A promoter-hijack strategy for conditional shutdown of multiply spliced essential cell cycle genes

Samejima et al. 10.1073/pnas.0712083105.

Supporting Information

Files in this Data Supplement:

SI Figure 5
SI Text
SI Figure 6
SI Figure 7
SI Movie 1




Fig. 5. No kif4a knockout cells were obtained by the conventional method. (a) The conventional knockout strategy. (b) A physical map of the kif4a locus and the targeting constructs. (All of the physical maps are based on the ENSEMBL chicken genomic DNA sequence.) Black boxes on the genomic map indicate the positions of exons. Lines and arrows indicate (putative) genes in the region and the direction of transcription. (c) Southern blotting confirmed the correct first-allele targeting. EcoRV digested-genomic DNA was hybridized with the 5' external probe shown in (b). I and II correspond to each of the steps described in a.





Fig. 6. Immunoblot of kif4a heterozygote cell lines. (a) Immunoblot of four kif4a heterozygotes established by the conventional strategy, expressing kif4a cDNA and kif4apro-tTA2/tTA3 in the presence or absence of doxycycline for 3 days. Second targeting was performed by using heterozygotes 1 and 2. a-Tubulin was used as a loading control. (b) Immunoblot of kif4a heterozygotes established by the promoter-hijack strategy, expressing kif4apro-tTA2 in the presence or absence of doxycycline for 24 h. Second targeting was performed with heterozygotes 1, 2, and 3. a-Tubulin was used as a loading control. Asterisk marks an unspecific signal. (c) kif4a cDNA silent mutation used to distinguish expression of rescue construct from endogenous ones.





Fig. 7. Multiply spliced forms of KIF4A are expressed in DT40 cells. The ENSEMBL database records 30 exons for the kif4a gene, whereas the NCBI database records 29 exons that match the dominant transcript of DT40 cells. RT-PCR using primer pair a/b copies the canonical transcript. However, we confirmed the expression of an extra exon, a long exon 6 and a long exon 7 using reverse primers c/d/e specific for each sequence based on the ENSEMBL or EST database. Forward and reverse primers are shown as black and red arrowheads, respectively.





SI Movie 1

Movie 1. TrAPGFP-KIF4A integrated cell line. TrAPGFP-KIF4A protein localizes on chromosome axes and diffusely in cytosol throughout mitosis. During anaphase, part of KIF4A transfers to the central spindle and later concentrates at the midzone and midbody. A midbody remnant from the previous division also appears. Because two movies following the same cell continuously were fused, two time = 0 points are shown. The first, from the beginning to the onset of anaphase, shows one focal plane. The second, from the onset of anaphase to late cytokinesis, is a projection of 10 focal planes. Time interval, 1 min.





SI Text

Construction of the Targeting Vector and First Allele Targeting by the Conventional Method. The kif4a locus resides on chromosome 4 at location 512.766-496.350 of the chicken genome sequence database. We initially used a conventional strategy in attempts to knock out the gene (see SI Fig. 5a). Screening of a DT40 genomic library by hybridisation with two probes located in the 5' untranslated region (UTR) and middle of the kif4A gene yielded a phage genomic clone containing a 19.1 kb insert, which covers chromosome 4 at location 523.828-504.712. A targeting vector was designed to replace 3.2 kb, including the predicted kinesin motor domain, with a puromycin resistance cassette flanked by Lox P sites (loxP puro) (kinesin motor deletion vector; see SI Fig. 5b). The kif4a cDNA was originally cloned by expression-vector screening by using an antibody made to a gel band present in mitotic chromosome scaffolds (1) and subsequently completed by RT-PCR.

First-allele targeting by this deletion vector occurred in 16% (3/19) of drug-resistant clones, as verified by Southern blotting (see SI Fig. 5c). Next, the puromycin cassette was successfully excised by transient transfection with a plasmid encoding Cre-recombinase. This allows the original vector to be reused for second-allele targeting.

Failure to Obtain a kif4a Knockout by the Conventional Method. The knockout process broke down when we tried to express a kif4a "rescue" cDNA in the heterozygotes. Cells were transfected with vector pairs in which KIF4A expression was driven by tTA2, tTA3, or tTA4 (the latter are variant forms with transactivation activities of 98%, 39%, and 10% relative to the canonical tTA (2). Although a few clones expressing KIF4A under the control of CMV-tTA4 were obtained, they were unhealthy, and expression of the rescue construct was lost after 2-3 days in culture (data not shown). This suggested that continuous overexpression of KIF4A protein is toxic to cells. Furthermore, all attempts to target the second kif4a allele in these cells failed, only random insertion events or retargeting of the first allele were observed. These experiments appeared to confirm RNAi studies indicating that kif4a is an essential gene (3-6).

Because KIF4A overexpression appeared to be toxic, we tried another approach and obtained cell lines expressing KIF4A relatively stably when the cDNA expression was driven indirectly by its own promoter. We cotransfected kif4a heterozygotes with vectors encoding kif4apro-tTA2 or kif4apro-tTA3 together with the tetO-kif4a cDNA and established lines stably expressing KIF4A using both vector pairs (see SI Fig. 6a). We then tried to disrupt the kif4a second allele in these clones but obtained no knockouts in 464 drug-resistant clones screened. This result suggests that multiple splice-variants of KIF4A are necessary for the life of the cell.

Methods

Library Screening and Construction of Targeting Vectors. Plaques of lambda Fix II carrying a genomic DNA fragment from DT40 cells were screened with two 300- and 330-bp probes amplified from DT40 genomic DNA with two primer pairs (gtttagaaacatgctcca and atccagcccctgttgcta; ccaatgtacattaaagat and cagcttgtgatgaggaaa). The phage insert covers from the 5' UTR to the middle of the kif4a gene. Kinesin motor deletion and Disruption targeting vectors were assembled in pBS (Stratagene).

Kinesin motor deletion vector. The 5' arm (4518bp EcoRI/AatII) was obtained from kif4a phage DNA. The 3' arm (2956bp SpeI/NotI) was amplified from DT40 genomic DNA with primers (agcactagttcttcatgcagcaagaatagatctatccttg and atggcggccgcagtgaagatatgcccctgataagtctgtgc). A LoxP puro resistant cassette was inserted into the BamHI site and linearized by PvuI digest. The 5' probe was amplified from genomic DNA with primers (cacgtgtggtgcttgggc andacctgagccaggtg).

Disruption vector. Arms were amplified from kif4a heterozygote genomic DNA with primers (catggtacccggtgcgtggcgctgcgctgccg and catgcggccgcgtgaagatatgcccctgaraagt) and transferred into the NotI/Acc65I site of pBS. A Puromycin resistance cassette (7) was inserted into the BamHI site in the opposite orientation from the kif4a open reading flame.

