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Fig. 1. Northern blotting of additional KIFs. KIF2B is expressed ubiquitously in 2-week-old mouse tissue. KIF16B mRNA is detected in testis as a 4.1-kb band and a 3.3-kb band in brain. KIF18A was found in adult brain and embryonic head. KIF18B expression is dominant in testis. KIF19A is detected in testis, lung, and brain. KIF24 bands are seen in testis and spleen lanes. Ts, testis; Si, small intestine; Kd, kidney; Ht, heart; Br, brain; Sp, spleen; Sc, spinal cord; Lv, liver; Lu, lung; Pa, pancreas; Eh, embryo head; E, embryo; Sm, skeletal muscle; St, stomach; Ty, thymus; Ov, ovary.

exhaustive TBLASTN search was conducted to detect KIF transcripts in the complete human genome. After obtaining all KIFs, the amino acid sequences between IFAY and LAGSE motifs were subjected to phylogenic analysis. In Fig. 2, human and mouse homologs were aligned with CLUSTAL W (13) software by the neighbor-joining method (14). The phylogenic tree was drawn with MACVECTOR software (Oxford Molecular, Cambridge, U.K.). For Fig. 3, maximum parsimony was calculated (15), and the phylogenic tree was drawn by using TREEVIEWPPC (16). Bootstrap values were assessed by 10,000 random samplings. Classification of all KIFs were carried out as described (2). Sequences used in this study can be obtained from our supplemental data or through the Celera database public segment (found on the web at www.celera.com).

The KIFs presented here were identified by the following criteria: conservation of upstream Walker A ATP-binding motifs and a LAGSE or similar sequence ˜150–200 aa residues downstream, a YXXXXXDLL motif where X is any amino acid, and a SSRSH motif located between the Walker A and LAGSE sequences. Two predicted transcripts contained only the LAGSE consensus, and two transcripts had only IFAY sequences. In other organisms, a gene encoding only the LAGSE region and another encoding only IFAY were found in Drosophila. The prediction of KIFs using conventional software will automatically predict LAGSE-containing proteins to be KIFs. However, in a previous study it has been implied that the motor domain cannot be separated into modules (17). This indicates that IFAY and LAGSE sequences must be present at an appropriate order and spacing. Therefore, sequences lacking conserved motifs may not function as molecular motors and were excluded from this study. Additionally, genes from the same locus were considered to be splice variants and were omitted.

Results and Discussion

KIFs Previously Identified in Our Laboratory. Previously, we have identified 25 KIFs in mice (11, 1822). Most were found by using molecular biological approaches. This study presents all KIFs in mouse and human and concludes the search for further unidentified KIFs.

Identification of 13 Additional KIFs. We report 10 previously unidentified KIFs. KIF18B, KIF19A, KIF23, and KIF24 were identified in adult mouse brain, spinal cord, and small intestine cDNA by PCR. KIF23 has been reported in humans (23), but we have isolated it from mice by using PCR. KIF2B and KIF18A were found in embryonic cDNA by PCR. KIF4A and KIF4B, and KIF19A and KIF19B have a highly homologous motor domain. Therefore, it was difficult to discriminate the differences between them by PCR. These paralogs were found by using KIF4A and KIF19A amino acid sequences, respectively, as a template for BLAST searches. KIF26A and KIF26B also were discovered by BLAST searches using ScSMY1 as a template. SMY1 has amino acid motifs similar to KIF motor domains, although it may not be functional (24). With the exception of KIFs with motor domain sequences similar to that of SMY and therefore having low conservation in amino acid motifs, we were able to identify all KIFs by PCR.

KIF2C (25), KIF20B (26), and KIF25 (27) have been reported in humans and not in mice and were found in mice by our database search.

KIFs presented in this paper and previously identified KIFs were found by cross-hybridization methods or PCR using degenerate primers (11, 18, 28). These methods are laborious, hazardous, and time consuming. With this report there is no further need to search for new KIFs. However, the actual number of functional KIFs can only be determined after completing endogenous protein purification, peptide sequencing, and motility assays.

Tissue Distribution of Additional KIFs. As part of our preliminary results concerning novel KIFs, Northern blotting results of KIF2B, KIF16B, KIF18A, KIF18B, KIF19A, and KIF24 are shown in Fig. 1. KIF2B is ubiquitously expressed in 2-week-old mice at 2.8 kb. KIF16B displays an intense 4-kb band in the testis lane and a 3.3-kb band in the brain lane. KIF18A is expressed in adult lung and embryonic head. The band migrates at 4.6 kb. The 3.9-kb band corresponding to KIF18B is most intense in adult testis. It is also highly expressed in the spleen and thymus and weakly in kidney, liver, lung, skin, small intestine, and stomach. No signal is detected in adult brain, heart, and muscle. KIF19A transcripts are found in adult testis, lung, and brain. A strong doublet band can be seen in the ovary lane. There are also bands in the embryo and spleen lanes. The higher band in ovary corresponds to a protein of 4.6 kb, the lower band to a protein



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