as a transporter of mitochondria (19). Mitochondria KIFs in axonal transport, dendritic transport, and intracellular are ubiquitous organelle, which is consistent with the distri- trafficking in epithelial cells remain elusive. The functional bution of KIF1B. However, cell division is also a process specificity of each KIF is believed to reside in the nonmotor observed in all cells except certain highly differentiated cells, domain of the molecule. This belief is based on the evidence such as neurons. In new born animals, when the actual number that the amino acid sequences of the nonmotor domain are of cells in the body is increasing, a certain population of almost quite distinct between different members. Initially, the only every tissue should be undergoing cell division. But in rela- tools to further devide the nonmotor domains into different tively grown up animals cell division events should be limited subdomains were the prediction of three-dimensional struc- to specific area of the organism, such as testes, lymphatic ture by analysis softwares based on the primary amino acid organs (spleen), and certain epithelial tissues (alimentary duct, sequence such as the coiled–coil algorithm (41, 42). However, dermis, etc.). Our materials for the Northern blot analyses as more and more information on full-length cDNA sequence were obtained from 4-week-old mice. By the criteria above, accumulated, subdomains in the nonmotor domains became KIF15 may be involved in cell division. KIF6, -7, and -9 are clear for some of the new KIFs. One example is the AF6͞cno dominantly transcribed in testes compared with other tissues, domain (43). The function of this domain is unknown, but the but it may be premature to conclude that they are involved in domain exists in the region flanking the motor domain toward cell division. We believe the situation in testes is complicted. the carboxyl terminal in KIF1A and -1B. The question is In this tissue active spermatogenesis (involving cell division) whether this domain exists in KIF1C. The full cDNA sequence Cell Biology: Nakagawa et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9659

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