Promoter-hijack vector. The promoter-hijack targeting vector was assembled in pTre-tight (Clontech_Takara) with an insertion of double stranded primers (aggacgtcccaccgcggtggcctaggactagtagatctgacgtcag and ctgacgtcagatctactagtcctaggccaccgcggtgggacgtcct) into the AatII site. Next, the lox P puro cassette was inserted into the BglII site in both orientations (modified pTre-tight). The 5' arm (2344 bp SpeI/SpeI) and most of the 3' arm (3,832 bp XmaI/EcoRI) were obtained from phage DNA. Double stranded primers (ctagtacgcgtatggtgcgggaggaggagaaggggatcccggtgcgcgtggcgctgcgctgccgcccgctggtgccgaaggagacgagcgagggctgccagatgtgcctgagcttcgtgc and ccgggcacgaagctcaggcacatctggcagccctcgctcgtctccttcggcaccagcgggcggcagcgcagcgccacgcgcaccgggatccccttctcctcctcccgcaccatacgcgta) were added to the 3' arm. The 5' and 3' arms were transferred into the SpeI and MluI/NotI sites within modified pTre-tight, respectively. The resultant targeting vector was linearized by using PvuI. A 5' probe (500 bp) was amplified from phage DNA by using primers (tcagtagcttcatttcct and agttatgcacagttacacag).

Construction of kif4a cDNA and Introduction of Silent Mutations. A partial cDNA was obtained by screening an expression library with an antibody raised again chromosome scaffold band 3 (1). The 5' half of the cDNA was obtained by RT-PCR from DT40 total RNA with primers ccgcgggtcgacatggtgcgggaggaggag and aagcttcagcagctcatactgcct. Silent mutations were introduced in the "rescue" cDNA to distinguish its expression from that of the endogenous locus by RT-PCR (see SI Fig. 6c). To introduce the silent mutations into the cDNA, an EcoRI/HindIII fragment within the cDNA was replaced with a DNA fragment amplified with primers (ctgaagcttgagcgagactttcagaagcaggctagcgttcttcgg and ttggaattcagcctccatttcggtcgcatggtttcgctcttcaat). The kif4a cDNA was cloned into the tet/doxycycline-repressible vector pUHD10.3 (a gift from H. Bujard, University of Heidelberg, Heidelberg, Germany). KIF4A expression was detected by RT-PCR with either wild-type (tttcagaagcaggccagt and tcagcctccatttctgtt) or mutant (tttcagaagcaggctagc and tcagcctccatttcggtc) primer pairs.

PDK11 Expression Vector Construction. A chicken EST (ChEST871e12 obtained from the MRC Gene Service) contained the full-length pdk11 cDNA. The cDNA was transferred to pECE containing a histidinol resistance cassette.

Promoter Construction. It is a major undertaking to define a promoter region precisely including all enhancers and suppressors, particularly because elements such as enhancers can sometimes be placed far apart. Thus, we used an operational approach to clone fragments of gene-specific promoters containing essential elements. In the case of the survivin gene, a region of the 5'UTR was cloned from a DT40 genomic DNA library and sequenced. The survivin promoter fragment was selected from this based on the published analysis of the human survivin promoter (8), and all essential elements identified in that study were included. The smc2 promoter fragment (3,823 bp) was chosen based on the experimental evidence that the smc2 cDNA preceded by 3.8 kb of the 5' UTR is able to rescue an smc2 conditional KO cell line much more efficiently than expression of the cDNA under a CMV promoter (D.H., Shinya Ohta, Tina Freisinger, F.M., Lau Sennels, Flavia Alves, F.L., A.K., Juri Rappsilber, and W.C.E., unpublished data). In the case of kif4a and incenp, we subcloned the longest 5' UTR fragments obtained from the DT40 genomic DNA library. Individual sequences are attached below.

The kif4a promoter (6015 bp) was generated from kif4a phage DNA (SpeI/EcoRI) using double stranded primers (ccgccgcgccgcgggggctgctgagagcctcacgggccccttccgcgcctctcggcccgctctgcccgtccctcagttggcccgaatctagaatg and cattctagattcgggccaactgagggacgggcagagcgggccgagaggcgcggaaggggcccgtgaggctctcagcagcccccgcggcgcggcgg). The survivin promoter (1957 bp) was amplified from a phage DNA containing the survivin gene isolated in our laboratory with primers (cttcacaagttacaaatg and ggagcactggcaggattc) (A. Carvalho, C. Morrison and WCE, unpublished). The incenp promoter (5668 bp) was generated from a phage DNA containing the incenp gene isolated in our laboratory partly amplified with primers (cgagcggtaggtcctgaat and cccctcgagcgctccggcttacctcagc).

kif4a promoter sequence (from ENSEMBL chicken genomic DNA sequence database): GGATCCAAAAGGTAACTGGTGCTGCTGGACTTGTGGTGTGGTGATGCAGATTCTGGTCACAGTGGAAATCGGGCTCATAAGCCAAGAGTGCATCTACATTACACGGCACTACTCTGTTCCAGACCCTTTGGTTAATGTTAATGCCTCAGCTTGCTATCAGAGCTGTTGGATACTTGGTAGAAATAGAGCCACTTTCTGGCGCAATAACTTCTGGTTTTCTTCCCAACATTTATTAGGGTCGTTGCCAAAGTGGGTTGTGAATAAAGCCTCGCAGTACCTGGTGCCGCAGGTGAGTGCAGCATTTCTATGGTGTGTATGCCAAGTGCCGCCCCACCGGCTGGAGCACAGGGCTGAGGAGAGTGGGTGTCCCTGGCAGCGGAGCTGCACTAGGAATGTGCATCGTGGCTCACCTCAGTGTGGCAGTACATTCTGTCAGCCTTACCAGTCCGGCAGCTGGCTGATTCATATCCGAAGCCAGAGGGAGGTGAATAATGCCACTATTCCGGTTTCCGCTTGTGCAGTGTCCTCCCTCACCTCTGCTGTTCATCTGAACAGGACCTCAAGGCTCAGGAGCTTTTGGAGCTCCTGAACCAAGCAGCAGGGCAGGACAGGCACAGAAATACCCTTGGTTTACAGGAAGTTTGATTTCACTTTCCTAAAGCAGCTGATGTCATGAGGGGATCTGAGCAATTAGAAGATGGTCCTACAAGGGAGTTAAAAACTGGCCTTTCCTGTGCTCCTATGCAGCCAGAAAATAAATGCAGTAGCTGTCAGTAGTGGCCAGATTGCCAAGAATTCTGATAAGCTGCCATAGTTGTTTCCCCCTATGCTTCCCTGCTTTTGATCAGATGTTAAAGAAACTGCACAAGGCCTGTGTGCAGTACCCTGCCTGGAAGCAGCAGCACAACGTGAACACGAAGCCCTGGCTGTACCCTGAGCAGAACAAGCTTCCCGTGCTGGCGCTGTCAGAGCTGGCACTGCAGCGCGCTGCGTCGCTGGAGAACATCGACGAGAGCAGCCTGGCTGAAGAGAAGGATGAGAGCAGTGACCATGGTGGCTTGGAGAACTGATGTTGCTCAGGCTTGTCTAGTGGCTTACAATAAGGGGGGAAGTTTTTTACTTAAAAAAGAAATCCTCGTGTCTGTAGCCTTTTGTCATGAACCAGCTGCATGGCTCCTGGCTTGCTGGCCCTGCTTAGTGCAAGGCACGCGGGCTGACAGTCCCGACCTGGTAATGTCATCTGTGGGAGAATTCATACCATGTATTTCTCAGGTGAAAACTGCCCTTACAGAATGCAGCTTGTTCTGAGCCTGTTGCTCCAAGAGGTCGAGTTACCTGCAGTACCTCTGCCAGGCTGCTCTACCAGCTGGTGAGTTACTCATTGACAGACTGCTCTTTGCCTCTGTTCTGTGAGTGGCTGAAATATCTTGTGTTGCTGGAGCTGTTGTTTTCCCAAAGGGAATCAATAACTACCTGGTGTTTCCCTCCAGCTCTGAACCATGAAGTGACAAGTACAACCCATGCTACTGCTCTGTCCATTAAAGATACTTAAGCCATGAATGGTCCTTGAAAAGAATCCACTCCTTCCTTGTTAGCCTACAGCCAGCTGTGGGGGCTGCAGAAGCACCTGAGCAATTCTGAGTATTAGCAGAGGAAGCTGCCCTGGGCAGGGCTGATTTTTCTGGAGCTCCTGTCAGGCTCTTAAGTAGGATTAAAAGGTGGGAGTTGTATTTGCAGTCCTCTGTGCAGTGAGGTCAAAGCACTCTTTGCTCCCAGGCAGTGTGGTTGTGCCGGAAGCCTGATGTGTGCAGCTGTTTGCTGTGCCTTCCACCAGGCAGAGCACAGGGGGGGCTTTGGGGCCAGGGATTGGCCCTTGAAACATCAGCCTGGCAAGCCTGAGCAGAACACCAGGGAAGACAGAGCAACATGGAATGTTATCTTGTAATTTGATCATCCTCCTTCCCCAAGAAACATGTAGGTGCTGTAAAGCTGGTGTGAGACTGGAAGCATACATGCTCTGCATCTCCATAGGCCTCTTACTTTACGTCTCAGGGTGCTACAGCCTGGATGCTTATTTCCCTGGTAGAATAACTGACTTCTTTATGGGACTGAAAGTCTTTTCTTCCTAGCGTTCTTAATTCTACTTCAGCTAAGGCAAATCCAGCAGGCAGAGCTGGAGCCACATTGGGCTGCTCCTAGCTGTCAAGTGAGGTGACAAAGACAAATCCTTCTGTTGCAGCCCTTCTTCTTTCTGGCAGCACTGAAATGCCACTGCAAACTAATCACAGGCTGACTTCCATCCCAGTCAGAGGAAAAACACCCTTATAATAGGGCTGCACCTGGTATGGGTTCGCTGCATTTTTCCTGCAGGCTGTAAAGGTAATTGGAGCCACGCATTTCTCAGGGGAGTGCTGACGGTGGCAACAAACCCTTCAGAGACCTCCTGAGTGAGAATAAGATGAAGTCGTGTTGCTGTATTAGTTTTATTATTTCAGTTATTACAGTGAGTGGAAAACACCGCATAGAAAGCCAGAGCAAGGGCTGTCCTAATACCATTTACAGTATGATACTATCCTACATCTGTGTCCAGCTTTGCTGATAAACTGTAGTGTTTTCATCTGTGCCCTCCCAGCACAAGGCACGGCAGGCTTTGTCCCCTGTCTCAGTAGAACAGCTACCATGTCACCATTTCAGCAGCAGGAGGGTGAATTGTCCCAGCAGGAGAGAACGTACCCAAGGCAGAGGAAAAGGGGTAGGGAGCTCTGAGGGGAGCACCTGAAAGGGAGGGGGCTGTGCCAAGTGAAGGAAGCAAGTTCAGCAAGTGAAACTTTAACAGTGAAAGCACAACATGTAAGGGAACCTGACTATGTGCTACTGCAGTTTAGAAACATGCTCCAAGCATCTTGGTCCTGTATCACAGGAGTGGATAAGCAGCAACAAGGTATGCACATGCCTTGGCAGACTAGTGCAGCTAGAGTTGTGCATTGAGTTTGACTGGATTTTACTCCTGTTTGAGTACCAAGCAAACCCAGTAGTGTAGAGCAGAGGTAGAATAGTGGAGAAGGAAAGGACTTCATATCACCAGAGAAGTGAACAGGTTGATCTCTTAGAGAAGGCCATGCTCTGCTAAAGTGCACTCGGGCACTCCTGGCGTTACATCCTAGCAACAGGGGCTGGATCCAGAACCAGCAGTGCCCTCCTTTTAACTTATATCCTACTAGAATAGAAGCATAGTTCTGTCAGGACAAGCTGCCCCCTGGTACCCACTTTCATGCCAGCACCACCAACTTGTTTCATCATCCAGTTCCAGGAGAACAGACTGAAGCCCCATCCATCTTCCAGCTAAATAGGCTGTGGTGTTCCTTCAAGCAGGGAGCAGAGCTGATACCTGTGCAGTTGTGCATCCAGTGACAACTGAAACTTAATACATCATCTCAGATGTTGAGGAAAGGTAGTGGAGAGTTTCTTGCAGGACAGTTCCTCCAGAGAGGACACAGCAGACAGCTGCCTATTAGTGAACAGTTCTTTCTTTCTGTCTCTGGTAATCTGGACAGGAAAAAGAAGGCAAGGCTGAAGGGGCAGGAGAGCAGTTGCTGATGGTGAGGAGAAACTAGCCAAGATGTTACAAGAAAACAAATTCTGCTTATACTTCATTAATAGATGCAGTGTTTGCTGCCTTCATAAATAGCAGCTGGTTTTAAAATAGCACATTCACATCTCAGCTAGTTGTTTCAAGTTATTTCAGTGCTTGTTTTGAAGTCCTCTCTTCCCTTGCTGCTTATTTTCCTATACTTACATTTAAGGGTCAGTTCCCTATTTTATGAAGAATCCTTGTTTGGCTTCATTTGACCTTTCCCCACTCCTCCTTTCTCCCTCTGAGTATCTCTGGACATTATGGGTTCAGAAATCAATAGCTAAGAAGTTTTTCCCCCTTGGCAATATTTAAACATCCTTATAGAAATGGACTGAAAACACAGTGTCTATCTACCATCAGCATAGGTCCGCTAGCAACAGAACTGGTTCCTGTGTTACTCAAGGATAAGCAGGTTCTTGTACACTTCCATGAGAAACCATCTCAGCTTTTAGCACTGTTTTCTTAAGTCCTGCTCTACAAGATTCTATGCGTGAGCAGTTGAAGCTAATCCCTGCTGTTTTGAATCAGTAACTTTTCTGATCCCCAGACTGAAGACCAGTGTTTTCTGATCTGAACACTGCTTGTGTGACAACACCTACAATTGCAGACTCATCACTGCTAAGCCCAGGTGGATCAGCAGGGATGCAGAGCAGTCTTGCCCAAGGAGAAACGGTGCTAGGTGACCAGATTTGGAGCTAGCAGGCACTTACTGTAAGGGAAGTAACGCACACGCATGGTGATTTCACGGGCTGTCTTCAGGATCTCCACAGCCTGAAAGCAGAAAGAGTTGATCAATGCTCAAACTGTTTTGGAGGATAGGAAACAGCAGTGCATCAGTTACACAGCGCCCCCAGCTGAGCTGGTGTGAAGGAGAAGGCTATGCACCACTTCCCATCTTCCTGCTTGGGAGGAGAGCGCGTAGTGGGGAGCAGAACTCACCTTGCTGTGCTCGATGTCCTGGAATCCACATCATTTACAGACAGCACCTGATCTCCTTCTTGCAGCCCAGCTCTGTGTGCATCTGAGTCAGGGATCACCTGCAAAGGGCAGAGCAGATGCAGCAGTGCTGACACTCTATGGTCAGTACTGCAGGTCCTGGAGCTTGCACAATGCGCCCGTGCAGCGTGTTCACACGGGGAGGGGGGGGGTCATGTGGAGAACAGGGACTGCAGTGCTCCCTGCAGGCTAGGGAGGGGTAAGCAAACGGTGTAGCATTGCCTGCCTGCTACCTGGAAAGTGGCAGATTACAGCTCTGAGTCTGGAGGTTCATGCAAAGGCAGATGACAGCCTCCAAGCACACCCAGCTGGCTCTGCAACACCCAAGGGAACAGACAGAACACAGGGGGGGGGGGAAGGAGAGCACGAACACACCTTAGAGATGAAGATCCCCAGCTGCGAGGCCTTTCCTCCCCGGATGTTGAAGCCGAGCTGTGAGACAAACCACGTCAGCAGCGAAACACCACTTCTACGGCAATCCCCCCCAGTGCCCGCAGGGTCGTTCCCTCGCGCCCGTCCCAGCCCCTCTCACACGACGTTGCCGTTCCGTACAGCTTTGTCCCTCCTCCCCGCCCGGCGCTCCCAGCAGGGCAGCAGCCCCCCTCACCCCACAGCCCCCGCCGACCACCCGTGGTGACGCTCTACGCGCTCTCTTTGCGCTCAGTCAGACCTTATCACGCCCTCGCAGCTCTGCCCGCCTCTTTGCCAATTTAGGGAAAACAACTTGAGTTCAACGGGAAAAAAGGCCGCGCCGAGGTCAAGAGCGACCCGCGCGGGGCCGCCGTGGGGTCCCGCAGCACTCACCTGCGCCCCGGGAGGCTTCTTGAGGACGACGGTGCGGGGCAGGAACTGCGTGAGCTCGTTGTTGTAGTCGGGGTGGTAAACCCTCTGCAAAACAAAGCGGCGGGCCTGAGGCGCTGCCCCGAGCCGAGCCCCGCCGAGCCGCAACCCCCCCCTCGGAGCCTTCCCTCACCTCGTGAGGCGGCACCCAGGCGGGCGGGCTCTCGTAGGGCGGCAGGAAGACCACCGGGAAATCATCGTAGGGCAGCCGGCCCTCCATGCCGCCTCCCGCGCGCGCCGCGCCCTTTCGGCCTCCCTCGGTTCCGCCTCTCGCGCGGCGCCGCGCGCCGTGATGACGTCGAGCCCCGTGACTGGCGGAGCGGCGCGCGGCACCGGTTTGAACGCGGACGGTTGGGGCGCCGCCATCTGCGATCCAACGGGTGGCGGCGGGGCGCGGGGTGGGTCCGGCGGGGCGGCCCTGAGGGTGTGCGGGCTGCAGGCCGCTCTGCGCCTTCCCCGCCGCGCCGCGGGGGCTGCTGAGAGCCTCACGGGCCCCTTCCGCGCCTCTCGGCCCGCTCTGCCCGTCCCTCAGTTGGCCCGAA

survivin promoter sequence (A. Carvalho and W.C.E., unpublished data): TTACTCTGTCACCAGGGTGTAGCCCTTTTATCCAGTCTAGCAGCTTCTCTCTCTCCGATTTTTTTTTCTGCAGAAGAGTAGGAAGTTGGCACTTTTCTCTGCTTTCTGACTGAAGGTTGGTCAGTGTGTTCTGAGTATGAATCGTATCCACTGTCTTGTTCCTTTCGCATCAAAGCCCACACAGAGCTACACAACAAATATAATGTATTGGTTTTAAATCTACTGTGTATACCTCTTAGGTACTTGGCTATAATGTTGCCTTAATGGACTCTTCATGTTGGTAACAGCAGGAAGGTGGGCATGTATTACGCTTTTGTGTAGCTGATGTACATCTTCATCCAGTCAAAACCAGTGTTAGTGGGAGGAAATAAGAACCCATTCAAGAATTGTGAACTAACATCCATGCACATTTCAGCCTGCTTTATGACTCTGTGAAAATTTTCCATGTTACTGGTGTTAGGAATTAGCTGTACTTGGAAAGGAAAAAAATGTCTTCATGAATGGAATTAAAGCCCTTCCCTTGAACAAGCCTATCTCTAGTTATCCTGTTTGCTAAGCTGACGTTCTGTTGTCTTTCTGCACTTGGCAAACTGAAATATGGCCAATAGAGGGATGCTTCCTGCTTCTCTGAGATGAAATCCCAGAGGAGCACTGGCAGGATTCTCCTGCAACTACTTCTCTACTAGGCATCCACTCAGAGGCCACATACGTTCAGGACCTAGCAGGCTCTATCCTTGCCAGCCAGAGCAGGCATAAAAGATAATCTGTAGAAGAGCAGTTCTGCACCGCATTCTCAAAGATGGCTGTGAAGAGTTTGAGGAGCTTCTGGAATGGAACCTTCAGTAAAGGTTATAGGGGCTGGAGATCACTATTGCTGTTCTGCGGGCTGCAGGAACAAGAAATGGCTCTGCAACTTTGAAGTCCACCTGCGTATCTGAGGAGATAACTTGTTTCATACTTATTTGCCTCTATTTCTTGGAAAATCAGCCAGTTAACTGTTGTATCTGTTGCATTCCACATCTGTGAGACTGTAGCCTTTAATTTCGTTCCCTTGGTATTATTTGAACGTGCTCTGGGAAGCTGTACCCTGTTCCTTAGTGTTGCTTGTGAAAGTCTTAAATGGCTTTGAACTGTTCCCTATGAAGTCCTTTCCCATTGATCCTAATGGCTTTGGTGAGAATAAGGTGCAGCTGTCTTGAGGATCATGTTGCTGATAGTTTGAGCGAAAACAAATGGTCATGACCTAAAAAGACATTCGTAACTTGTGAAGAGGTCCTAGCAAACCAGATCCGAAGGGCACTGATGAAGCTGGTGGTATCTGTTCAGAATATGCAGGTGGGACATCTTGGATACTCCAGATGTCTGTTAGAGATAGTATCTTCTTCTCTGCTGGCTGTGTGGGTGAGCAGAGCATGCTAAGGAGGCACTGTTTGAGCAGAAATTCACCCCTTGCCTTCAGCATGAATCTTCTGAGTATGAGAGGATGAGGCCAGACTATTTAACAGGCACTGTGTGAGACCAGGTGTGTGCTCTGATACGGGCTGGCAGCCCTGCGAAAACACACTAGGGATGCCAGGGAGTTCTGCCTGCCCTCTCTTGGTGAGCGCCCGGATGAGGGAGCTTTTGAAGCAGCTGTAAAGCAGCTGTGGGGCAGCTGAGCACCAAATGTCAATTGTGCCTCTTGACCTATTGCTGTTACATGGAAGCAGAGGCTGAGCCCAGCGAACCCCAGACCTCTCCAAGTGCTTGTGTGTGTTTTGATGCCTCATAACCAGGGAGCTGCTAGGGATCGGTTTGTCCTGCCTGTTCATCAACACTGGGTGGGCGTTAAGGTTCCCTCCACGCTAAGCCGCGCCAGCAAGATGGCTGCCGCCCCGGGGCGGACTACGTCTCCCAGCAGCCCTCGCGCCGCTCCGTTCCCGCCTTGCCCCGCGCGCCCAACCGCCCTGGTTTGAAAAATG

incenp promoter sequence (from ENSEMBL chicken genomic DNA sequence databases): GAGCTCAGGTCTCTGTGCAGACTTCTCCCAGGGAACACGCTGATTGCTCATCTCAACTTTCCACATGAGCACTACCACCTCCCTCAAGTGTCACTGACAAACTGGTGTAGGCTGGGAAGGCAGAGAACTGAAGCAGCAGCTCCTCTGTCTGCAGACTGCTCCAGCTGCACAAACACACTCATGCCTGTGTCACAGAGATATCATTTCCCAGTCAATTGACTACGGTTTCTGCCTCCCTGCACAATTTTATGCAGAGATTTGGTCGATTTGGGCTGATTCTATTTCCCCCCCTTCCCCAACATCACTTAGATGAAAGTGTTTTAACAAATGCCTAGATACAGTGTGACTCAAAGCTGACAAAACTAAATACCAAAGGCATTAGCACTTTATTAGAAGCCAATCTGGTTTCAGAAGCAATGATTTTTAGAAAGCCCAGATCTGAAAGCCTTCTAGCCTTCTCAGGAGCAGCAAGAGCAAGGGATGTGAAAGATTTGGTGCTCGACTTTTGAATCTCGTTTCCTCGGGTGACTGTGCTAGTTAGTTCTTTGTCCATGAATTTTATCTTCACCCAAAAAGCATTTGATTTCCTCTGCCAATTTCAGAGAGCAGACACATTCATTGTCTTCCAGTTCCTCAAAAGAATCCACAGAAGGCACTCATACTGAAAGACCCTCTGCAGGAAATGCAATAGCCTCAGGGCAATAAGCTACCAAACCACACTCGTACCTAGAACCAGCAAGGGTGCACTTTCCTTTCTACTCTGCATTTATTTGCTTTTTAAAATTCATCTGCAACTTCTGTGACCCATTTTCCAATACCTGTGCCCCAGCTTATACTCCTTCCACAGCAAAACAGAGCCTGAGGCTCACCCTGGCAGCAGATGAGAACCCGAAGATTGCACCCTAAATGGATAGAAGCCACAAAACCTACTGCTGAGGAAACTAGGAGGAACTGCAGCTAATATGGATGAGCAGCTCTATAGCATAGCTGAGAAGCTGACTTAGCCAACAGAACGCACTCAATTAACAGAAAACTTACAGCTACTCTGCTGGAGGCAGAGGAGGATTCTTGTGCCGCTGCCTTCTTTGCAATGGCCCTGCTGATAGATCTCCGGATCATGCTCTCTCTTTTGCTGGCCAGAGAATATTTTTCTGCCAAGGAAGTCTTATGGCCTTTGTGGGTGCCACCTACATAGCTTCGGCGTCCAGAACGTCGTGTTTTGGATATCACCGTGGCCATTTTGTCGTTGGAGTCCTGGAAAAGCACCTTCTCTAGGCTTTGAGACAACAGATCTCCATGGAGCATGGCATGATCTCTATTTCTGGTAGCCTTGGGAGTGGAGGTACTTGATTTTTGTTTGGTGGCCTCTTCTTCCTCTCCTGCTGTCTGGTCATCAGGGACTGCGCTGACAGCAGGCACCTCGTGCAGTTCATTCATTGCACCCTCCTTGGACACCTGCTCTGCTGGAACAGCCTCACAGGCTGCATCACCACAATCAGCACTGCCTTGCTGTGGAGGTAACTCAGTCTGAGGACAGTCACTGCAATCCACTTTCACCATGGGACTTTGTGCTTCTGCTGGCAATTTCTCAAGCAGTGCTGGAAGCTCCATTTTAGTGTTGGCCTGAGACCTCACCTCTTCCTCCTGACAGCCAGGGTTCTGGGGTTGCTCTTTGCTGCAGCGTCTTTGGGAACTTGGCCTGGAAGACGCTAGCTTGATGCTGCTTCTCCTTCTGGACAACCTTAAATGAGAACAACTTCAAATGAAACAATGCAGGAATCTCCCACTCCTCAGCTGTCTGCGTCTTGTTTTGAAACAAGTTTTACATTGAACCTACAGCCTTCCTCCCCACATCCACTTCCCCTTACAACACACCTTTTATCATTGGCTAAATGCAGGAATAAAAGAAACCTTGACAGCTCTGGCAGATCTAGCCAGGCATACTACATTTACTCGCAACTAAAGGCTGCATCGTAGAAACACCAACTGCAGTGGCTGAGGTACCAGGACACTTGCAGTTCTTTGTGGATATTAACCCACTGCCCCACCTGGAGCTGATGTCAGGGGATAGCAAAGTCATACTGATACAACCCACTCCACTCTGTTTGCCCCAGGTGAAGCACAGACACCCACACAGGAAGGAATTTTGGATTAAGTTTCCCTAGCAAGATAGGACAAACTATGCAGTGGTTTGGGGAAGCTCCACAGCAAATTCAGCTTTGAAATGCTCTGCATTAGCTAATTTAACAAGAGATAACAGAGCCATGTACTTGATCAGCAAGTCGAGGAGATTAGCTGGGCAAAACCAGAAGATGACCTCAGTGTTTCAGACATGAAACAAAGTGGAGATCCAAGGTGGAAGTGCCTAACAGCAAGAAAACAGGGCTACAAAAACCCAAGGCAGCATAAACTAGTTAAATGAGCGAGGGTTTACAGTTCCTGGTGGAAGAACACTTGCTACTGGCCTTCAAGTTGATACCCCCTGCCTTCCTTCCAGTTTTGTTCTCTTAATTACTCATTCTGACCTCTAGCAAAAAATATCACTTCCTGCTAAAGTTCACAAAAACAAGGCTATTAATTGGGAAACTCATCAACATGCTAATAAATGGTTTCTAAAATATGTACTGAGGAAACCATAGCAATGGAAACAACAGTGGCTTGACCCTGCAGGATTAATGTGCTTAACTCCAACACGAACATCCTGTTGACGATTATCCAACCTTTCAGACATTGCCACATCAGCAGATCTTTCTCAAAGCTAAGTTTAAATTAGGAAAAAAACACATGCAAGATTTGTCTTCTTTACGGGAGCAATCAAGCTGCTATCTAGCTGCTACCTGAAAACTAACAGCATGGCTTCAGAACTCTCCCTGTTTAGATTTGCAGTATTTTCTTCTCCTGCCCTCCCTCCTTCAGCACACACATGCATGAACTTCCCACGCTTCCTTACTATTCTTGCTGTTCTCTCCATCCTCCATGTCTAGTTTTTGTCAGTCTTTCAGTCCTAACGCTTCTCTGGGGTTTTTCATCACAGTTATCAAAAGGTAATGGCCTTACCACACAGTTTATTACTGTATATCCAGTTACTGGGTTAGGTCACACTAAAGCAATTACCAGCACGCCTACCCTTGGATTACAGCATATGCTTCTCTGAAAAGCAGGTTATTAACCACCTTGTCCCAACCACAAGATGAAAGCAGATGCAGTTGGCCACCATGAAGGTATGATGTCAAGGAAGTTAATTAGCATCAAAGCCAAATCACTCCCCAAACAATCCCTCCTTTGCATCCATTTGCAGACAACTGTTGTCACGAAGAAAGGCATTCCTCCATTTATCCTCCTTCTTACCTTTTCTACTTAATGAGCTTTGGCTTACATCCTGGTGAATCTCTTTAGTGGTACAGGAAAGGGAAAGAAGAAAAAGAAGTTTGGCTGCTTGCCATTACTACGGGCAGGCTGCGTGACAGAACAGTGGCACTGTCTACGCAGAAAGCTATTAAGAGCACAAGGTAAAAAGCACAATGCAAGCTACATTTCTTCTGCAGTCTACTTGCAATGGATAAGATGCACAGTTTTAGGTTCTGTCTATTTAGGGCTAAGCTTCAAAAAAAAAAAACCCAACAGTAAGGGTGGTTATATACCAAATCACTTGAAACTTTTACTTGTTTCATTCCAAACCCCTCTGAGTCCTTTCTGTCTGCTAGCTTAAATAACACCAATAGACATGACACTTTTGCACATTCCTCGTAGCAGGCTACATTCTCAGATGAGTGAACACGGGTACAACCTCAGGTCGAAAGCTGTGCTGAAAAAAAGCATGAAATTGAACAAAAAGAAAACTCAAGCAAACAAGAAAGTAGAAAAAGATGTTTATTGTTTTTAAACAAACAAAAGAACACGAAGGGACAACTGACTCTACCAAATTTTCACAAGAATTTAAAGTGGTGATTGACCTTGACACACCCTTTGAAATTTAATATTTACTAAATCAAGAAGCAAGCAGCTCTTCTGAGTGCACTTACGCAGAAACAGTTAAAATTTCATTTCACTGTTAAATGCCACACGCAGCAGAGCCAGACTCACCCACCTCCTCCTGGTTGGCTCATTCTCATCTTTCAGAGCTGAAGACTGCCTCTTCCTAGGCCGTCTCCTCTGGGATGGCGTTTTGGGCATCAGGCCAGGTTCAGCTCTGAAGTTGCTGCTGAAATGCAGAAAGCAGAGGCTGCATTCCACTGCTATCTGCAAGTCACGCTGCCCACCACCCAGCCGTAGCAATGTTCACCCATTAAGAGCAACAGGGGAGAAGCAATTGGGGTGTAACACCACCTAGCACTAATGCCTCAATGAAGCTGCTAAACACAGTAAATCCCTCACTCAGCTGAACAGTGCACTATCCCTAGGCTCACGCCAGGGCTGTGCTACCAGCAGCCAGGAGCACACACAGCCTTATGCTCTCAGTGTGTGAGAGCATCCTGGGAGGCTCCCTGGAGGGCAGCCCCTACCTATCCAGCATCCTCAGGCCCTGCTCCTCCACCTCCCGCAGCCAGGCCAAGTGCTTGTGCTCAGCATCGTGAAGGAACCTGGAGAGTCTCTGCCCACACACTTCCAGCAGCCGCATGGGACCGTCGGCTGCCGCCATGACCCTGCCTGAAACACAAGAGACAAAATGCAAACCTTCGAGGATACAGAGGGTGACCAAAGCCCACTTTGTGCCTCACAGCAGGGCACAAATGAGCCCAGCCGCAGCTCTAAGGCAGACGCAAACCCAGAGGACCGCCCTACTAGCTTTCCCCCAAGCCCGCAGGTCCAGACCCCAAGATGCCAACCCACTTCGCGCTCCCCTCACCACTCTCAGCAAGAAGCACCATACCCTCACCACTACGGCTCTGTCCCCCCCCTTTCCACTCCTGGTCCCAAATACCGTGGCTTCCACCAATCACGGTGCTTCCTCAGCGGAGCCCTCCCCACTACTGGCTGTCTCCCGCGTCCATCACTTCCCTACTGTTAAAATCCAGTGCCTATTGGTCCTCTACCCCAAGCCCCGCCTTCCACGTCCTCCACAACTACGGAGGCGAGCGAGCAGAAGGGAAGCAGCTTTTGAGAGTGCCGCGCCCGCCTCCTGACCAATGAGAAGGGCGGGCGGCGGGGAGCGCGGCGGCGATAGGCCAGCGCGGCCGTCACGATGGCAGAGTGGGAGGCGAGCGGTAGGTCCTGAATTCCAGGCGGACAGAGCTGCTGCGCGCGCGGTGGCGGCAAGCGAGGGGAGTGGGGTTGGGCCTGGTGTGCGCGCAGGGGGCGGTTGGGGCTGAGGTAAGCCGGAGCG

smc2 promoter sequence (from ENSEMBL chicken genomic DNA sequence databases): GACAGGAATTCGGATACATATGCATTACATTCCCGAGAGTCCATTAGCACGCAGTCCCTCCCCCCTTGCGCTTACATAGTTAAGTCGTAAAGGATGAAGGCTCATAGTCTTCCTCACTGCAAACTTCTGACCCCAAAGGGGGGTATCCCCCAAACCGGTTTCAGCTTGCCATGTGCACATCTAATCACCTCCCTGTCAAATGTTTTCGAGCTACTTTCCAGTCCTTCTCTTTATGGCCTTCATCCCCCTTGTTATTCCAGACCCAGTGTCTGTGAAAGTGCTTGGAATCCCTTATTTTCTAACACTCTCTACATAAACTATCTCTATTTGCCCCTATCTCAGTTTCTGTGAAGCTCCTTTCCCTTTTCTTACTGTGAGGCCCTTCCCTCTTTTACTGCAGAGTCCTGTTCTCATTCTTCACTACTCCAGCAAGCCCACTGTAGGATCATGTGGAAAATATAACTGATGTAGAGAAACTTAAAAAGTGGTTTTGTCCCCAGAAACAAAAACTACTGGAGTGTGGGATTTAAACATAAGGGATTAAAAAATGCAGGTCTCCTCTTACCTCGGCTTTGCAGATGGAGACCCACAAGATCCATATGATAAGCAGACAAAAGAGGAGTCTTGTGGTGCCAGGATGCTCCTGTTTTGGTTGAAGGATAAGGCAGATAATGGTTTCAGCATAGAGTCTTGAAGGCTTTGGTGTCCAGAATGAGAATGGTTTGTTATCTGTGAAATTAATATCGAAGTTAATTATTTGTAAATACGAATATGAGCTCTAAGAAGAACCTGACTATAATACTTGATGCTCTACCCAAGTATTGCTAAGGTTAGGAAAGTATTTTAGGGAGGCATGGGGACTGTAAAGGGGAAGGGAAAAGAGACTAAATGACAGAAGCAAGGAGAGAGACAGAAGATGTGGGAAATTCTGCTCTTCCTCTTCCACTATAACAGAAATGCCTTCCTGGTAAGCACTGTGCCCTCACCCCTTGGTGAAGAGCACACAGGACAGCATTTATTTTGCTAACACTGTTATCATATATTAGCACAATTGCACCTAATATATTTATCAACGGCAGTCCTGTGTCTGTTGTATCTGTCAGTTCAACAAATGTTCTATGTTTTTCACATGTTTGTGAAGCTGTTCCAGTGAAAGTCTGTGGGGGTGCTTACATACAAAATCAAAATACTCAATGCCATAAGGGGAGATTTGCAGCTTTCCACAGATGCTTAGTAATGAGAGTACAGAGAAAATGGAAATCAACCCATCTGAGGAGAGTGCATAGCAGTGGAGTAAATTTGCAAAACAGGAATTTCCAGTGAGATGCAGAAAGTTTTGTCTTTTATCCCTCAAACCACGAAATGATCAGATATCAGAACAGGTTGCCTGAAGAGATTGTAAAATCTCTGTCTTCTGAAATGTTCCTAGCTTGACAGGCTTGTGAACAGTTTGAGTTGTCTGGCTTGGTTTTGACCATGTGCTGAGGCTAGAAGACTTCCAGAAATGCCTATCAACTTAAATTATTCTATAACTATGGCATAATGGAAACTGTACTCACAACTTATAATTAGCTCACTGTATAACAAAGAGTGCTTGCTTGGTATAAGCAACTGCAAACATTAACACACTGTGAACTAAAGCAAGTGTTAGGAGCCAGGAAAGTTGTGACTTCATATTAATTTATATTGTTTTTATTTATTAAAAGTTTCTAGGTGACACTTCTCAAATCAGCTGAAGAAACAGAAAAACCCCATGGGAAATTTACCCTAGTGGCACAGACTTTAATATCTTTCTTCAGAACCAAGTCCCGAAAGGAAAAGGAACAATTATCTGTTCTATACAATTAACAGCATTACAGTATGATTTTCAAAGAAACCCCAAACCATTAAAATTACAACATTTGTCTACCCAAAAGGCTTTGTTCCATCACACACACAAAAAAAGGTGTTGCAGTACCAAAATAGCACATTTTCTTCTCTGTATCTACTGTAACATGCCTTTAATTCTTATGAGATACAACATCTGCCAGTGAAAAAAAGGAAAAGAAAAAAATAAGGATCTTCCTGAATTTATCATATCACTAACCTGTGCTTTATTATCATGTATTTATTAAAATCAACTCCATTCTCAATCCCTAAAATATTCTTTTCATAAAGCAATGAAGCCATACTATATGTTTACCTCGCCAGAATATCTATTTGTGACTTCAGAGTTAGTATCTGATAAGTATTTTGTATAATGCACATTATGGTGTAATAAAACAAAAAATGTGCCAAAATAAACGGTTTCTCCTTCTTCCTCAAACAATGTGCTATATTAATTTCATCATACTAATTAGATGTTAAGTAGCTGCAACGTTTTCTCCTTAAGACAGAGCACTGTCATTAAAAGAGTACATTTTATCACGACATCAAACAAAGTACCCAAACTTGTACGACATTTGAGACTACAAACATAATACAAAAACACTTAAGTAAGGATGAAATTATCCCTCCTGCACACGTCACTTACTCTAGCTGTGAAGTCCCTGGTTAAGGATAGAAAGTACTTATTTTTCTGATGCCTAACACAACCATAGCTCAGCTTGTTTGTAATGAGCCTTCAGCAATACCAGAAATAGTTCTTCCTTATTTCCATTTTTCTTTCCTCCAGGTAGGATTTTCAAATTGGTAACACTAAAGCCCAGCGGGCAAAGGAATACAGAACTCCATCTGCAATACACCGGTCTATTTTTACAGTGTAACAGCAGCTTCGAGGAAACGCCTAGACCTAAAGAACACAGCCTAAGCGGGACGCTGAAACTCCCAATTATACTCACCCCACCTCCCTCAGGGGCGGAGATCCGCCATTTTTGCGCCTTCCCGCCCAGACTTGGGAGGACCTCGCCGCGCCTTTTCGCCCAGCAGGAGTGCAGGGGACCTTCAGCCTTTGGGGCTGCTCTCCATACTCGCCTGATGTTATGGCTCCTTCCGCCCCAGGAAACAGAGCCTCTACTTTCTCACTATGCCCTATTTTTATTTTAAAAGAATAAGTAAGTGGATTGAACATCTCTCTTCCCGTCCTGGGGGAAGTAGACCAGACAGACCGAGACCTCTTTGTCAGGCAAAGTTTTGGGCAGCCATAGAAGGGCACCTCCTCCAAGGTCCGCTTCCTCTCGGCGCCAAAATTCAAACGTTAGTTGTGACGAGCATGCGAGAGGGGTTCGGTGGCTGCTGGCGGTTCACTGAGGGCTCCCTTCGGTTGGTACCTGCAGGGCTGCGAGGCGGTTTGGTGGGGGCAAAGTGCAGTCGCGGGGCTGTGTTGGTCGAGGTGCATGGCAGCGTCTCCCTGGGCTCCAGTGCGGTAGGCGGGAATCTGCGGTGGGTGAGTGGGGAGAGACCCGTCCTTTTGGCCCCCAGATCGTACTGGAGTCAGAGTAACCCGAACGGGGAGGGGGATCATCGAGTCCAACTCCCGGCCCCACACGGGATAACAACCGTAATGACCAGCGTTATGACAAGAGTGGGCTGTAGCTATAGCGATGAACAGTGGGTGAAGACACGTTGTTTAAAGAATTTAACGTACACAGGGTTGTTGGGATGCTCACTGGATCTGCGGTCCTTCGTGGTTTTGTTTAAAAATGCATGCTATTTTTGTTCTCAGACGTGTAGCTGTGTTAGGCATACGGTACAACTTGTTTCTTTTGTATGTGTGGGTTTTTTTATTATTTTTGTTTTTGTGGTTTTTTTTTTTTTTTTCCTTCAGAGCTCGAAGAGTTACAGTCCAGTCCTTTCCTTGTACTGCTGGGGGAAGCACCAACGTGGCCCCCTGACAGATTAAAGA

Southern Blotting. Typically, 10 mg of genomic DNA was digested overnight with the desired restriction enzyme and loaded on an 0.8% agarose gel. The DNA was then transferred to Hybond-N membrane (Amersham Pharmacia) and blotted with each probe.

Quantitative RT-PCR Analysis. Quantative RT-PCR analysis was performed as previously described (9). Primer pairs detecting expression of kif4a (gacgccaccgtgtgtgag and ctcaccagcagggaagca) and actin (tgctgtgttcccatctatcg and ttggtgacaataccgtgttca) were used as a control.

Detection of KIF4A Alternative Spliced Forms by RT-PCR. DT40 cDNA was prepared from total DT40 RNA by using an oligo-dT primer. The first PCR reaction was performed by using exon 1 forward primer, a (cttacgactacgtgttcgaa) and ENSEMBL exon 10 reverse primer, b (ctcagccctctgctcagc), producing a single band. Next, a second PCR was performed to amplify each splice-variant by using the first PCR product as template. Primers were as follows: exon 1 nested a single forward primer, a' (cgtcggttgagcaggagg); ENSEMBL exon 8 specific reverse primer, c (ctgaaccaaaaggaatgtctc); ENSEMBL exon 9 specific reverse primer, d (tgaagtaatttgctccccc); EST extra exon-specific reverse primer, e (taatctgaattcaaagtctctgagg). All PCR products were gel purified, cloned into pGEM-T easy (Promega), and sequenced.

Annexin V-PE. After addition of doxycycline, cells were harvested at the indicated time points. Reactions were performed according to the manufacturer's instructions (BioVision) and analyzed by flow cytometry (Becton Dickinson).

Immunoblotting Analysis. Total cell extracts were separated by SDS/PAGE and blotted onto nitrocellulose membranes (Amersham Pharmacia). After blotting, the membranes were stained with Ponceau S. Membranes were blocked with 5% skimmed milk in PBS and processed for enhanced chemiluminescence by standard methods.

Indirect Immunofluorescence and Microscopy. Cells were fixed by using 4% paraformaldehyde (PFA) for 5 min in cytoskeleton buffer (CB; 137 mM NaCl, 5 mM KCl, 1.1 mM Na2PO4, 0.4 mM KH2PO4, 2 mM MgCl2, 2 mM EGTA, 5 mM Pipes and 5.5 mM glucose), permeabilized with 0.15% triton in cytoskeletal buffer, blocked in blocking buffer (1% PBS/BSA) and incubated for 30 min with the specific antibodies: first antibodies in blocking buffer (anti-Kif4A 1:500, B512 1:1000). Cells were washed three times in PBS, fluorescence-labeled secondary antibodies applied (1:1,000 for alexa 594 and 1:500 for alexa 448 Molecular probe_Invitrogen) and counterstained with DAPI. Alternatively, cells were rinsed with PBS and fixed with cold methanol: acetic acid and incubated for 30 min with the specific antibodies: first antibodies in TEEN buffer [1 mM Triethanolamine·HCl (pH 8.5), 0.2 mM Na-EDTA, 25 mM NaCl] with 0.1% Triton and 1% BSA; anti-SBP 1:20, anti-Kif4A 1:500. Cells were washed three times in KB buffer [10 mM Tris·HCl (pH 7.7), 150 mM NaCl. 0.1% BSA], fluorescence-labeled secondary antibodies applied (1:1,000 and 1:500, 448 Molecular Probes_Invitrogen), and counterstained with DAPI. For live imaging, cells were grown on coverslips treated with 0.25 mg/ml Con A (Calbiochem). Images were taken every minute at 0.5-sec exposure and 32% neutral density. One focal plane was imaged from metaphase to anaphase onset. Subsequently, 10 focal planes (0.4 mm each) were imaged from anaphase until cytokinesis of the same cell. The two datasets were then combined. Three-dimensional datasets were collected with a Delta Vision system (Applied Precision) based on an Olympus IX-70 with a Chroma Technology Sedat filter set driven by the SoftWorx software under standard conditions. All of the image files are obtained as raw (r3d) and deconvolved (d3d) files readable by the SoftWorx software. Three-dimensional datasets were converted to Quick Projections in SoftWorx and then converted to TIFF files and imported into Adobe Photoshop for final presentation. Levels were adjusted across each entire image to lower nonspecific background haze by using the standard Photoshop adjustment of levels.

Construction of Triple-Affinity Purification with GFP tag (TrAPGFP). pTrAP plasmids containing a His tag, S-tag, and streptavidin-binding pepide (SBP) tag (10) were designed in our laboratory. Double stranded primers (gtcgacgctagcatgcatcaccatcaccatcacatggacgagagaccaccggctggcaaggcggccacgtggtggagggcctggccggcgagctggagcagctgcgggccaggctggag and gctgtccatgtgctggcgttcgaatttagcagcagcggtttctttgccgcgccgccgggctcccgctggccctgagggtggtgctccagcctggcccgcagctgctccagctcgccggc) were amplified by PCR with primers (gtcgacgctagcatgcatc and gtcgagggatccgctgtccatgtgctggcg). The PCR product was cloned (NheI/BamHI) into the NheI/ BglII sites of pEGFPC1. The NheI/XmaI fragment of the pTrAP was cloned into NheI/AgeI sites of pEGFPC1 to generate pTrAPGFP. The pTrAPGFP PCR fragment amplified with primers (gtcgacgctagcatgcat and gtcacgcgttctagatccggt) was inserted into Acc65I(blunt)/MluI sites within the kif4a promoter hijack vector.

1. Earnshaw WC, Halligan B, Cooke CA, Heck MMS, Liu LF (1985) Topoisomerase II is a structural component of mitotic chromosome scaffolds. J Cell Biol 100:1706-1715.

2. Baron U, Gossen M, Bujard H (1997) Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. Nucleic Acids Res 25:2723-2729.

3. Mazumdar M, Sundareshan S, Misteli T (2004) Human chromokinesin KIF4A functions in chromosome condensation and segregation. J Cell Biol 166:613-620.

4. Mazumdar M, et al. (2006) Tumor formation via loss of a molecular motor protein. Curr Biol 16:1559-1564.

5. Kurasawa Y, Earnshaw WC, Mochizuki Y, Dohmae N, Todokoro K (2004) Essential roles of KIF4 and its binding partner in organized central spindle midzone formation. EMBO J 23:3237-3248.

6. Zhu C, Jiang W (2005) Cell cycle-dependent translocation of PRC1 on the spindle by Kif4 is essential for midzone formation and cytokinesis. Proc Natl Acad Sci USA 102:343-348.

7. Sugawara H, Kurosaki M, Takata M, Kurosaki T (1997) Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor. EMBO J 16:3078-3088.

8. Li F, Altieri DC (1999) Transcriptional analysis of human survivin gene expression. Biochem J 344 Pt 2:305-311.

9. Vagnarelli P, et al. (2006) Condensin and Repo-Man-PP1 co-operate in the regulation of chromosome architecture during mitosis. Nat Cell Biol 8:1133-1142.

10. Keefe AD, Wilson DS, Seelig B, Szostak JW (2001) One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, SBP-tag. Protein Expr Purif 23:440-446.

This Article

  1. Published online before print February 8, 2008, doi: 10.1073/pnas.0712083105 PNAS February 19, 2008 vol. 105 no. 7 2457-2462
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